Soil was treated with 6.7 kg MSMA ha-1 to induce straighthead.

†† Yield d ifference = Treated yield - Untreated yield, and *P* is probability of *t* test for the difference.

‡‡ Lodging 1-9 scale: 1 as no plants lodged and 9 as over 80% plants lodged.

**Table 2.** Nineteen Chinese rice germplasm accessions had no significant yield reductions from straighthead induced by MSMA (monosodium methanearsonate) at 6.7 kg hn-1 in 2001 (Yan et al., 2005).

The soil to induce straighthead by application of MSMA for research purposes was studied by Yan et al. (2008) (Table 3). In the straighthead evaluation soil amended by MSMA, pH and Mehlich-3 extractable P, Ca, Mg, Fe, Zn and As concentrations are significantly lower, while S, Mn and As are higher than those in the native soil where MSMA has never been applied. However, soil electronic conductivity, organic matter and K, Na and Cu concentrations are not affected by the amendment of MSMA. Decreased soil pH resulted from the MSMA is significantly associated with decreased Ca (r=0.92), Mg (r=0.78), and P (r=0.41), but increased As (r=-0.87), S (r=-0.73), and Mn (r=-0.59) concentrations in the soil.


† EC, soil electrical conductivity.

‡ SOM, soil organic matter.

§ Means in each column with the same letter are not significantly different at the 0.05 probability level

**Table 3.** Soil properties and minerals for samples collected from the straighthead designated field before (Before MSMA) and after (After MSMA) the application of 6.7 kg MSMA ha-1 in comparison with native soil sample which never receives MSMA application (No MSMA) in 2004 and 2005. (Before MSMA soil received MSMA application previously for straighthead studies) (Yan et al., 2008).

## **3.2. Prevention methods in rice production**

The sporadic nature of straighthead and the lack of a specific and definite causal factor have made straighthead difficult to be prevented. Since 1950s, rice researchers had tried to prevent straighthead using chemical application. Evatt and Atkins (1957) applied Feralum, a mixture of ferric and aluminum sulfates to soil for controlling straighthead. In Portugal, Cu deficiency was found to be associated with straighthead (Karim and Vlamis, 1962), and application of copper sulfate to the soil when seedlings were transplanted was reported to prevent or greatly reduce straighthead (Cunha and Baptista, 1958). Ricardo and Cunha (1968) studied copper sulfate as a supplier of Cu for straighthead control since soil organic matter may bind Cu and reduce its availability for uptake by plants. However, chemical prevention never reaches applicable scale because an effective chemical has never been developed, so the control effects are not stable.

A water management practice that is called 'Draining and Drying' was developed by farmers in the early 1900s (Atkins et al., 1957; Slaton, 2001), and is currently used as the only recom‐ mended method to prevent straighthead in rice through DD50 Computerized Program and agricultural extension system in the USA (Wilson et al., 2010b; 2010c). Rice fields are drained about 2 weeks after a permanent flood, dried thoroughly until cracks appear in the soil and rice leaves begin to curl and exhibit yellowing as drought stress symptoms, and then re-flooded for the remainder of season. The drying must be completed about 10 to 14 days before the internode elongation starts (Wells and Gilmour, 1977), and the best timing could be predicted by the online DD50 Program http://dd50.uaex.edu/dd50Logon.asp. Fields that favor straight‐ head are permanent, which means each time when rice is planted, straighthead will develop at some level to cause yield losses if the flood is not drained for the soil to be aerated at appropriate time (Wilson et al., 2010c). Soil aeration is believed to speed the decay of soil organic matter (Editor's Note, 1946) and help oxidize arsenic (As) into arsenate, which is biologically inactive (Marin et al., 1992). Therefore, once straighthead occurs in a field, growers will keep using the Draining and Drying method permanently because of unaffordable consequences.

Table 1 shows cultivar variation on yield recovery of the Draining and Drying from the traditional-continuous flood. Long grain type cultivar Cocodrie and medium Bengal are high recovery cultivars with about 80% of the recovered yield. Cypress, Drew and Madison are the intermediate recovery cultivars with more than 40% of the yield to be recovered by the Draining and Drying. Jefferson, Priscilla and Wells are the low recovery cultivars because they display certain resistance to straighthead.

Currently, the Draining and Drying method is applied to more than one third of the rice acreage in Arkansas as a preventative measure (Wilson, per. Comm.). Using Arkansas rice harvested area of 723,000 hectares in 2010, K.B. Watkins, agricultural economics professor in the Uni‐ versity of Arkansas, Rice Research and Extension Center, made the following estimates: \$ 9.21/ ha for additional labor cost to open levee gates for the draining, \$ 20.93/ha for power cost to water the dried fields afterwards, and \$ 56.77/ha for additional application of fungicide to control blast since blast disease is known to be more severe in fields or parts of fields in which the water in paddies falls below recommended levels (TeBeest et al., 2007). As a result, straighthead prevention added either \$ 7.264 million for the draining and reflooding only or \$ 20.945 million for the draining, reflooding and blast control to rice growers in Arkansas. Furthermore, an additional 308.4 m3 of water are required to re-flood each hectare after drying, which resulted in an extra 74.324 million m3 of water utilized for straighthead prevention in Arkansas in 2010. Wasting water is becoming a public concern because Lonoke, Prairie, Arkansas, and Jefferson counties with 150,317 hectares of rice in 2010 have been designated as having critical levels of groundwater (Riley, pers. comm.). Thus, preserving the natural resource of water is important for the long term economic viability of these counties. Therefore, the Draining and Drying method for straighthead prevention is costly for rice growers and wasteful of natural resources, and results in drought-related yield loss.

## **3.3. Resistant germplasm for straighthead breeding**

significantly associated with decreased Ca (r=0.92), Mg (r=0.78), and P (r=0.41), but increased

As (r=-0.87), S (r=-0.73), and Mn (r=-0.59) concentrations in the soil.

§ Means in each column with the same letter are not significantly different at the 0.05 probability level

**Table 3.** Soil properties and minerals for samples collected from the straighthead designated field before (Before MSMA) and after (After MSMA) the application of 6.7 kg MSMA ha-1 in comparison with native soil sample which never receives MSMA application (No MSMA) in 2004 and 2005. (Before MSMA soil received MSMA application

The sporadic nature of straighthead and the lack of a specific and definite causal factor have made straighthead difficult to be prevented. Since 1950s, rice researchers had tried to prevent straighthead using chemical application. Evatt and Atkins (1957) applied Feralum, a mixture of ferric and aluminum sulfates to soil for controlling straighthead. In Portugal, Cu deficiency was found to be associated with straighthead (Karim and Vlamis, 1962), and application of copper sulfate to the soil when seedlings were transplanted was reported to prevent or greatly reduce straighthead (Cunha and Baptista, 1958). Ricardo and Cunha (1968) studied copper sulfate as a supplier of Cu for straighthead control since soil organic matter may bind Cu and reduce its availability for uptake by plants. However, chemical prevention never reaches applicable scale because an effective chemical has never been developed, so the control effects

A water management practice that is called 'Draining and Drying' was developed by farmers in the early 1900s (Atkins et al., 1957; Slaton, 2001), and is currently used as the only recom‐ mended method to prevent straighthead in rice through DD50 Computerized Program and agricultural extension system in the USA (Wilson et al., 2010b; 2010c). Rice fields are drained about 2 weeks after a permanent flood, dried thoroughly until cracks appear in the soil and rice leaves begin to curl and exhibit yellowing as drought stress symptoms, and then re-flooded for the remainder of season. The drying must be completed about 10 to 14 days before the internode elongation starts (Wells and Gilmour, 1977), and the best timing could be predicted by the online DD50 Program http://dd50.uaex.edu/dd50Logon.asp. Fields that favor straight‐ head are permanent, which means each time when rice is planted, straighthead will develop at some level to cause yield losses if the flood is not drained for the soil to be aerated at

† EC, soil electrical conductivity. ‡ SOM, soil organic matter.

224 Rice - Germplasm, Genetics and Improvement

are not stable.

previously for straighthead studies) (Yan et al., 2008).

**3.2. Prevention methods in rice production**

Varietal resistance is regarded as the most efficient, economical, and environmentally friendly strategy for straighthead prevention (Wilson et al., 2001; Yan et al., 2005; Dunn et al., 2006). The earliest attempt at breeding for straighthead resistance in the USA started in 1950s (Atkins et al., 1957), but little progress had been made because the inheritance of straighthead resist‐ ance had not been well understood because of limited resistant germplasm until 2002 (Yan et al., 2002).

In 2001, 124 accessions of germplasm including 109 *indica*and 15 *japonica* cultivars introduced from China were evaluated for straighthead resistance, and 19 showed resistance to straight‐ head (Table 2) (Yan et al., 2005). Seven had increases of grain yield from 134 to 1115 kg ha-1 under the influence of straighthead, and the other 12 had reductions from 7 to 1197 kg ha-1, but all the increases and decreases due to straighthead were not significant. Their straighthead


**Table 4.** USDA core collection number, plant introduction (PI), cultivar name and country of origin, average rate of straighthead in 2003 (SH03) and 2004 (SH04) for resistant accessions rated 4 or less on a 1-9 scale and their positions in principal component analysis (PCA) (Agrama and Yan, 2010).

ratings ranged from 1 to 3 while susceptible check Cocodrie and Mars were rated 8 and 7, respectively.

All the resistant cultivars are *indica*. In terms of the cultivar 'Jing 185-7', ('Jing' means *japoni‐ ca* in Chinese), a study has indicated that Jing185-7 is an *indica*(Agrama and Yan, 2010). Nine accessions of the resistant germplasm are in the very early group having 63 - 69 days to heading except Dian No. 01, two in the early group having 83 days to heading, two in the intermediate group having 89 - 90 days to heading, and all six in the late group having 90 or more days to heading except Jing 185-7. Preliminary observation of days to heading had incorrectly classified Dian No. 01 in the very early group. Plant heights vary from 89 cm for Tie 90-1 in the very early group to 133 cm for Sheng 12 in the late group. Two accessions, Zanuo No1 and Jinnuo No6, are waxy endosperm type containing no amylose, and the other seventeen nonwaxy accessions have amyloses ranging from 14.8% for Shufeng 121 to 27.0% for Shufeng 109 in their endosperms. Aijiaonante is the first semi-dwarf cultivar bred in 1956 in China (Qian and Liu, 1993), and Zhenshan 97 is a popular maintainer line of hybrid rice in China (Virmani, 1994).

In 2002, 1002 accessions selected from 1794 accessions of the USDA Rice Core Collection (Yan et al., 2007; 2010b; Agrama et al., 2010) were evaluated for straighthead resistance in Arkansas (Agrama and Yan, 2010). These selections have proper maturities ranged from 48-110 days and plant heights ranged from 65-150 cm because the maturity and height largely affect the assessment of panicle fertility, which is essential for straighthead infestation. Those rated 4 or less in the 2003 straighthead evaluation were verified in larger plots and more replications in 2004. In total, 42 accessions (4.2%) displayed resistance (Table 4).

The 42 resistant cultivars originate from 15 countries in ten geographic regions worldwide, with the most (24 or 57%) from China, are classified into 5 clusters (Fig. 3) (Agrama and Yan, 2010). Cluster K1 includes three references, indicating none of the resistant cultivars belong to *Deep water*, *Australian* and *Aromatic* type. K2 includes 13 *indica* cultivars referenced by Zhe733, all from China except entry 488 from an unknown country in Africa. Referenced by IR64, K3 consists of another group of 12 *indica* cultivars originated from six countries of five regions: China, South America, South Pacific, Southeast Asia and the Subcontinent. Four Chinese cultivars, entry 1467, 1475, 1502 and Shufeng109, are positioned between K2 and K3. K4 has two *Tropical Japonica* references only and K5 contains 11 *Temperate Japonica* cultivars originating from seven countries of four regions: Centeral Asia, China, and Eastern and Western Europe. Two cultivars are positioned between K4 and K5: entry 46 (GPNO 254) developed in Louisiana, U.S.A. and entered in the germplasm collection in 1977; and entry 1198 (WC 6570) developed in Spain and entered the collection in 1975.

ratings ranged from 1 to 3 while susceptible check Cocodrie and Mars were rated 8 and 7,

**Table 4.** USDA core collection number, plant introduction (PI), cultivar name and country of origin, average rate of straighthead in 2003 (SH03) and 2004 (SH04) for resistant accessions rated 4 or less on a 1-9 scale and their positions

All the resistant cultivars are *indica*. In terms of the cultivar 'Jing 185-7', ('Jing' means *japoni‐ ca* in Chinese), a study has indicated that Jing185-7 is an *indica*(Agrama and Yan, 2010). Nine accessions of the resistant germplasm are in the very early group having 63 - 69 days to heading except Dian No. 01, two in the early group having 83 days to heading, two in the intermediate group having 89 - 90 days to heading, and all six in the late group having 90 or more days to heading except Jing 185-7. Preliminary observation of days to heading had incorrectly classified Dian No. 01 in the very early group. Plant heights vary from 89 cm for Tie 90-1 in the very early group to 133 cm for Sheng 12 in the late group. Two accessions, Zanuo No1 and Jinnuo No6, are waxy endosperm type containing no amylose, and the other seventeen nonwaxy accessions have amyloses ranging from 14.8% for Shufeng 121 to 27.0% for Shufeng 109 in their endosperms. Aijiaonante is the first semi-dwarf cultivar bred in 1956 in China (Qian and Liu, 1993), and Zhenshan 97 is a popular maintainer line of hybrid rice in China (Virmani,

respectively.

in principal component analysis (PCA) (Agrama and Yan, 2010).

226 Rice - Germplasm, Genetics and Improvement

1994).

**Figure 3.** Unrooted neighbor-joining tree based on C.S. Chord (Cavalli-Sforza and Edwards, 1967) for 42 accessions resistant to straighthead rated 4 or less in a 1-9 scale and derived from the USDA rice core collection (Core entry num‐ ber used in the chart) and reference cultivars (McNally et al. 2006) (AUS-Australia, ARO-Aromatic, IND-Indica, TRJ-Tropical Japonica, TEJ-Temperate Japonica) genotyped with 72 molecular markers (Agrama and Yan, 2010).

## **4. Gene mapping and development of DNA markers for breeding**

## **4.1. Association mapping of quantitative trait loci (QTL) for straighthead**

Because of the sporadic nature of straighthead and its unidentified causes, molecular marker assisted selection is essential for improvement of resistance in breeding programs. To take advantage of recent advances in gene-mapping technology, we executed a genome-wide association mapping study to identify genetic markers associated with straighthead using 547 accessions of germplasm from the USDA rice core collection and 75 simple sequence repeat (SSR) markers covering the entire rice genome (Agrama and Yan, 2009). A mixed-model approach combining the principal component assignments with kinship estimates proved to be particularly promising for association mapping. The extent of linkage disequilibrium was described among the markers. Seven marker loci are highly-significantly associated with straighthead at a significance level of 0.0001 = 4.0 value of –log10q (Fig. 4).

The SSR markers RM263, RM105 and RM277 on chromosomes (chr) 2, 9 and 12, respectively, show very strong association with straighthead (*p*< 9.83x10-8, *q*< 1.31x10-6). Four other loci, RM490, RM413, RM116 and RM224 are highly associated with the disorder (*p*< 0.0001). Three alleles, each of marker RM490 (87 bp), RM413 (105 bp) and RM277 (122 bp), and two alleles (182 bp and 183 bp) of RM263 show significantly low straighthead rates of resistance. Only three accessions (core entry 748, 1344 and 1402) carrying allele 105 bp of RM413 have the lowest straighthead rate with the average of 3.9. Nine accessions with the allele 122 at RM277 on chr 12 (57.2 cM) have a significantly low straighthead rate (4.1). The rates of 15 accessions with allele 182 at RM263 (chr 2) are lower (4.6), on average, than the accessions with other alleles. Moderate straighthead rates are associated with alleles 87 bp at RM490 (23 accessions), 183 bp at RM263 (15 accessions) and 137 bp at RM105 (59 accessions).

**Figure 4.** Marker loci significantly associated with straighthead disease with a value of - Log *q* = 4 which indicates a correlation probability 0.0001 among 547 accessions of germplasm in the USDA rice core collection, which were phe‐ notyped in Arkansas and genotyped with 75 genome-wide SSR markers (Agrama and Yan, 2009).

#### **4.2. Identification of a major QTL for straighthead resistance**

We mapped the QTLs for straighthead using two recombined inbred line (RIL) F9 populations, one with 170 lines genotyped with 136 SSRs and another with 91 lines genotyped with 159 SSRs (Pan et al., 2012). These lines were evaluated for straighthead in both 2008 and 2009 with three replicates per year.

**4. Gene mapping and development of DNA markers for breeding**

Because of the sporadic nature of straighthead and its unidentified causes, molecular marker assisted selection is essential for improvement of resistance in breeding programs. To take advantage of recent advances in gene-mapping technology, we executed a genome-wide association mapping study to identify genetic markers associated with straighthead using 547 accessions of germplasm from the USDA rice core collection and 75 simple sequence repeat (SSR) markers covering the entire rice genome (Agrama and Yan, 2009). A mixed-model approach combining the principal component assignments with kinship estimates proved to be particularly promising for association mapping. The extent of linkage disequilibrium was described among the markers. Seven marker loci are highly-significantly associated with

The SSR markers RM263, RM105 and RM277 on chromosomes (chr) 2, 9 and 12, respectively, show very strong association with straighthead (*p*< 9.83x10-8, *q*< 1.31x10-6). Four other loci, RM490, RM413, RM116 and RM224 are highly associated with the disorder (*p*< 0.0001). Three alleles, each of marker RM490 (87 bp), RM413 (105 bp) and RM277 (122 bp), and two alleles (182 bp and 183 bp) of RM263 show significantly low straighthead rates of resistance. Only three accessions (core entry 748, 1344 and 1402) carrying allele 105 bp of RM413 have the lowest straighthead rate with the average of 3.9. Nine accessions with the allele 122 at RM277 on chr 12 (57.2 cM) have a significantly low straighthead rate (4.1). The rates of 15 accessions with allele 182 at RM263 (chr 2) are lower (4.6), on average, than the accessions with other alleles. Moderate straighthead rates are associated with alleles 87 bp at RM490 (23 accessions), 183 bp

**Figure 4.** Marker loci significantly associated with straighthead disease with a value of - Log *q* = 4 which indicates a correlation probability 0.0001 among 547 accessions of germplasm in the USDA rice core collection, which were phe‐

We mapped the QTLs for straighthead using two recombined inbred line (RIL) F9 populations, one with 170 lines genotyped with 136 SSRs and another with 91 lines genotyped with 159

notyped in Arkansas and genotyped with 75 genome-wide SSR markers (Agrama and Yan, 2009).

**4.2. Identification of a major QTL for straighthead resistance**

**4.1. Association mapping of quantitative trait loci (QTL) for straighthead**

228 Rice - Germplasm, Genetics and Improvement

straighthead at a significance level of 0.0001 = 4.0 value of –log10q (Fig. 4).

at RM263 (15 accessions) and 137 bp at RM105 (59 accessions).

**Figure 5.** Straighthead phenotypes in parents of mapping populations,resistant parents Zhe733 and Jing185 with fully developed panicles while susceptible parents R312 and Cocodrie with severely distorted spikelets (Pan et al., 2012).

**Figure 6.** Four QTLs for straighthead resistance are identified from RIL F9 population of Zhe733/R312 (a) and two QTLs from RIL F9 population of Cocodrie/Jing185, are marked by black bar (Pan et al., 2012).

Four QTLs were identified to be associated with straighthead resistance in the Zhe733/R312 population on chr6, 7, 8 and 11 (Fig. 6a). The QTL on chr8 had the largest LOD (23.0), highest additive effect (-2.1) and smallest marker interval (1.0 cM) between RM6838 and RM72, and explained the most total variation (46%) for straighthead among the identified QTLs. From the Cocodrie/Jing185 population, two QTLs were identified (Fig. 6b), one on chr3 (LOD=3.8), and another on chr.8 (LOD= 27.0). The chr.8 QTL is within a 1.9 cM interval between RM22559 and RM 72, has a -2.1 additive effect, and explained 67% of total variation. RM72 at 6.76 Mb is the most distal marker of the chr8 QTL identified in both populations. RM6838 in Zhe733/R312 and RM22559 in Cocodrie/Jing185 are physically located very close to each other at 5.85 Mb and 5.70 Mb, respectively. The overlapping intervals on chr.8 identified in both populations indicate the presence of a major QTL at this location, designated as *qSH-8* (Fig. 5a for Zhe733/ R312 and 5d for Cocodrie/Jing185).

## **4.3. Fine mapping of** *qSH-8***, a Major QTL for straighthead resistance**

Within the putative region of *qSH-8*, four recombinants (RIL12, 112, 174, and 306) are identified in Zhe733/R312 and four recombinants (RIL418, 423, 480, and 533) are identified in Cocodrie/ Jing185 population for fine mapping according to the substitution strategy described by Paterson et al. (1990). Using an additional 16 SSR markers derived from the Gramene database http://www.gramene.org/, and 9 InDel markers designed from the MSU rice genome browser http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/to compare the sequence of Nippon‐ bare with 93-11 in the targeted region,qSH-8 is fine mapped in a 290 kb interval between RM22573 and InDel 27 in the Zhe733/R312 population, and a 690 kb region between InDel 11 and RM22613 in the Cocodrie/Jing185 population (Fig. 7).

Three markers, SSR AP3858-1, InDel 11 and InDel 5 are in the 290 kb interval, and should cosegregate with *qSH-8* to predict either resistance or susceptibility of a rice line to straighthead. Both RIL 12 and 306 in the Zhe733/R312 population have the R312 genotype at AP3858-1, InDel 11 and InDel 5 loci, which matched up with the R312 phenotype, susceptible to straighthead with high ratings (8.7±0.5 for RIL 12 and 6.8±1.3 for RIL 306). Conversely, both RIL 112 and 174 have the Zhe733 resistant genotype at these loci, and have low straighthead ratings (1.6±0.9 for RIL 112 and 1.3±0.5 for RIL 174) as well. These results prove the hypothesis that cosegregation exists between *qSH-8* genotype and straighthead phenotype.

## **4.4. Marker development for marker-assisted breeding of straighthead resistance**

We have tested 72 accessions of global germplasm for a match between straighthead pheno‐ type and *qSH-8* genotype indicated by the markers AP3858-1 and InDel 11. The 72 accessions originated from 28 countries, and a large portion of them (22 accessions) were from China, followed by the Philippines and the USA. Forty of the tested accessions are resistant to straighthead with ratings of 4 or less, and the remaining 32 are susceptible with straighthead ratings of 6 or more based on previous studies by Yan et al. (2002; 2005) and Agrama and Yan (2009; 2010). For InDel 11, 30 accessions have either no alleles of or alleles different from parental Zhe733, R312, Cocodrie and Jing185. The remaining 42 have the parental alleles for InDel 11, where 32 genotypes have a good match with the expected phenotype (Table 5). For

Rice Straighthead Disease – Prevention, Germplasm, Gene Mapping and DNA Markers for Breeding http://dx.doi.org/10.5772/56829 231

Four QTLs were identified to be associated with straighthead resistance in the Zhe733/R312 population on chr6, 7, 8 and 11 (Fig. 6a). The QTL on chr8 had the largest LOD (23.0), highest additive effect (-2.1) and smallest marker interval (1.0 cM) between RM6838 and RM72, and explained the most total variation (46%) for straighthead among the identified QTLs. From the Cocodrie/Jing185 population, two QTLs were identified (Fig. 6b), one on chr3 (LOD=3.8), and another on chr.8 (LOD= 27.0). The chr.8 QTL is within a 1.9 cM interval between RM22559 and RM 72, has a -2.1 additive effect, and explained 67% of total variation. RM72 at 6.76 Mb is the most distal marker of the chr8 QTL identified in both populations. RM6838 in Zhe733/R312 and RM22559 in Cocodrie/Jing185 are physically located very close to each other at 5.85 Mb and 5.70 Mb, respectively. The overlapping intervals on chr.8 identified in both populations indicate the presence of a major QTL at this location, designated as *qSH-8* (Fig. 5a for Zhe733/

Within the putative region of *qSH-8*, four recombinants (RIL12, 112, 174, and 306) are identified in Zhe733/R312 and four recombinants (RIL418, 423, 480, and 533) are identified in Cocodrie/ Jing185 population for fine mapping according to the substitution strategy described by Paterson et al. (1990). Using an additional 16 SSR markers derived from the Gramene database http://www.gramene.org/, and 9 InDel markers designed from the MSU rice genome browser http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/to compare the sequence of Nippon‐ bare with 93-11 in the targeted region,qSH-8 is fine mapped in a 290 kb interval between RM22573 and InDel 27 in the Zhe733/R312 population, and a 690 kb region between InDel 11

Three markers, SSR AP3858-1, InDel 11 and InDel 5 are in the 290 kb interval, and should cosegregate with *qSH-8* to predict either resistance or susceptibility of a rice line to straighthead. Both RIL 12 and 306 in the Zhe733/R312 population have the R312 genotype at AP3858-1, InDel 11 and InDel 5 loci, which matched up with the R312 phenotype, susceptible to straighthead with high ratings (8.7±0.5 for RIL 12 and 6.8±1.3 for RIL 306). Conversely, both RIL 112 and 174 have the Zhe733 resistant genotype at these loci, and have low straighthead ratings (1.6±0.9 for RIL 112 and 1.3±0.5 for RIL 174) as well. These results prove the hypothesis that co-

R312 and 5d for Cocodrie/Jing185).

230 Rice - Germplasm, Genetics and Improvement

**4.3. Fine mapping of** *qSH-8***, a Major QTL for straighthead resistance**

segregation exists between *qSH-8* genotype and straighthead phenotype.

**4.4. Marker development for marker-assisted breeding of straighthead resistance**

We have tested 72 accessions of global germplasm for a match between straighthead pheno‐ type and *qSH-8* genotype indicated by the markers AP3858-1 and InDel 11. The 72 accessions originated from 28 countries, and a large portion of them (22 accessions) were from China, followed by the Philippines and the USA. Forty of the tested accessions are resistant to straighthead with ratings of 4 or less, and the remaining 32 are susceptible with straighthead ratings of 6 or more based on previous studies by Yan et al. (2002; 2005) and Agrama and Yan (2009; 2010). For InDel 11, 30 accessions have either no alleles of or alleles different from parental Zhe733, R312, Cocodrie and Jing185. The remaining 42 have the parental alleles for InDel 11, where 32 genotypes have a good match with the expected phenotype (Table 5). For

and RM22613 in the Cocodrie/Jing185 population (Fig. 7).

**Figure 7.** Fine mapping of *qSH-8* using Zhe733/R312 (a-c) and Cocodrie/Jing185 (d-f) F9 RIL populations. (a) *qSH-8* regionof 1.2 cM between RM6838 and RM72, (b) a 340 kb region between RM22573 and RM22589, (c) a 290 kb re‐ gion between RM22573 and InDel 27; (d) *qSH-8* regionof 2.8 cM between RM22559 and RM72, (e) a 710 kb region between AP3858-1 and RM22613 and (f) a 690 kb region between InDel 11 and RM22613 (Pan et al., 2012).

marker AP3858-1, 38 accessions do not have the parental alleles, and 25 out of the remaining 34 accessions have a good match between the genotype and phenotype. Because InDel 5 is monomorphic in the Cocodrie/Jing185 population, it is not desirable for screening the global germplasm collection. *χ<sup>2</sup>* test indicates a high association of InDel 11 with straighthead (*P*=0.0014), with 76.2% of the genotypes matching the phenotypes among those global accessions (Table 6). Similarly, AP3858-1 is highly associated with straighthead (*P*=0.0004) with a match of 73.5%. In the Zhe733/R312 population, all three markers (InDel 5, InDel 11, and AP3858-1) are verified by *χ<sup>2</sup>* test at the *P*<0.0001 level of significance for all where AP3858-1 has a slightly higher ratio of co-segregation (80.0%) than InDel 11 (79.6%) and InDel 5 (78.5%). InDel 5 is not polymorphic in the Cocodrie/Jing185 population, and the remaining two markers are verified at the *P*<0.0001 level of significance for both. InDel 11 has slightly higher ratio of co-segregation (85.1%) than AP3858-1 (81.2%) in the Cocodrie/Jing185 population.


\* Zhe733 and Jingl85 as the straighthead resistant parents for the RIL populations while

\*\* Cocodrie and R312 as the susceptible parents.

\*\*\* Core collection accessions with PI No. and C1or No, PVP as Plant Variety Protection.

\*\*\*\*\* A total of 42 accessions display parental allele screened by In Del 11. The 32 accessions listed above are those have genotype matched with phenotype, but there are other I 0 accessions which genotypes do not match with phenotypes.

\*\*\*\* 'a' as resistant, 'b' as susceptible, and 'h' as heterozygote genotype but still considered as resistant because straighthead is a dominant trait.

\*\*\*\*\*\* Straighthead rating using a 1-9 scale, with 4 or below being resistant and 6 or above being susceptible.

**Table 5.** Association of marker InDel 11 genotype with straighthead phenotype in a global germplasm collection (Pan et al., 2012).


\*The accessions or RILs selected for marker verification were either the resistance with straighthead rating 4 or below or the susceptibility with rating 6 or above in global germplasm collection and two F9 populations.

\*\*A total of 34 accessions were selected for verification of AP3 858-1 because remaining 3 8 had either no alleles of or different from parental Zhe733, R312, Cocodrie and Jingl85, and for the same reason, 42 accessions were applied for verification of lnDell 11.

**Table 6.** Association analysis between marker genotypes and straighthead phenotype (Pan et al., 2012).

## **4.5. Bridge germplasm for cultivar development**

are verified at the *P*<0.0001 level of significance for both. InDel 11 has slightly higher ratio of

co-segregation (85.1%) than AP3858-1 (81.2%) in the Cocodrie/Jing185 population.

\* Zhe733 and Jingl85 as the straighthead resistant parents for the RIL populations while

\*\*\* Core collection accessions with PI No. and C1or No, PVP as Plant Variety Protection.

\*\*\*\*\* A total of 42 accessions display parental allele screened by In Del 11. The 32 accessions listed above are those have genotype matched with phenotype, but there are other I 0 accessions which genotypes do not match with phenotypes. \*\*\*\* 'a' as resistant, 'b' as susceptible, and 'h' as heterozygote genotype but still considered as resistant because

**Table 5.** Association of marker InDel 11 genotype with straighthead phenotype in a global germplasm collection (Pan

\*\*\*\*\*\* Straighthead rating using a 1-9 scale, with 4 or below being resistant and 6 or above being susceptible.

\*\* Cocodrie and R312 as the susceptible parents.

232 Rice - Germplasm, Genetics and Improvement

straighthead is a dominant trait.

et al., 2012).

Since the susceptible parent Cocodrie is a widely grown cultivar in the USA (Linscombe et al., 2000), it will be important to improve Cocodrie for straighthead resistance. Among 162 SSRs used for mapping and fine mapping in Cocodrie/Jing185 population, 101 are monomorphic between parent Cocodrie and resistant line RIL506 which is resistant with straighthead rating 2.3. Thus, the genetic similarity between Cocodrie and RIL506 is 62%. In other word, 62% of marker loci are same between Cocodrie and RIL506 in the whole genome. Four other resistant RIL lines 404, 407, 479 and 480 have a genetic similarity of more than 50% with Cocodrie. These resistant lines can be used for improving straighthead resistance in long grain *tropical japoni‐ ca* cultivars like Cocodrie in the southern US. However, the susceptible R312 is not a commer‐ cial cultivar in the USA, so the improvement of straighthead resistance for R312 is not important in the USA.

## **Acknowledgements**

The authors thank everybody who has directly and indirectly contributed to this project, and every institution that has directly and indirectly supported this project.

## **Author details**

Wengui Yan1 , Karen Moldenhauer2 , Wei Zhou1,2,3, Haizheng Xiong1,2,4 and Bihu Huang3

1 United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Dale Bumpers National Rice Research Center, USA

2 University of Arkansas, Division of Agriculture, Rice Research and Extension Center, USA

3 University of Arkansas at Pine Bluff, USA

4 Zhejiang University, State Key Lab of Rice Biology, Institute of Nuclear-Agricultural Scien‐ ces, China

USDA is an equal opportunity provider and employer

## **References**


**Acknowledgements**

234 Rice - Germplasm, Genetics and Improvement

**Author details**

Wengui Yan1

ces, China

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The authors thank everybody who has directly and indirectly contributed to this project, and

1 United States Department of Agriculture, Agricultural Research Service (USDA-ARS),

2 University of Arkansas, Division of Agriculture, Rice Research and Extension Center, USA

4 Zhejiang University, State Key Lab of Rice Biology, Institute of Nuclear-Agricultural Scien‐

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## **Genes and QTLs for Rice Grain Quality Improvement**

## Jinsong Bao

[50] Yan, W. G, Correa-victoria, F. F, Marin, A, Marassi, J, Li, X, & Re, J. (2010a). Compa‐ rative study on induced straighthead in the U.S. with natural straighthead in Argen‐

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tina.Proc 33rd Rice Technical Working Group conference Feb: , 22-25.

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238 Rice - Germplasm, Genetics and Improvement

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56621

## **1. Introduction**

As a major cereal crop, rice (*Oryza sativa* L.) is crucial to food security for at least half the world population. New varieties with high yield potential, good quality and high resistance to biotic and abiotic stresses are needed in order to meet the demand for more food arising from the rapid human population growth and concurrent decrease in arable land. Improvement of rice quality has now become a foremost consideration for rice buyers and breeding programs.

Quality is defined as "the totality of features and characteristics of a product or service that bears its ability to satisfy stated or implied needs" (International Standard Organization (ISO) 8402 1986). Features are identified properties of a product which can be related to the quality characteristics. Grain quality of rice is the totality of features and characteristics of rice or rice product that meets the demand of end-user. The concept of grain quality covers many features ranging from physical to biochemical properties, and includes milling efficiency, grain shape and appearance, cooking easiness, eating palatability, and nutrition. Thus, rice grain quality generally includes four classes, i.e. milling quality, appearance quality, cooking and eating quality, and nutritional quality (Figure 1). Many countries have set up their own protocols to assess the respective quality. International organizations such as ISO, Association of Analytical Communities International (AOAC), and American Association of Cereal Chemists Interna‐ tional (AACCI) have set up methods to evaluate some quality parameters, for example, apparent amylose content (AAC). Rice is consumed mainly as milled, so eating quality mentioned in this article generally relates to the cooked milled rice. However, due to the impact of the western life style, whole grain rice or brown rice becomes popular worldwide, so that the nutritional quality has expanded to the nutrients of brown rice.

Grain quality and its assessment are not only important to consumers, end-users, processors, but also to rice breeders who are engaged in creating rice varieties haboring new features such as high quality, high yield potential, highly resistant to abiotic or biotic stresses. It is necessary

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

for rice breeders to understand how the quality traits are inherited from their parents. Genetic studies have revealed many genes and quantitative trait loci (QTL) for grain quality, though the grain quality traits are complex. Some major genes have been cloned, and their functions in a specific pathway, such as starch, protein, lipid, and flavonoids biosynthesis, have been characterized. Some QTLs have been finely mapped for further map-based cloning and functional characterization. The known genes or QTLs have been successfully applied in breeding programs for marker-assisted selection (MAS) to improve the breeding and selection efficiencies.

This chapter highlights the genes and QTLs available for grain quality of rice, summarizing how many QTLs and genes have been mapped or characterized, and how many could be used in marker assisted selection (MAS), which could help breeders to in-deep understand the genetics of grain quality of rice and apply the knowledge in their breeding practices.

**Figure 1.** Four facets of grain quality

## **2. Four facets of grain quality**

## **2.1. Milling quality**

Milling quality determines the final yield and the broken kernel rate of the milled rice, which is of concern for consumers and farmers. Three main parameters, brown rice recovery (the percentage of brown rice to rough rice), milled rice recovery (the percentage of milled rice to rough rice), and head rice recovery (the percentage of head rice to rough rice) are used to evaluate the quality and efficiency of the milling process. Brown rice is the de-hulled rice with the palea and lemma removed. Brown rice itself is a type of whole grain that could be used for cooking and eating. Removing all of the bran which consists of the aleurone and pericarp, and germ or embryo from brown rice results in white (or milled) rice. Some milled grains are broken during milling, head rice is a standard term for the whole milled grain. In calculation of head rice recovery, kernels longer than or equal to 3/4 full length of a kernel were considered as whole grains. Among all three parameters to determine the milling quality, head rice recovery is the main factor determining rice market value and one of the most important criteria of milled rice.

## **2.2. Appearance quality**

for rice breeders to understand how the quality traits are inherited from their parents. Genetic studies have revealed many genes and quantitative trait loci (QTL) for grain quality, though the grain quality traits are complex. Some major genes have been cloned, and their functions in a specific pathway, such as starch, protein, lipid, and flavonoids biosynthesis, have been characterized. Some QTLs have been finely mapped for further map-based cloning and functional characterization. The known genes or QTLs have been successfully applied in breeding programs for marker-assisted selection (MAS) to improve the breeding and selection

This chapter highlights the genes and QTLs available for grain quality of rice, summarizing how many QTLs and genes have been mapped or characterized, and how many could be used in marker assisted selection (MAS), which could help breeders to in-deep understand the

Milling quality determines the final yield and the broken kernel rate of the milled rice, which is of concern for consumers and farmers. Three main parameters, brown rice recovery (the percentage of brown rice to rough rice), milled rice recovery (the percentage of milled rice to rough rice), and head rice recovery (the percentage of head rice to rough rice) are used to evaluate the quality and efficiency of the milling process. Brown rice is the de-hulled rice with

genetics of grain quality of rice and apply the knowledge in their breeding practices.

efficiencies.

240 Rice - Germplasm, Genetics and Improvement

**Figure 1.** Four facets of grain quality

**2.1. Milling quality**

**2. Four facets of grain quality**

Appearance is one of the crucial properties of rice grain affecting its market acceptability. After milling, the appearance of the grain is associated with size, shape (long vs. round), chalkiness, and translucency. Grain length, width, thickness are used to describe the physical dimensions of rice kernels, while the grain shape is expressed as the ratio of length to width. Grain appearance is also largely determined the clarity, the vitreousness, and the translucency of the endosperm, which is specifically required by most segments of the rice industry. According to the location of the chalkiness in the endosperm, it could be classified into three groups, white belly (chalkiness on the dorsal side of the grain), white back (chalkiness on the ventral side) and white core (chalkiness in the center). Generally, the great the chalkiness, the lower the market acceptability. Percentage of chalky grain is the proportion of grains having a chalky spot on (or in) the endosperm. Chalkiness is measured visually with scales for 0 for none, 1 for small (<10%), 5 for medium (10-20%) and 9 for large (>20 % of the area). Grain transparency may be measured using a light permeation instrument or with an image analyzer, with which the size and shape may be measured simultaneously.

## **2.3. Cooking and eating quality**

Cooking and eating quality determines the easiness of cooking, as well as the firmness and stickiness of the cooked rice. Rice cooking and eating quality is highly related to some easily measurable physicochemical properties: apparent amylose content (AAC), gel consistency, gelatinization temperature (GT) and pasting viscosity. All these parameters are related to the properties of starch that makes up 90% of milled rice. Starch consists of two kinds of molecules, the linear and helical amylose and the branched amylopectin. Amylose content is measured with a simplified procedure using I2-KI solution. Due to the binding ability of long chain of amylopectin with I2, the amylose content measured with I2-KI solution is also termed as apparent amylose content (AAC). The AAC of milled rice may be classified as waxy (1-2%), very low (5-12%), low (12-20%), intermediate (20-25%) and high (>25%). Gelatinization is the disruption of molecular orders within the starch granule manifested in irreversible changes in properties such as granular swelling, native crystallite melting, loss of birefringence, and starch solublization. The gelatinization temperature determines the time and energy input required for cooking. Gel consistency was developed as a parameter to index the tendency of cooked rice to harden on cooling, and is normally classified as hard, medium, and soft. Pasting viscosity is another useful parameter to differentiate rice with similar AAC, and is popularly measured by a Rapid Visco-Analyser (RVA) developed by Newport Scientific Pty Ltd., Australia. RVA records the viscosity continuously as the temperature is increased, held constant for a time, and then decreased.

The above mentioned are objective parameters for the cooking and eating quality. However, eating quality is quite subjective and thus is difficult to define as it depends on consumer preferences. Sensory quality of cooked rice could be evaluated by a trained sensory panel (Champagne et al. 2010). Four steps are used to evaluate the cooked rice texture (Table 1). In addition to texture, the flavor (aromatics, taste, mouthfeel) of cooked rice can also be evaluated by the sensory panel.


**Table 1.** Sensory descriptive texture attributes and their definitions used to evaluate cooked rice texture1

## **2.4. Nutritional quality**

measured by a Rapid Visco-Analyser (RVA) developed by Newport Scientific Pty Ltd., Australia. RVA records the viscosity continuously as the temperature is increased, held

The above mentioned are objective parameters for the cooking and eating quality. However, eating quality is quite subjective and thus is difficult to define as it depends on consumer preferences. Sensory quality of cooked rice could be evaluated by a trained sensory panel (Champagne et al. 2010). Four steps are used to evaluate the cooked rice texture (Table 1). In addition to texture, the flavor (aromatics, taste, mouthfeel) of cooked rice can also be evaluated

**Phase I** Place 6–7 grains of rice in mouth behind front teeth. Press tongue

Initial starchy coating Amount of paste-like thickness perceived on the product before

Slickness Maximum ease of passing tongue over the rice surface when saliva starts to mix with sample.

**Phase II** Place 1/2 teaspoon of rice in mouth. Evaluate before or at first bite. Springiness Degree grains return to original shape after partial compression. Cohesiveness Degree to which the grains deform rather than crumble, crack, or

Hardness Force required to bite through the sample with the molars.

Moisture absorption Amount of saliva absorbed by sample during chewing.

**Table 1.** Sensory descriptive texture attributes and their definitions used to evaluate cooked rice texture1

Cohesiveness of mass Maximum degree to which the sample holds together in a mass while chewing.

Roughness Amount of irregularities in the surface of the product.

Stickiness between grains Degree to which the kernels adhere to each other.

Stickiness to lips Degree to which kernels adhere to lips.

**Phase III** Evaluate during chew.

**Phase IV** Evaluate after swallow.

1Adapted from Champagne et al. (2010).

Chewiness Amount of work to chew the sample. Uniformity of bite Evenness of force throughout bites to chew.

Residual loose particles Amount of loose particles in mouth.

Toothpack Amount of product adhering in/on the teeth.

over surface and evaluate.

mixing with saliva (3 passes).

break when biting with molars.

constant for a time, and then decreased.

242 Rice - Germplasm, Genetics and Improvement

**Phases/attributes Definition**

by the sensory panel.

As one of the most important staple food in the world, nutritional quality is closely related to human health, and thus is highly valued by consumers. Protein is the second most abundant constituent of milled rice, following starch. Lysine is the first limiting essential amino acid in rice based on the human requirements. Protein and lysine content are two important param‐ eters determining nutritional value of rice. With social development, diverse people eating rice as staple food may require rice with distinct nutritional quality. For those in the underdevel‐ oped region where micronutrient deficiency (Vitamins and minerals, such as iron and zinc) is apparent, genetics study for and biofortification of micronutrients by breeding are necessary to improve the nutritional quality of rice. For those with improved living standards, consuming of brown rice as one kind of whole grains becomes popular to combat chronic diseases, such as diabetes. Whole grain rice (brown rice) provides more minerals, vitamins, dietary fibers, and phenolics to human health than milled rice (Bao 2012a).

## **3. Genes and QTLs for grain quality**

## **3.1. Milling quality**

Milling quality is assessed by brown rice recovery, milled rice recovery and head rice recovery, which is one kind of complex quantitative trait whose genetic control is poorly understood. Up to date, no major gene has been genetically identified and functionally characterized. However, many studies have been carried out to search quantitative trait locus (QTL) for the milling quality (Table 2). These researches improve our understanding of the genetic control of milling quality, and provide molecular markers that are useful in breeding for improvement of milling quality in rice.



1: BC=backcross; BIL=backcross inbred line; DH=doubled haploid; I=*indica* subspecies; J=*japonica* subspecies; RIL=re‐ combinant inbred line; W=wild rice. IL: introgression lines.

2: The value in this column indicates the number of chromosome; the two or three same values in the same line indicate two or three QTLs in the same chromosome.

3: Percentage of total variation explained by a single QTL (%).

**Table 2.** Summary of main-effect QTLs for milling quality traits mapped on rice genome

#### *3.1.1. Brown rice recovery*

A total of 20 QTLs have been identified in eight studies, covering all chromosomes except chromosome 2 (Table 2). A major QTL at the interval between markers RM42 and C734b on chromosome 5 is also responsible for grain width (Tan et al. (2001). A QTL on chromosome 3 likely shares the same genomic region for grain length (Lou et al. 2009). These results indicate that brown rice rate relates to the grain shape and size of rice kernel. Five QTLs were detected in the study of Li et al. (2004a), of which three were expressed in two years, indicating that there are QTL-by-environment interactions effects.

## *3.1.2. Milled rice recovery*

**Population Property1 No. of QTLs**

244 Rice - Germplasm, Genetics and Improvement

combinant inbred line; W=wild rice. IL: introgression lines.

3: Percentage of total variation explained by a single QTL (%).

there are QTL-by-environment interactions effects.

**Table 2.** Summary of main-effect QTLs for milling quality traits mapped on rice genome

two or three QTLs in the same chromosome.

*3.1.1. Brown rice recovery*

Head rice recovery

**Chromosome**

Zhenshan 97/Minghui 63 I/I, RIL 2 3, 5 4.8, 7.0 Tan et al. 2001 Nipponbare/Kasalath J/I, BIL 4 4,9,10,11 7.6-19.9 Li et al. 2004a Asominori /IR24 J/I, RIL 2 11, 12 7.7, 12.2 Dong et al. 2004 Caiapo/ *O. glaberrima* I/W, DH 2 5,7 5.3, 6.1 Aluko et al. 2004 Teqing/Lemont I/J, IL 5 1,2,5,6,7 11.5-30.7 Zheng et al. 2007 Chuan7/Nanyangzhan I/J, RIL 1 3 6.7 Lou et al. 2009 L204/01Y110 J/J, RIL 3 1, 4, 9 6-9 Nelson et al. 2012

Zhenshan 97/Minghui 63 I/I, RIL 1 3 10.1 Tan et al. 2001

Nipponbare/Kasalath J/I, BIL 3 3, 6,7 9.7-12.2 Li et al. 2004a Asominori /IR24 J/I, RIL 3 1, 3, 5 8,7-22.1 Dong et al. 2004 Caiapo/ *O. glaberrima* I/W, DH 5 1,3,6,8,11 7.6-54.1 Aluko et al. 2004 Zhenshan 97/WYJ-2 I/J, DH 2 3, 8 10.1, 16 Jiang et al. 2005 Teqing/Lemont I/J, IL 3 1,5,6 5.8-5.9 Zheng et al. 2007 Chuan7/Nanyangzhan I/J, RIL 1 3 29.7 Lou et al. 2009 Cypress/RT0034 J/I, RIL 2 6,9 12, 16 Nelson et al. 2011 Cypress/ LaGrue J/J, RIL 4 1,5,9,10 8, 12 Nelson et al. 2011 L204/01Y110 J/J, RIL 7 6, 6, 8, 9, 9, 10, 11 3-8 Nelson et al. 2012

IR64/*O. rufipogon* I/W, BC2F2 3 1,2,5 5.2-5.5 Septiningsih et al. 2003

1: BC=backcross; BIL=backcross inbred line; DH=doubled haploid; I=*indica* subspecies; J=*japonica* subspecies; RIL=re‐

2: The value in this column indicates the number of chromosome; the two or three same values in the same line indicate

A total of 20 QTLs have been identified in eight studies, covering all chromosomes except chromosome 2 (Table 2). A major QTL at the interval between markers RM42 and C734b on chromosome 5 is also responsible for grain width (Tan et al. (2001). A QTL on chromosome 3 likely shares the same genomic region for grain length (Lou et al. 2009). These results indicate that brown rice rate relates to the grain shape and size of rice kernel. Five QTLs were detected in the study of Li et al. (2004a), of which three were expressed in two years, indicating that

**distribution2 PVE3 Reference**

A total of 19 QTLs have been identified in seven studies, covering all chromosomes except chromosome 8 (Table 2). There are no strong or reproducible QTLs for the milled rice recovery. Three independent studies detected QTL for the milled rice recovery on chromosome 5 (Tan et al. 2001; Aluko et al. 2004; Zheng et al. 2007), but there are actually not at the same region. Li et al. (2004a) reported that two of four QTLs were detected in two years, indicating that the QTL-by-environment interactions effects exist.

## *3.1.3. Head rice recovery*

Up to date, a total of 34 QTLs locating at all the chromosomes have been reported in ten studies with the number of QTLs varied from 1 to 7 in different studies. A major QTL located on chromosome 3 is also a major QTL for grain length (Tan et al. 2001), suggesting that genetic relationship exists between grain size or shape and the percentage of head rice. Other studies frequently identified the QTL at chromosome 3 (Li et al. 2004a; Dong et al. 2004; Aluko et al. 2004; Jiang et al. 2005; Lou et al. 2009), proving that there might be a major gene for head rice. In addition, QTLs on chromosome 1, 5 and 6 are also detected by at least three independent studies. Li et al. (2004) detected three QTLs for head rice, but all of them were detected only in a specific year, suggesting that the head rice is largely affected by the environment. However, Nelson et al. (2011) showed that more variance of head rice yield was explained by main-effect QTL than QTL × environment effect in the Cypress/RT0034 RIL population, whereas the maineffect QTLs contributed a little less to genetic variation than those of QTL × environment effect in the Cypress/ LaGrue RIL population. There is a clear coincidence of QTLs for head rice recovery with early-heading QTLs in the hotter growing location, hinting an environmental effect (Nelson et al. 2011). Note that some genetic populations were derived from cultivated rice and wild rice (Septiningsih et al. 2003; Aluko et al. 2004), but all milling-yield-increasing effects came from the cultivated parent.

## **3.2. Appearance quality**

## *3.2.1. Grain size and shape*

Grain shape is not only key determinant of grain quality but also of grain yield potential. A long, slender grain of rice is generally preferred by consumers in Southern China, the USA, and South and Southeast Asian countries, whereas consumers in Japan, Korea, and Northern China prefer short or round grain of rice (Huang et al. 2013).

Grain length, grain width, length-to-width (grain shape) are the most stable properties of the variety, so they are highly heritable. Genetically, a lot of QTLs have been identified for grain length, grain width, and grain shape (Figure 2a). The chromosome 3 harbors more QTLs than others (Figure 2b). Some are major genes that have been map-based cloned with their function characterized (Table 3), some are finely mapped (Table 4), while many are with minor effects and are waiting for further characterization. The finely mapped QTLs provide potential markers for molecular breeding to modify grain shape while use of functional markers derived

**Figure 2.** Number (A) and distribution (B) of QTLs for appearance quality (data from Gramene: http:// www.gramene.org/).

from the cloned genes would lead to precise phenotype in breeding. QTL mapping studies also suggest that many QTLs exhibit pleiotropic effects; they control not only grain length, but also grain width, grain shape or grain yield (Bai et al. 2010; Fan et al. 2006; Guo et al. 2009; Li et al. 2011; Song et al. 2007; Wang et al. 2012). Functional characterization of the cloned genes provides evidence underlying the pleiotropic effects (Fan et al. 2006; Li et al. 2011; Song et al. 2007; Wang et al. 2012). A good review of Huang et al. (2013) has summarized the current progress in the genetic base of grain shape of rice.


**Table 3.** The cloned genes for grain appearance quality (grain shape and endosperm chalkiness)

#### *3.2.1.1. Grain length*

from the cloned genes would lead to precise phenotype in breeding. QTL mapping studies also suggest that many QTLs exhibit pleiotropic effects; they control not only grain length, but also grain width, grain shape or grain yield (Bai et al. 2010; Fan et al. 2006; Guo et al. 2009; Li et al. 2011; Song et al. 2007; Wang et al. 2012). Functional characterization of the cloned genes provides evidence underlying the pleiotropic effects (Fan et al. 2006; Li et al. 2011; Song et al. 2007; Wang et al. 2012). A good review of Huang et al. (2013) has summarized the current

(B)

**Figure 2.** Number (A) and distribution (B) of QTLs for appearance quality (data from Gramene: http://

(A)

progress in the genetic base of grain shape of rice.

www.gramene.org/).

246 Rice - Germplasm, Genetics and Improvement

A total of 47 QTLs for grain length have been detected in different populations. Among them, the Chromosome 3 harbors more QTLs than other chromosomes (Figure 2). Up to date, two QTLs have been map-based cloned (Table 3), and seven QTLs have been finely mapped (Table 4).

GRAIN SIZE 3 (GS3) is the first QTL that has been map-based cloned for grain length. It was detected in the RIL population derived from Minghui 63 and Chuan 7, displaying a major role for grain length and weight and a minor role for grain width and thickness and functioning as a negative regulator for grain size (Fan et al. 2006; 2009). The GS3 protein contains an organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR)-like domain, and a von Willebrand factor type C (VWFC) domain in the C terminus. The OSR domain functions as a negative regulator of grain length and deletion mutants of this domain result in the formation of longgrain rice. The C-terminal TNFR/NGFR and VWFC domains act as positive regulators of grain length and loss-of-function mutations of these domains lead to the development of very short grain (Mao et al. 2010; Takano-Kai et al. 2009).

GRAIN LENGTH 3 (qGL3) is a major grain length QTL recently identified in three mapping populations (Zhang et al. 2012; Hu et al. 2012; Qi et al. 2012). qGL3 encodes a putative protein phosphatase with Kelch-like repeat domain (OsPPKL1). A rare allele, i.e a single nucleotide substitution (C→A) leads to a long grain phenotype by an aspartate-to-glutamate transition in a conserved AVLDT motif of the second Kelch domain in OsPPKL1 (Hu et al. 2012; Zhang et al. 2012; Qi et al. 2012). Genetic analysis of a near-isogenic line (NIL) for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection (Hu et al. 2012). A new variety containing the new allele shows increased grain yield, which indicates that GL3 is a powerful tool for breeding highyield crops (Qi et al. 2012).

## *3.2.1.2. Grain width*

A total of 48 QTLs for grain width have been detected in different populations with more QTLs on chromosome 3 and 5 (Figure 2). Up to date, four QTLs have been map-based cloned (Table 3).

GRAIN WIDTH 2 (GW2) is a major QTL for rice grain width and weight, which was initially detected from a cross between a large-grain japonica rice variety (WY3) and a small-grain *indica* rice variety (Fengaizhan-1). GW2 encodes a RING-type E3 ubiquitin ligase (Song et al. 2007). WY3 has a 1-bp deletion resulting in the introduction of a premature stop codon in its exon 4, causing the large-grain phenotype. GW2 negatively regulates cell division by targeting its substrates to proteasomes for regulated proteolysis; loss of GW2 function results in an increase in cell number in the spikelet hull and acceleration of the grain-milk filling rate, thus enhancing grain width, weight, and yield.

GRAIN WIDTH 5 (GW5) is a major QTL for seed width on chromosome 5 (qSW5) (Wan et al. 2008; Weng et al. 2008; Shomura et al. 2008). A survey of GW5/qSW5 polymorphisms in various rice landraces has revealed that deletions in this gene may have played an important role in the selection of increased grain size from artificial and natural crossings during rice domesti‐ cation (Shomura et al. 2008). The GW5/qSW5 gene encodes a nuclear protein of 144 amino acids with an arginine-rich domain. Because GW5/qSW5 physically interacts with polyubiquitin, it is likely to act as a regulator in the ubiquitin–proteasome pathway and regulates cell division of the outer glume of the rice spikelet (Wan et al. 2008; Weng et al. 2008; Shomura et al. 2008).

GRAIN SIZE ON CHROMOSOME 5 (GS5) is a major QTL affecting grain width, grain filling, and grain weight (Li et al. 2011). It encodes a serine carboxypeptidase and functions as a positive regulator of grain size. Analysis of genomic DNA sequences and promoter swaps in transgenic plants reveals that nucleotide changes in three segments of the GS5 promoter seem to be responsible for the variations in grain width (Li et al. 2011).

GRAIN WIDTH 8 (GW8) is a major QTL affecting grain width and grain yield from the cross between HXJ74 and Basmati385 (Wang et al. 2012), which encodes SQUAMOSA promoterbinding protein-like 16, referred to OsSPL16, belonging to the protein family of SBP domaincontaining transcription factors. Six polymorphisms in the DNA sequence of OsSPL16 exist in the parents HXJ74 and Basmati385. Among them, a 10-bp deletion in the promoter region has been shown to be responsible for the slender grain trait of Basmati385 (Wang et al. 2012).

GS3, GL3, GW2, and GW5/qSW5 are negative regulators of grain size, but GS5 and GW8 are positive regulators of cell proliferation. Other genes associated with grain shape including the SMALL AND ROUND SEED (SRS) loci have been well reviewed in Huang et al. (2013).


Functional markers developed from these major genes and finely mapped QTL resources allow breeders to efficiently manipulate grain size and shape (Tables 3 and 4).

**Table 4.** Fine mapped QTLs associated with appearance quality of rice

GL: grain length; GW: grain wideness; GS: grain shape and PGWC: percentage of grains with chalkiness

#### *3.2.2. Grain chalkiness*

a conserved AVLDT motif of the second Kelch domain in OsPPKL1 (Hu et al. 2012; Zhang et al. 2012; Qi et al. 2012). Genetic analysis of a near-isogenic line (NIL) for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection (Hu et al. 2012). A new variety containing the new allele shows increased grain yield, which indicates that GL3 is a powerful tool for breeding high-

A total of 48 QTLs for grain width have been detected in different populations with more QTLs on chromosome 3 and 5 (Figure 2). Up to date, four QTLs have been map-based

GRAIN WIDTH 2 (GW2) is a major QTL for rice grain width and weight, which was initially detected from a cross between a large-grain japonica rice variety (WY3) and a small-grain *indica* rice variety (Fengaizhan-1). GW2 encodes a RING-type E3 ubiquitin ligase (Song et al. 2007). WY3 has a 1-bp deletion resulting in the introduction of a premature stop codon in its exon 4, causing the large-grain phenotype. GW2 negatively regulates cell division by targeting its substrates to proteasomes for regulated proteolysis; loss of GW2 function results in an increase in cell number in the spikelet hull and acceleration of the grain-milk filling rate, thus enhancing

GRAIN WIDTH 5 (GW5) is a major QTL for seed width on chromosome 5 (qSW5) (Wan et al. 2008; Weng et al. 2008; Shomura et al. 2008). A survey of GW5/qSW5 polymorphisms in various rice landraces has revealed that deletions in this gene may have played an important role in the selection of increased grain size from artificial and natural crossings during rice domesti‐ cation (Shomura et al. 2008). The GW5/qSW5 gene encodes a nuclear protein of 144 amino acids with an arginine-rich domain. Because GW5/qSW5 physically interacts with polyubiquitin, it is likely to act as a regulator in the ubiquitin–proteasome pathway and regulates cell division of the outer glume of the rice spikelet (Wan et al. 2008; Weng et al. 2008; Shomura et al. 2008). GRAIN SIZE ON CHROMOSOME 5 (GS5) is a major QTL affecting grain width, grain filling, and grain weight (Li et al. 2011). It encodes a serine carboxypeptidase and functions as a positive regulator of grain size. Analysis of genomic DNA sequences and promoter swaps in transgenic plants reveals that nucleotide changes in three segments of the GS5 promoter seem

GRAIN WIDTH 8 (GW8) is a major QTL affecting grain width and grain yield from the cross between HXJ74 and Basmati385 (Wang et al. 2012), which encodes SQUAMOSA promoterbinding protein-like 16, referred to OsSPL16, belonging to the protein family of SBP domaincontaining transcription factors. Six polymorphisms in the DNA sequence of OsSPL16 exist in the parents HXJ74 and Basmati385. Among them, a 10-bp deletion in the promoter region has been shown to be responsible for the slender grain trait of Basmati385 (Wang et al. 2012).

GS3, GL3, GW2, and GW5/qSW5 are negative regulators of grain size, but GS5 and GW8 are positive regulators of cell proliferation. Other genes associated with grain shape including the SMALL AND ROUND SEED (SRS) loci have been well reviewed in Huang et al. (2013).

to be responsible for the variations in grain width (Li et al. 2011).

yield crops (Qi et al. 2012).

248 Rice - Germplasm, Genetics and Improvement

grain width, weight, and yield.

*3.2.1.2. Grain width*

cloned (Table 3).

Chalkiness is a major concern in rice breeding because it is one of the key factors in determining quality and price. The chalky endosperm consists of loosely packed, round and large com‐ pound starch granules while the translucent endosperm comprises tightly packed, polyhedral and small single starch granules. The chalky grains show significantly different physicochem‐ ical, morphological, thermal, cooking and textural properties from translucent grains. Per‐ centage of grains with chalkiness (PGWC) is one of the main indices of rice-determining appearance quality, which is easily determined.

Many factors contribute to the formation of chalkiness in the rice grain. Environmentally, rice grown at the higher temperature contains more chalky grains. Genetically, defect in genes affecting starch biosynthesis, starch granule structure, and grain filling may lead to endosperm chalkiness. These genes include *starch branching enzyme IIb* (*BEIIb*), *branching enzyme I* (*BEI*), *starch synthase IIIa* (SSIIIa), *floury* and *sugary* genes, etc. It should be noted that many of the genes characterized show pleiotropic effects on other traits in addition to chalkiness.

A rice genic male-sterility gene *ms-h* is recessive and has a pleiotropic effect on the chalky endosperm (Woo et al. 2008). Fine mapping and nucleotide sequencing analysis reveal a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyro‐ phosphorylase 1 (*UGPase1*) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. Overexpression of UGPase1 in *ms-h* mutant plants restored male fertility and the transformants produced T1 seeds that segregated into normal and chalky endosperms (Woo et al. 2008).

The grain incomplete filling 1 (gif1) mutant defects in grain-filling capacity, but its grains are with more chalkiness as a result of loosely packed starch granules. A frameshift mutation caused by a 1-bp nucleotide deletion in GIF1 results in premature termination of its open reading frame. GIF1 encodes a cell-wall invertase required for carbon partitioning during early grain filling (Wang et al. 2008a).

Two white-core genes have been characterized with knockout mutants. A floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophos‐ phate dikinase (PPDK) (Kang et al. 2005). Other two additional alleles, flo4-2 and flo4-3 also showed the same white-core endosperm phenotype. OsPPDKB was mainly expressed in the endosperm, aleurone, and scutellum of the developing kernel, suggesting that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling. Ryoo et al. (2007) characterized another white-core floury endosperm mutant (flo5) caused by T-DNA insertion into the SSIIIa.

A floury mutant, flo(a), exhibits floury characteristics in the innermost endosperm, while the outer layer of the endosperm appeared normal (Qiao et al. 2010). The *FLO(a)* gene was isolated via a map-based cloning approach and predicted to encode the tetratricopeptide repeat domain containing protein, OsTPR. Three mutant alleles contain a nucleotide substitution that generated one stop codon or one splice site, respectively, which presumably disrupts the interaction of the functionally conserved TPR motifs (Qiao et al. 2010). The OsTPR motifs may play a significant role in rice starch biosynthetic pathways, which causes the formation of chalkiness. Yang et al. (2012) identified a mutant 'Jiangtangdao 1' which had chalky endosperm with resistant starch content up to 11.67%. The putative gene *starch branching enzymne 3* on chromosome 2 was finely mapped and a cleaved amplified polymorphic sequence (CAPS) marker for marker assisted selection was developed (Yang et al. 2012).

For the naturally occurring chalkiness, earlier studies (Li et al. 2004; Tan et al. 2000; Wan et al. 2005) identified 24 QTLs from three crosses among Asian cultivars (Figure 2). Recently, Wan's group in China (Guo et al. 2011; Liu et al. 2010; Wan et al. 2005; Zheng et al. 2012; Zhou et al. 2009) and others (Yamakawa et al. 2008; Liu et al. 2012) have identified many more QTLs for grain chalkiness. Among them, two QTLs have been finely mapped (Table 4).

qPGWC-8 is a major QTL for the percentage of grains with white chalkiness in the interval G1149-R727 on chromosome 8 which was identified using a chromosome segment substitution line (CSSL). Guo et al. (2011) narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. qPGWC-8 accounted for 50.9% of the difference in PGWC between the parents.

qPGWC-7 is a QTL for the percentage of grain with chalkiness (PGWC) on 7 which was identified using a set of chromosome segment substitution lines, made from a cross between PA64s and 9311. Segregation analysis of the F2 population from the cross between C-51 (a CSSL harboring qPGWC-7 and having a chalky endosperm) and 9311 showed PGWC is a semidominant trait, controlled by a single nuclear gene. Fine mapping of qPGWC-7 with a large F2 population constructed from the cross C51 × 9311 delimitated it to a 44-kb DNA fragment, containing thirteen predicted genes (Zhou et al. 2009).

The markers tightly linked to qPGWC-8 and qPGWC-7 facilitate cloning of the gene underlying the QTLs and is of value for marker-assisted selection for endosperm texture. However, it is still far away from clear understanding the mechanism of formation of the grain chalkiness. First, the QTLs mapping results show low coherence in different genetic populations, sug‐ gesting many minor QTLs affecting chalkiness exist in different rice germplasm that we do not know. Second, in addition to the major genes or QTLs we have known, how their interac‐ tions with each other, and with the major genes for amylose and protein synthesis (Liu et al. 2010; Zheng et al. 2012) that may affect chalkiness are unknown. Third, effect of environment on the formation of chalkiness is well known, but how its effect on the gene expression that leads to the formation of chalkiness is largely unknown.

## **3.3. Eating and cooking quality**

The grain incomplete filling 1 (gif1) mutant defects in grain-filling capacity, but its grains are with more chalkiness as a result of loosely packed starch granules. A frameshift mutation caused by a 1-bp nucleotide deletion in GIF1 results in premature termination of its open reading frame. GIF1 encodes a cell-wall invertase required for carbon partitioning during early

Two white-core genes have been characterized with knockout mutants. A floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophos‐ phate dikinase (PPDK) (Kang et al. 2005). Other two additional alleles, flo4-2 and flo4-3 also showed the same white-core endosperm phenotype. OsPPDKB was mainly expressed in the endosperm, aleurone, and scutellum of the developing kernel, suggesting that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling. Ryoo et al. (2007) characterized another white-core floury endosperm mutant (flo5) caused by T-DNA insertion

A floury mutant, flo(a), exhibits floury characteristics in the innermost endosperm, while the outer layer of the endosperm appeared normal (Qiao et al. 2010). The *FLO(a)* gene was isolated via a map-based cloning approach and predicted to encode the tetratricopeptide repeat domain containing protein, OsTPR. Three mutant alleles contain a nucleotide substitution that generated one stop codon or one splice site, respectively, which presumably disrupts the interaction of the functionally conserved TPR motifs (Qiao et al. 2010). The OsTPR motifs may play a significant role in rice starch biosynthetic pathways, which causes the formation of chalkiness. Yang et al. (2012) identified a mutant 'Jiangtangdao 1' which had chalky endosperm with resistant starch content up to 11.67%. The putative gene *starch branching enzymne 3* on chromosome 2 was finely mapped and a cleaved amplified polymorphic sequence (CAPS)

For the naturally occurring chalkiness, earlier studies (Li et al. 2004; Tan et al. 2000; Wan et al. 2005) identified 24 QTLs from three crosses among Asian cultivars (Figure 2). Recently, Wan's group in China (Guo et al. 2011; Liu et al. 2010; Wan et al. 2005; Zheng et al. 2012; Zhou et al. 2009) and others (Yamakawa et al. 2008; Liu et al. 2012) have identified many more QTLs for

qPGWC-8 is a major QTL for the percentage of grains with white chalkiness in the interval G1149-R727 on chromosome 8 which was identified using a chromosome segment substitution line (CSSL). Guo et al. (2011) narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. qPGWC-8 accounted for 50.9% of the difference in

qPGWC-7 is a QTL for the percentage of grain with chalkiness (PGWC) on 7 which was identified using a set of chromosome segment substitution lines, made from a cross between PA64s and 9311. Segregation analysis of the F2 population from the cross between C-51 (a CSSL harboring qPGWC-7 and having a chalky endosperm) and 9311 showed PGWC is a semidominant trait, controlled by a single nuclear gene. Fine mapping of qPGWC-7 with a large

marker for marker assisted selection was developed (Yang et al. 2012).

grain chalkiness. Among them, two QTLs have been finely mapped (Table 4).

grain filling (Wang et al. 2008a).

250 Rice - Germplasm, Genetics and Improvement

into the SSIIIa.

PGWC between the parents.

Great progresses have been made in the understanding of the genetic basis of cooking and eating quality (Bao 2012b; Chen et al. 2012). Starch properties play important role in deter‐ mining the cooking and eating quality, which is highly associated with starch biosynthesis related genes. Starch biosynthesis pathways and genes or enzymes participating in have been well clarified (Figure 3). Amylose is synthesized mainly by GBSSI, and the amylopectin synthesis process is governed by a combination of multiple isoforms of SS, BE, and DBE to produce a uniform number of chains per amylopectin cluster. *Wx* encoding GBSSI is mainly responsible for the natural variation of amylose content, gel consistency and RVA pasting viscosity, while the SSIIa is mainly for gelatinization temperature, thermal properties, and amylopectin structure (Bao 2012b).

## *3.3.1. Apparent amylose content, gel consistency and RVA pasting viscosity*

*Wx* locus on chromosome 6 is a major QTL for amylose content, gel consistency and RVA pasting viscosity (He et al. 1999; Bao et al. 2000; Bao 2012; Wan et al. 2004; Fan et al. 2005; Septiningsih et al. 2003; Aluko et al. 2004; Lapitan et al. 2009; Lanceras et al. 2000; Tan et al. 1999). Map-based cloning of the qGC-6, a locus for gel consistency, indicates that *Wx* is the major gene controlling it (Su et al. 2011). Five functional markers in the *Wx* gene, a (CT)n microsatellite (or simple sequence repeat, Ayres et al. 1997; Bligh et al. 1995;), a 23bp insertion/ deletion sequence (Inukai et al. 2000; Wanchana et al. 2003; Teng et al. 2012)., and three single nucleotide polymorphism (SNP) markers (Bligh et al. 1998; Cai et al. 1998; Hirano et al. 1998; Isshiki et al. 1998; Larkin and Park 2003) are well characterized, with different alleles differing in AAC (Ayres et al. 1997; Bligh et al. 1995; Chen et al. 2008a; Inukai et al. 2000; Larkin and Park 2003), and RVA pasting viscosity (Bao et al. 2006a; Chen et al. 2008b; Larkin et al. 2003; Larkin and Park 2003). Among them, the (CT)n microsatellite in the *Wx* gene located 55 bp upstream of the putative 5′-leader intron splice site has many alleles with n ranging from 8 to 22 in diverse rice germplasm (Ayres et al. 1997; Bergman et al. 2001; Bao et al. 2006a; Chen et al. 2008a; Bao et al. 2002a; Han et al. 2004). Another locus, the G/T single nucleotide polymor‐ phism (SNP) at the putative leader intron 5′ splice site, and a G to T mutation at this site reduces

**Figure 3.** Starch biosynthesis pathway in rice endosperm (modified from Jeon et al. 2010). Starch biosynthesis consists of two distinct phases: the glucan initiation process and the starch amplification process. The plastidial starch phos‐ phorylase (Pho1) extends the chains of the initial priming sites such as free chains of malto-oligosaccharides in the presence of Glc-1-P. The subsequent mechanisms underlying the glucan initiation process remain to be established. Branched dextrins are putatively processed by the coordinated activities of SS, BE, and/or DBE to produce the proto‐ type of an amylopectin cluster structure, which further develops into amylopectin to establish the basic structure. AG‐ Pase, ADP glucose pyrophosphorylase; BE, starch branching enzyme; DBE, starch debranching enzyme; GBSSI, granulebound starch synthase; Pho1, plastidial starch phosphorylase; SS, soluble starch synthase; DBE includes isoamylase (ISA) and pullulanase (PUL).

the efficiency of *Wx* pre-mRNA processing and thus results in the lower level of spliced mature mRNA, Wx protein, and AAC (Wang et al. 1995; Bligh et al. 1998; Cai et al. 1998; Hirano et al. 1998; Isshiki et al. 1998). Waxy, low amylose, and some intermediate amylose rice have the T SNP allele, while some intermediate and high amylose rices have the G allele (Ayres et al. 1997; Bligh et al. 1998; Cai et al. 1998; Isshiki et al. 1998). The G/T SNP explained 80% (Aryes et al. 1997) to 90 % (Bao et al. 2006a) of the total observed variation in AAC in the nonwaxy rice accessions.

Rice with similarly high AAC still differs in cooking and eating quality due to potential effect of amylopectin structure and other factors. Gel consistency and RVA pasting viscosity are effective to differentiate rice with high AAC. Genetic studies show that the exon 10 SNP of *Wx* is responsible for the genetic basis for the gel consistency, the proportion of amylose bound to amylopectin, the proportion of amylose able to leach, gel hardness (Tran et al. 2011) and RVA pasting viscosity (Traore et al. 2011). Tran et al. (2011) indicated that the rice with SNP allele C at exon 10 produces soft, viscous gels, has a soft texture when cooked, but with high retrogradation, and the rice with SNP allele T gives a short, firm gel, and has a firm texture when freshly cooked with little change in texture over storage. In a cross between two varieties having similar high AAC, but with different paste viscosity properties, Traore et al. (2011) indicated that the exon 10 SNP marker is associated with most RVA pasting measurements and the proportion of soluble to insoluble apparent amylose.

#### *3.3.2. Gelatinization temperature, thermal properties*

SSIIa locus on chromosome 6 is a major QTL for gelatinization temperature and amylopectin structure (Aluko et al. 2004; He et al., 1999; Bao et al., 2004; Fan et al., 2005; Wang et al. 2007; Tian et al. 2005; Lapitan et al. 2009; Umemoto et al. 2002). Map-based cloning of the alkali degenerate locus gives evidence that the gene encoding SSIIa is the major gene responsible for gelatinization temperature (Gao et al. 2003). Nakamura et al. (2005) revealed that the function of SSIIa is to elongate the short A and B1 chains with degree of polymerization (DP) < 10 to form long B1 chains of amylopectin. Genetic engineering by introduction of *indica* active *SSIIa* gene into *japonica* rice increases GT and gives longer amylopectin side chain length (Nakamura et al. 2005; Gao et al. 2011).

Four functional SNPs in the SSIIa gene have been revealed (Umemoto et al. 2004; Umomoto and Aoki 2005; Nakamura et al. 2005; Bao et al. 2006b; Waters et al. 2006). The first one is at 264 bp in Exon 1 of AY423717, where a change from G to C results in change of glutamate to aspartate. The second site is at 3799 bp, where glycine encoded by GGC is replaced by serine encoded by AGC. The third site is at 4198 bp, where valine encoded by GTG is replaced by methionine encoded by ATG. The fourth site is at 4330 bp, glycine-leucine encoded by GGGCTC is replaced by glycine-phenylalanine encoded by GGTTTC. *SSIIa* gene fragments shuffling experiments by Nakamura et al. (2005) show that only the third and fourth SNPs are functional, and the third SNP (G/A) is crucial for SSIIa activity, the enzyme is inactive when it is A SNP (coding for methionine) no matter which SNP at 4229/4330 bp (GC/TT) is present. The GC/TT is most common and is strongly associated with GT (Waters et al 2006; Bao et al. 2006b). This GC/TT polymorphism alone can differentiate rice with high or intermediate GT (possessing the GC allele) from those with low GT (possessing the TT allele), explaining 62.4 % of the total variation in pasting temperature (Bao et al. 2006b). Few rice accessions with GC allele have low GT phenotype, which can be explained by their carrying the A SNP allele in the third SNP (Umemoto et al. 2004; Waters et al. 2006; Lu et al. 2010). However, it should be mentioned that the A allele of third SNP (G/A) is quite rare in natural populations. The frequency of A at is 1 in 30 rices (Bao et al. 2006b), 9 in 180 rices (Chen et al. 2003), 127 in 1543 rices (Cuevas et al. 2010), 5 in 65 rices (Umemoto et al. 2004), and 13 in 73 rices (Waters et al. 2005). It should also be noted that genetic control of intermediate GT rice starch remains unknown. Intermediate GT rice is characterized by more chains of DP24-35, which may be synthesized by other enzymes (Cuevas et al. 2010).

#### *3.3.3. Contributions of other starch biosynthesis related genes*

the efficiency of *Wx* pre-mRNA processing and thus results in the lower level of spliced mature mRNA, Wx protein, and AAC (Wang et al. 1995; Bligh et al. 1998; Cai et al. 1998; Hirano et al. 1998; Isshiki et al. 1998). Waxy, low amylose, and some intermediate amylose rice have the T SNP allele, while some intermediate and high amylose rices have the G allele (Ayres et al. 1997; Bligh et al. 1998; Cai et al. 1998; Isshiki et al. 1998). The G/T SNP explained 80% (Aryes et al. 1997) to 90 % (Bao et al. 2006a) of the total observed variation in AAC in the nonwaxy

**Figure 3.** Starch biosynthesis pathway in rice endosperm (modified from Jeon et al. 2010). Starch biosynthesis consists of two distinct phases: the glucan initiation process and the starch amplification process. The plastidial starch phos‐ phorylase (Pho1) extends the chains of the initial priming sites such as free chains of malto-oligosaccharides in the presence of Glc-1-P. The subsequent mechanisms underlying the glucan initiation process remain to be established. Branched dextrins are putatively processed by the coordinated activities of SS, BE, and/or DBE to produce the proto‐ type of an amylopectin cluster structure, which further develops into amylopectin to establish the basic structure. AG‐ Pase, ADP glucose pyrophosphorylase; BE, starch branching enzyme; DBE, starch debranching enzyme; GBSSI, granulebound starch synthase; Pho1, plastidial starch phosphorylase; SS, soluble starch synthase; DBE includes isoamylase

Rice with similarly high AAC still differs in cooking and eating quality due to potential effect of amylopectin structure and other factors. Gel consistency and RVA pasting viscosity are effective to differentiate rice with high AAC. Genetic studies show that the exon 10 SNP of *Wx* is responsible for the genetic basis for the gel consistency, the proportion of amylose bound to amylopectin, the proportion of amylose able to leach, gel hardness (Tran et al. 2011) and RVA pasting viscosity (Traore et al. 2011). Tran et al. (2011) indicated that the rice with SNP allele C at exon 10 produces soft, viscous gels, has a soft texture when cooked, but with high

rice accessions.

(ISA) and pullulanase (PUL).

252 Rice - Germplasm, Genetics and Improvement

Cooking and eating quality is a complex trait which is not only determined by the *Wx* and *SSIIa* genes, but also other genetic factors, such as other starch biosynthesis related genes. Three evidences show the effect of other genes in determining the cooking and eating qualities. First, In a population derived from two parents having similar intermediate AAC, QTLs rather than *Wx* locus are associated with the RVA pasting viscosities, and two of which might be located close to the *starch branching enzyme 1* (*SBE 1*) and *SBE3* loci (Bao et al. 2002b). Second, in an association mapping with all the starch biosynthesizing genes, additional five genes (AGPlar, PUL, SSI, SSIIa, and SSIII-2) with minor effects were detected when the effect of *Wx* gene was eliminated. Again, with the model controlling for SSIIa, a further search identified *Wx*, *SBE3*, *ISA*, and *SSIV-2* as minor genes that affect GT additively (Tian et al. 2009). Third, what factors will determine the cooking and eating quality of waxy rice is complex, because the GBSS is not active in waxy rice. It is expected that genes other than *Wx* are to control the genetic basis of pasting and thermal properties of waxy rice. Comparing starch physicochemical properties among different microsatellite groups in *starch branching enzyme 1* (*SBE1*) and soluble starch synthase 1 (SSS), waxy rices with the *SBE*-A allele have higher peak viscosity (PV), hot paste viscosity (HPV) and cold paste viscosity (CPV) than those with other alleles, and those with the *SSS*-B allele have higher HPV and CPV than other alleles (Bao et al. 2002a). Han et al. (2004) indicated that nucleotide polymorphisms in both *SBE1* and *SBE3* loci account for 70% of the observed variation in HPV and CPV, and for 40% of the observed variation in PV. Yan et al. (2011) conducted association analysis for pasting viscosity parameters of waxy rice using starch synthesis-related gene markers, showing that 10 gene markers were involved in controlling the pasting viscosity parameters. Among these, the *pullulanase* gene plays an important role in control of PV, HPV, CPV, breakdown viscosity, peak time, and pasting temperature (PT) in glutinous rice.

To date, there are many markers resources derived from starch biosynthesis related genes available for molecular breeding for the purpose of improving the cooking and eating quality (Tian et al. 2010; Bao et al. 2006b; Jin et al. 2010; He et al. 2006; Yan et al. 2011).

## **3.3.4 Other traits related to cooking and eating quality**

In addition to the amylose content, gelatinization temperature, gel consistency and pasting viscosity, other parameters, such as water absorption, volume expansion and cooked rice elongation have been set up to evaluate the cooking characteristics of rice (Bao et al. 2009).

Ahn et al. (1993) identified a QTL on chromosome 8 for cooked rice elongation. Rani (2011) found that a functional marker targeting an SNP in the GS3 is associated with kernel elonga‐ tion. Tian et al. (2005) detected 3, 2, and 2 QTLs for water absorption, volume expansion and cooked rice elongation, respectively in a DH population. While no QTL on chromosome 3 and 8 was detected, one common QTL for all the traits is at the *Wx* locus on chromosome 6, suggesting that the *Wx* gene plays a major role in determining these cooking characteristics in addition to other cooking and eating quality traits (Tian et al. 2005).

The aroma of cooked rice contributes to consumer sensory acceptance of rice. The aromatic compound 2- acetyl-1-pyrroline (2-AP) reportedly is the primary component of the popcornlike smell of aromatic rice. Fragrance (fgr) is a recessive trait that is controlled by a major gene on chromosome 8 (Lorieux et al. 1996; Jin et al. 2003). Bradbury et al. (2005a; 2005b) reported that the *badh2* gene could most likely be the *fgr* gene since it has an 8-bp deletion and three SNPs in its exon 7 compared to the functional *Badh2* gene which encodes putative betaine aldehyde dehydrogenase 2 (BADH2), and developed molecular markers for fragrance genotyping. Shi et al., (2008) found a novel null *badh2* allele (*badh2-E2*), which has a sequence identical to that of the *Badh2* allele in exon 7, but with a 7-bp deletion in exon 2. By map-based cloning strategy, Chen et al. (2008c) confirmed that the full-length BADH2 protein encoded by *Badh2* renders rice nonfragrant by inhibiting biosynthesis of 2-acetyl-1-pyrroline (2AP), a potent flavor component in rice fragrance. Functional markers derived from fgr are sufficient to carry out molecular marker assisted breeding to improve the sensory quality of rice (Shi et al. 2008; Chen et al. 2008c; Jin et al. 2010). So far as we are aware, there is no genetic report on the other sensory characteristics of rice.

## **3.4. Nutritional quality**

evidences show the effect of other genes in determining the cooking and eating qualities. First, In a population derived from two parents having similar intermediate AAC, QTLs rather than *Wx* locus are associated with the RVA pasting viscosities, and two of which might be located close to the *starch branching enzyme 1* (*SBE 1*) and *SBE3* loci (Bao et al. 2002b). Second, in an association mapping with all the starch biosynthesizing genes, additional five genes (AGPlar, PUL, SSI, SSIIa, and SSIII-2) with minor effects were detected when the effect of *Wx* gene was eliminated. Again, with the model controlling for SSIIa, a further search identified *Wx*, *SBE3*, *ISA*, and *SSIV-2* as minor genes that affect GT additively (Tian et al. 2009). Third, what factors will determine the cooking and eating quality of waxy rice is complex, because the GBSS is not active in waxy rice. It is expected that genes other than *Wx* are to control the genetic basis of pasting and thermal properties of waxy rice. Comparing starch physicochemical properties among different microsatellite groups in *starch branching enzyme 1* (*SBE1*) and soluble starch synthase 1 (SSS), waxy rices with the *SBE*-A allele have higher peak viscosity (PV), hot paste viscosity (HPV) and cold paste viscosity (CPV) than those with other alleles, and those with the *SSS*-B allele have higher HPV and CPV than other alleles (Bao et al. 2002a). Han et al. (2004) indicated that nucleotide polymorphisms in both *SBE1* and *SBE3* loci account for 70% of the observed variation in HPV and CPV, and for 40% of the observed variation in PV. Yan et al. (2011) conducted association analysis for pasting viscosity parameters of waxy rice using starch synthesis-related gene markers, showing that 10 gene markers were involved in controlling the pasting viscosity parameters. Among these, the *pullulanase* gene plays an important role in control of PV, HPV, CPV, breakdown viscosity, peak time, and pasting

To date, there are many markers resources derived from starch biosynthesis related genes available for molecular breeding for the purpose of improving the cooking and eating quality

In addition to the amylose content, gelatinization temperature, gel consistency and pasting viscosity, other parameters, such as water absorption, volume expansion and cooked rice elongation have been set up to evaluate the cooking characteristics of rice (Bao et al. 2009). Ahn et al. (1993) identified a QTL on chromosome 8 for cooked rice elongation. Rani (2011) found that a functional marker targeting an SNP in the GS3 is associated with kernel elonga‐ tion. Tian et al. (2005) detected 3, 2, and 2 QTLs for water absorption, volume expansion and cooked rice elongation, respectively in a DH population. While no QTL on chromosome 3 and 8 was detected, one common QTL for all the traits is at the *Wx* locus on chromosome 6, suggesting that the *Wx* gene plays a major role in determining these cooking characteristics in

The aroma of cooked rice contributes to consumer sensory acceptance of rice. The aromatic compound 2- acetyl-1-pyrroline (2-AP) reportedly is the primary component of the popcornlike smell of aromatic rice. Fragrance (fgr) is a recessive trait that is controlled by a major gene on chromosome 8 (Lorieux et al. 1996; Jin et al. 2003). Bradbury et al. (2005a; 2005b) reported that the *badh2* gene could most likely be the *fgr* gene since it has an 8-bp deletion and three

(Tian et al. 2010; Bao et al. 2006b; Jin et al. 2010; He et al. 2006; Yan et al. 2011).

**3.3.4 Other traits related to cooking and eating quality**

addition to other cooking and eating quality traits (Tian et al. 2005).

temperature (PT) in glutinous rice.

254 Rice - Germplasm, Genetics and Improvement

Few molecular genetics studies have been conducted for nutritional quality (Table 5), but many molecular breeding activities through transgenic engineering to improve nutritional quality of rice have been reported (see 4.4).

## *3.4.1. Protein and amino acid content*

There are nice reports about QTL mapping for protein content (Table 5). A total of 43 QTLs have been identified covering all 12 chromosomes. Chromosomes 1, 2 and 7 harbor more QTLs than other chromosomes. In addition to the total protein content, Zhang et al. (2008) detected 2, 4, 3 and 4 QTLs for protein fractions, albumin, globulin, prolamin and glutelin, respectively. The QTLs affecting contents of different protein fractions may locate at the same chromosomal region.

Wang et al. (2008c) identified 18 chromosomal regions for 19 individual amino acids, one of which at the bottom of chromosome 1 is a relatively strong QTL cluster, consisting of up to 19 individual QTL. A wide coincidence was found between the QTL and the loci involved in amino acid metabolism pathways, including N assimilation and transfer, and amino acid or protein biosynthesis (Wang et al. 2008c). Hu et al. (2009) identified a total of 12 QTLs for individual amino acid content and total amino acid content on chromosomes 1, 4, 6, 7 and 11. A QTL cluster on chromosome 1 was associated with the content of eight amino acids. The results are useful for candidate gene identification and marker-assisted breeding targeting the development of improved rice amino acid composition for human nutrition.

## *3.4.2. Fat content*

Fat content affects eating quality and nutritional values, and storage stability of rice as well. Apparently, 48 QTLs for fat content have been reported. Chromosome 1, 3 and 6 harbor more QTLs than other chromosomes (Table 5). Liu et al (2009) reported 14 QTLs for crude fat content in brown rice distributing on chromosomes 1, 3, and 5-9. One of which is a major QTL, qCFC5, locating on chromosome 5, which have been detected simultaneously among three popula‐ tions. Shen et al. (2012) characterized two stably expressed QTLs on chromosome 7, and they were detected in all three environments and were further confirmed by additional lines across


1: BC=backcross; BIL=backcross inbred line; DH=doubled haploid; I=*indica* subspecies; J=*japonica* subspecies; RIL=re‐ combinant inbred line; W=wild rice. IL: introgression lines.

2: The value in this column indicates chromosome number, the two or three same values in the same line indicate two or three QTLs in the same chromosome.

3: Percentage of total variation explained by a single QTL (%).

**Table 5.** QTLs for protein content and fat content in the rice grain

six environments. The stably expressed QTLs and major QTLs are suitable candidates for the improvement of FC via marker assisted breeding. Dynamic expression of QTLs for fat content during grain filling was detected by Wang et al. (2008b). Eleven unconditional QTL and 10 conditional QTL for FC were identified with more QTL expressed in the early developmental stages. The results suggested that accumulation of fat was governed by time-dependent gene expression. Ying et al. (2012) identified QTLs for fatty acid composition, and 29 associated QTLs were identified throughout the rice genome, except chromosomes 9 and 10. Nine rice orthologs of *Arabidopsis* genes encoding key enzymes in lipid metabolism co-localized with 11 mapped QTLs. A strong QTL for oleic (18:1) and linoleic (18:2) acid is associated with a gene encoding acyl–CoA:diacylglycerol acyltransferase, while another one for palmitic acid (16:0) is possibly associated with the acyl–ACP thioesterase gene.

## *3.4.3. Minerals*

Stangoulis et al. (2007) mapped the QTLs for inorganic phosphorus (P), total P, Fe, Zn, Cu and Mn concentrations. Norton et al. (2010) mapped 41 QTLs for the concentration of 17 elements in rice grain. Du et al. (2013) identified 23 and 9 QTLs for Ca, Fe, K, Mg, Mn, P, and Zn contents in brown rice in two environments of China, Lingshui of Hainan and Hangzhou of Zhejiang, respectively. Only 2 QTLs for Mg accumulation have been detected in both environments, indicating that mineral accumulation QTLs in rice grains are largely environment-dependent. Garcia-Oliveira et al. (2009) identified 31 putative QTLs for Fe, Zn, Mn, Cu, Ca, Mg, P and K contents with introgression lines derived from a cross between an elite *indica* cultivar Teqing and the wild rice (*Oryza rufipogon*). It was found that wild rice contributed favorable alleles for most of the QTLs (26 QTLs), and chromosomes 1, 9 and 12 exhibited 14 QTLs (45%) for these traits.

Phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) in rice grain may form complexes with mineral ions, such as Fe, Zn and Ca, leading to be low bioavailability of minerals to humans. A set of low phytic acid rice mutant lines with the aim of increasing the bioavailability of the minerals of rice (Liu et al. 2007) have been isolated. Functional markers have been developed from some mutants (Zhao et al. 2008; Tan et al. 2013), and candidate genes such as multi-drug resistance-associated protein ABC transporter gene 5 (Xu et al. 2009) have been revealed. These mutant and markers tagged for the mutation may help develop new rice with increased mineral bioavailability.

#### *3.4.4. Phenolics*

six environments. The stably expressed QTLs and major QTLs are suitable candidates for the improvement of FC via marker assisted breeding. Dynamic expression of QTLs for fat content during grain filling was detected by Wang et al. (2008b). Eleven unconditional QTL and 10 conditional QTL for FC were identified with more QTL expressed in the early developmental stages. The results suggested that accumulation of fat was governed by time-dependent gene expression. Ying et al. (2012) identified QTLs for fatty acid composition, and 29 associated QTLs were identified throughout the rice genome, except chromosomes 9 and 10. Nine rice

**Population Property1 No. of QTLs**

Protein content

256 Rice - Germplasm, Genetics and Improvement

Fat content

Samgang/Nagdong J/I, DH and

Zhenshan 97B/Wuyujing 2 I/J, DH and

or three QTLs in the same chromosome.

RIL

BC1F1

combinant inbred line; W=wild rice. IL: introgression lines.

3: Percentage of total variation explained by a single QTL (%).

**Table 5.** QTLs for protein content and fat content in the rice grain

**Chromosome**

8 1,1,3,5,6,6,7,9 6-19 Qin et al. 2010

10 1,1,3,3,5,6,7,7,8,9 3.8-21.3 Liu et al. 2009

Koshihikari/Kasalath J/I, BIC 7 2,2,3,4,7,7,10 4.5-15.8 Zheng et al. 2012 Asominori/IR24 J/I, RIL 3 1,3,8 8.5-13.9 Zheng et al. 2011 Zhenshan 97B/Delong 208. I/I, RIL 6 1,2,4,7,8,9 4-25.9 Zhong et al. 2011 Asominori/IR24 J/I, CSSL 8 1,1,2,3,6,8,8,11 3-54 Liu et al. 2011 Xieqingzao B/Milyang 46 I/I, RIL 5 3,4,5,6,10 4-19 Yu et al. 2009 Asominori/IR24 J/I, RIL 3 2, 7, 12 11-14 Zhang et al. 2008 Gui 630/02428 I/J, DH 5 1,4,5,6,7, 7-35 Hu et al. 2004 Zhenshan 97B/Minghui 63 I/I, RIL 2 6,7 6, 13 Tan et al. 2001 Caiapo/ *O. glaberrima* I/W, DH 4 1,2,6,11 3.3-5.8 Aluko et al. 2004

Fengaizhan-1/JZ1560 I/J, F2; F2:3 5 1,3,7,8,10 7.7-13.9 Ying et al. 2012

Sasanishiki/Habataki J/I, BC 7 1, 2, 3, 6 10, 11,12 5-21 Shen et al. 2010 Xieqingzao B/Milyang 46 I/I, RIL 4 3,5,6,8 7-13 Yu et al. 2009

Asominori/IR24 J/I, RIL 11 1,1,2,3,3,4,4,5,6,6,11 7-14 Wang et al. 2008b Gui 630/02428 I/J, DH 3 1,2,5 7.7-25.5 Hu et al. 2004

1: BC=backcross; BIL=backcross inbred line; DH=doubled haploid; I=*indica* subspecies; J=*japonica* subspecies; RIL=re‐

2: The value in this column indicates chromosome number, the two or three same values in the same line indicate two

**distribution2 PVE3 Reference**

Jin et al. (2009) found via linkage mapping that phenolic content, flavonoid content, and antioxidant capacity were individually controlled by three QTLs. Only one QTL on chromo‐ some 2 was shared by phenolic content and flavonoid content. Shao et al. (2011) identified QTLs for these traits via association mapping using a diverse set of rice germplasm including red rice and black rice. Four, six and six QTLs were found associated with phenolic content, flavonoid content, and antioxidant capacity, respectively. Among them, four QTLs for phenolic content were also shared for other two traits. *Ra* (i.e. *Prp-b* for purple pericarp) and *Rc* (brown pericarp and seed coat) were main-effect loci for rice grain color and nutritional quality traits. Association mapping for the traits of the 361 white or non-pigmented rice accessions (i.e. excluding the red and black rice) revealed marker (RM346) is associated with phenolic content.

Pigmented rice accumulates anthocyanins (black rice) and proanthocyanidin (red rice), which are benefit to human health. Genetically, the pericarp color of red rice was controlled by two complementary genes, *Rc* (brown pericarp) on chromosome 7 and *Rd* (red pericarp) on chromosome 1. When present together, these loci produce red seed color. *Rc* in the absence of *Rd* produces brown seeds, whereas *Rd* alone has no phenotype (Sweeney et al. 2006; Furukawa et al. 2007). A natural mutation in *rc* has reverted brown pericarp to red pericarp and resulted in a new, dominant, wild-type allele, *Rc-g* (Brooks et al. 2008). The color of dark purple pericarp was also controlled by two complementary genes, *Pb* and *Pp*, located on chromosome 4 and 1, respectively (Wang et al 2009). Wang and Shu (2007) mapped *Pb* gene and suggested that this gene may be *Ra* gene. Markers for these genes may be useful for pigmented rice breeding, especially useful if new rice expects to accumulate both anthocyanins and proanthocyanidin.

## **4. Molecular breeding**

Molecular breeding is the application of molecular biology tools in plant breeding, which is generally include marker assisted selection (MAS) and genetic engineering (genetic transfor‐ mation) in addition to QTL mapping or gene discovery. Both of MAS and genetic engineering have been applied in grain quality improvement in rice. MAS has been successfully applied for cooking and eating quality improvement because of available of the excellent markers, while the genetic engineering has been widely used to improve nutritional quality of rice.

## **4.1. Marker assisted selection**

QTLs underlying natural occurring variation in grain quality have been widely explored, however, only few of them have been applied in current rice breeding programs. To the best of our knowledge, most of reports in terms of improving grain quality simply mean to improve the eating and cooking quality. The most useful genes are *Wx*, *SSIIa*, and *fragrance* (Table 6). Functional markers developed from GS3 are also available for grain length improvement (Wang et al. 2011). There are two strategies to conduct MAS in the breeding program. One is to improve the grain quality for the rice with high yield potential or high resistance to abiotic or biotic stresses, but with low quality. This is referred to foreground selection, which means that selection of a marker for grain quality trait by MAS denotes a trait obtained. Foreground selection is particularly useful for traits that need laborious or time-consuming phenotypic screening procedures, such as grain quality traits. The other is to improve the yield potential and high resistance for good quality rice, such as basmati or jasmine rice. This is referred to background selection. The markers for grain quality are used as background selection, which is to avoid the loss of good quality traits during introduction of the other traits.

## *4.1.1. Wx, fgr and SSIIa*

Low quality of hybrid rice in China is mainly owing to its poor quality maintainer line. One of good ways is to improve the quality of maintainer line by MAS. Some important maintainer line, such as Zhenshan 97B (Zhou et al. 2003; Liu et al. 2006), Longtefu B (Liu et al. 2006), and II32B (Jin et al. 2010), G46B (Gao et al. 2009) have been the target of transferring the *Wx* allele conferring lower amylose content. The new hybrid rice derived from the improved maintainer line and restorer line is expected to have better quality because the restorer line generally has good quality. Furthermore, MAS with *Wx* gene marker for quality improvement of the conventional rice has been reported (Yi et al. 2009; Jantaboon et al. 2011; Jairin et al. 2009).

Consumers generally prefer fragrant rice to non-fragrant rice. Functional markers for *fgr* have been developed and successively used to transfer this gene from fragrance rice to the target non-fragrance rice (Yi et al. 2009; Jin et al. 2010; Salgotra et al. 2012; Jantaboon et al. 2011).

SSIIa is responsible for the variation of gelatinization temperature; the functional markers for SSIIa have been developed and used in MAS to improve the cooking quality (Jin et al. 2010; Jantaboon et al. 2011; Lu et al. 2010).).


**Table 6.** Useful PCR markers for MAS to improve cooking and eating quality of rice1

## *4.1.2. Combining grain quality with other traits*

Breeding is working for not only one trait, but all the traits for the formation of a new variety. In addition to grain quality traits, yield and other agronomic or resistance traits are also very important. For those rice cultivars already have good quality, the objective of MAS is to combine the important quality traits with other traits. There are special cases for basmati and jasmine rices which have premium grain quality, and have been widely accepted by consumers worldwide. MAS has been carried out to introduce bacterial blight resistance (Pandey et al. 2013; Win et al. 2012), blast resistance (Singh et al. 2012), brown planthopper resistance (Jairin et al. 2009), submergence tolerance (Jantaboon et al. 2011) and plant stature (Pandey et al. 2013) genes into the basmati or jasmine rices.

## **4.2. Transgenic engineering**

chromosome 1. When present together, these loci produce red seed color. *Rc* in the absence of *Rd* produces brown seeds, whereas *Rd* alone has no phenotype (Sweeney et al. 2006; Furukawa et al. 2007). A natural mutation in *rc* has reverted brown pericarp to red pericarp and resulted in a new, dominant, wild-type allele, *Rc-g* (Brooks et al. 2008). The color of dark purple pericarp was also controlled by two complementary genes, *Pb* and *Pp*, located on chromosome 4 and 1, respectively (Wang et al 2009). Wang and Shu (2007) mapped *Pb* gene and suggested that this gene may be *Ra* gene. Markers for these genes may be useful for pigmented rice breeding, especially useful if new rice expects to accumulate both anthocyanins and proanthocyanidin.

Molecular breeding is the application of molecular biology tools in plant breeding, which is generally include marker assisted selection (MAS) and genetic engineering (genetic transfor‐ mation) in addition to QTL mapping or gene discovery. Both of MAS and genetic engineering have been applied in grain quality improvement in rice. MAS has been successfully applied for cooking and eating quality improvement because of available of the excellent markers, while the genetic engineering has been widely used to improve nutritional quality of rice.

QTLs underlying natural occurring variation in grain quality have been widely explored, however, only few of them have been applied in current rice breeding programs. To the best of our knowledge, most of reports in terms of improving grain quality simply mean to improve the eating and cooking quality. The most useful genes are *Wx*, *SSIIa*, and *fragrance* (Table 6). Functional markers developed from GS3 are also available for grain length improvement (Wang et al. 2011). There are two strategies to conduct MAS in the breeding program. One is to improve the grain quality for the rice with high yield potential or high resistance to abiotic or biotic stresses, but with low quality. This is referred to foreground selection, which means that selection of a marker for grain quality trait by MAS denotes a trait obtained. Foreground selection is particularly useful for traits that need laborious or time-consuming phenotypic screening procedures, such as grain quality traits. The other is to improve the yield potential and high resistance for good quality rice, such as basmati or jasmine rice. This is referred to background selection. The markers for grain quality are used as background selection, which

is to avoid the loss of good quality traits during introduction of the other traits.

Low quality of hybrid rice in China is mainly owing to its poor quality maintainer line. One of good ways is to improve the quality of maintainer line by MAS. Some important maintainer line, such as Zhenshan 97B (Zhou et al. 2003; Liu et al. 2006), Longtefu B (Liu et al. 2006), and II32B (Jin et al. 2010), G46B (Gao et al. 2009) have been the target of transferring the *Wx* allele conferring lower amylose content. The new hybrid rice derived from the improved maintainer line and restorer line is expected to have better quality

**4. Molecular breeding**

258 Rice - Germplasm, Genetics and Improvement

**4.1. Marker assisted selection**

*4.1.1. Wx, fgr and SSIIa*

The advantage to conduct MAS is that abundant molecular markers are available for rice and many traits have been tagged with molecular markers. However, the disadvantage is that MAS is only effective when the target traits exist in rice germplasm, and becomes void when the traits of interest are not present in the rice germplasm. In this case, transgenic engineering is useful, which could introduce the new traits into rice by transferring the target gene from other species. Expression of exotic gene in rice could produce the target trait. Transgenic engineering has some successful examples to introduce new nutrient traits into rice grain, such as vitamine a (Va), that confers rice high nutritional and increased benefit to human health.

## *4.2.1. Resistant starch*

Consumption of resistant starch enriched foods is associated with decrease in the postprandial glycaemic and insulinaemic responses, accompanied by the production of fermentationrelated gases in the large bowel. A high-amylose transgenic rice line modified by antisense RNA inhibition of starch branching enzymes has a 8.05% of resistant starch content, which was shown to decrease the postprandial glycaemic and insulinaemic responses and promoted fermentation-related production of H2 in the large bowel of young and healthy adults who consumed the resistant starch-enriched rice meal (Li et al 2009).

## *4.2.2. Protein*

Expression of a gene encoding a precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein in transgenic rice plants results in the improvement of the nutritive value of rice; the crude protein content in rice grains was increased by 0.64-3.54%, and the methionine and cysteine contents of these transgenic rice grains were respectively elevated by 29-76% and 31-75% compared with those of wild-type rice grains (Lee et al. 2003). Over-expression of aspartate aminotransferase genes in rice results in altered nitrogen metabolism and increased amino acid content and protein contents in seeds (Zhou et al. 2009).

## *4.2.3. Va*

Vitamin A deficiency has been linked to night blindness, corneal scarring and permanent blindness. Vitamin A deficiency increases infant mortality rates and the incidence and severity of infectious diseases. Carotenoids, a precursor of Vitamin A, is an important lipid-soluble antioxidants in photosynthetic tissues, which are known to be completely absent in rice endosperm. The entire β-carotene biosynthetic pathway in rice endosperm has been intro‐ duced into rice by transformation of plant phytoene synthase, *Erwinia uredovora* carotene desaturase, and lycopene β-cyclase genes via *Agrobacterium*-mediated transformation. The transgenic rice, Golden Rice 1, can accumulate a maximal level of 1.6 μg/g total carotene in the endosperm. Insertion of the phytoene synthetase gene from maize and the carotene desaturase gene from *Erwinia uredovora* into rice resulted in the greatest accumulation of total carotenoids and β-carotene. Golden Rice 2 contains as much as 37 μg total carotenoids per gram of dry weight of grain, of which 31 μg/g is β–carotene (Paine et al. 2005).

## *4.2.4. Folate*

Folates are B vitamins (vitamin B9). Humans cannot synthesize folates and have to absorb them from the diet, with plants usually being the main dietary sources. Folates play roles in the prevention of neural tube defects and in reducing the risk of cardiovascular disease, colon cancer, and neuropsychiatric disorders. In the United States, folic acid is added to refined cereals and grain products; these products are major contributors to total folate intake. Rice is a poor source of folates (vitamin B9). Overexpressing two *Arabidopsis thaliana* genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway, Storozhenko (2007) obtained transgenic rice with a maximal folate content enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.

## *4.2.5. Minerals (Fe)*

has some successful examples to introduce new nutrient traits into rice grain, such as vitamine

Consumption of resistant starch enriched foods is associated with decrease in the postprandial glycaemic and insulinaemic responses, accompanied by the production of fermentationrelated gases in the large bowel. A high-amylose transgenic rice line modified by antisense RNA inhibition of starch branching enzymes has a 8.05% of resistant starch content, which was shown to decrease the postprandial glycaemic and insulinaemic responses and promoted fermentation-related production of H2 in the large bowel of young and healthy adults who

Expression of a gene encoding a precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein in transgenic rice plants results in the improvement of the nutritive value of rice; the crude protein content in rice grains was increased by 0.64-3.54%, and the methionine and cysteine contents of these transgenic rice grains were respectively elevated by 29-76% and 31-75% compared with those of wild-type rice grains (Lee et al. 2003). Over-expression of aspartate aminotransferase genes in rice results in altered nitrogen metabolism and increased

Vitamin A deficiency has been linked to night blindness, corneal scarring and permanent blindness. Vitamin A deficiency increases infant mortality rates and the incidence and severity of infectious diseases. Carotenoids, a precursor of Vitamin A, is an important lipid-soluble antioxidants in photosynthetic tissues, which are known to be completely absent in rice endosperm. The entire β-carotene biosynthetic pathway in rice endosperm has been intro‐ duced into rice by transformation of plant phytoene synthase, *Erwinia uredovora* carotene desaturase, and lycopene β-cyclase genes via *Agrobacterium*-mediated transformation. The transgenic rice, Golden Rice 1, can accumulate a maximal level of 1.6 μg/g total carotene in the endosperm. Insertion of the phytoene synthetase gene from maize and the carotene desaturase gene from *Erwinia uredovora* into rice resulted in the greatest accumulation of total carotenoids and β-carotene. Golden Rice 2 contains as much as 37 μg total carotenoids per gram of dry

Folates are B vitamins (vitamin B9). Humans cannot synthesize folates and have to absorb them from the diet, with plants usually being the main dietary sources. Folates play roles in the prevention of neural tube defects and in reducing the risk of cardiovascular disease, colon cancer, and neuropsychiatric disorders. In the United States, folic acid is added to refined cereals and grain products; these products are major contributors to total folate intake. Rice is

a (Va), that confers rice high nutritional and increased benefit to human health.

consumed the resistant starch-enriched rice meal (Li et al 2009).

amino acid content and protein contents in seeds (Zhou et al. 2009).

weight of grain, of which 31 μg/g is β–carotene (Paine et al. 2005).

*4.2.1. Resistant starch*

260 Rice - Germplasm, Genetics and Improvement

*4.2.2. Protein*

*4.2.3. Va*

*4.2.4. Folate*

Iron deficiency is the most widespread micronutrient deficiency world-wide that afflicts an estimated 30% of the world population, especially where vegetable-based diets are the primary food source. Expression of the soybean *ferritin* gene (Goto et al. 1999) or pea *ferritin* gene (Ye et al. 2007) in rice produced seeds with greater Fe contents. Especially, Vasconcelos et al. (2003) showed that expression of the soybean *ferritin* gene under the control of the glutelin promoter in rice has proven to be effective in enhancing grain nutritional levels, not only in brown grains but also in polished grains. Expression of a thermotolerant phytase gene from *Aspergillus fumigatus* in rice endosperm is expected to decrease the phytic acid and increase iron bioavailability (Lucca et al. 2001).

## *4.2.6. Flavonoids*

Flavonoids are lacking in the endosperm of rice. Expression of maize C1 and R-S regulatory genes driven by an endosperm specific promoter of a rice prolamin gene in rice grain resulted in dark brown pericarp of the C1/R-S homozygous lines, and the major flavonoids, dihydro‐ quercetin (taxifolin), dihydroisorhamnetin (3′-*O*-methyl taxifolin) and 3′-*O*-methyl quercetin were identified in the rice grain (Shin et al. 2006). These rice lines have the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.

## *4.2.7. Serotonin*

Serotonin derivatives such as *p*-coumaroylserotonin and feruloylserotonin, a family of plant polyphenol compounds, play roles in an array of biological activities including antioxidative activity, but neither their production nor identification has been reported in crop plants. Transgenic rice expressing the pepper hydroxycinnamoyl-CoA:serotonin N-(hydroxycinna‐ moyl) transferase gene produced on average 274 ng/g seed weight which was nine-fold higher than wild-type (30 ng/g seed weight) (Kang et al. 2005). Chemical treatments such as transcinnamic acid and tyramine increased the serotonin derivatives contents by two- to three fold in both wild-type and transgenic rice. The transgenic rice had higher radical scavenging activities than that of wild-type, suggesting that neutraceutical serotonin derivative could be enriched by transgenic engineering (Kang et al. 2005).

#### *4.2.8. Coenzyme Q*

Coenzyme Q (CoQ), also called ubiquinone, is an electron transfer molecule in the respiratory chain. CoQ is also a lipid-soluble antioxidant. Most cereal crops produce mainly CoQ9, which has nine isoprene units, whereas humans produce mainly CoQ10, with 10 isoprene units. CoQ10 is a very popular food supplement. Takahashi et al. (2009) produced CoQ10-enriched rice plants by introduction of the gene for decaprenyl diphosphate synthase. In CoQ10 enriched rice plants, seed CoQ10 content per weight was increased to up to 10 times that of wild-type rice, but its level is still insufficient for practical use. Combination of the transgene with giant embryo mutant lines produced giant embryo line-type CoQ10-enriched rice with seed CoQ10 content per weight increased to up to 1.4-1.8 times. It was found that CoQ was preferentially accumulated in bran and germ of rice seed.

## **5. Future directions**

Great progress has been achieved in our understanding of the genetic and molecular basis of grain quality of rice. This is especially true for grain appearance and grain shape, since they are not only linked with grain quality, but also with grain yield, a more important trait. Cooking and eating quality has a strong relation with starch biosynthesis pathway which has been well understood. Markers derived from the starch biosynthesis related genes have been widely applied in MAS. However, there are four major problem areas that challenge research‐ ers working on molecular genetics of grain quality.

## **5.1. Functional genes for milling quality and chalkiness**

Genetic understanding of milling quality is quite poor since only limited numbers of QTLs have been detected, and no QTL has been finely mapped or cloned. To make in-deep research into the area of milling quality, (1) rapid and accurate analytical tools are needed to measure the trait; (2) finely dissection of QTLs with large effect should be carried out; (3) because no mutants for milling quality have been reported, the mutants such as those induced by T-DNA insertion may provide a good start to characterize the genes responsible for milling quality. For grain chalkiness, two finely mapped QTLs await further characterization, and transcrip‐ tome for chalkiness formation during seed development have been described (Yamakawa et al. 2007; Liu et al. 2010). It looks optimism to see more progress from this area.

## **5.2. Molecular genetics studies for nutritional quality**

Nutrition quality of rice will be a new area for further research because people keep increas‐ ingly concern about the health benefit of the food they eat. Nutrition quality covers a wide range of traits, for example, protein, amino acids, fat and phenolics. In this area, naturally occurring variation for protein, amino acids, fat and fatty acid compositions have been under exploration, but only few genes have been characterized. Formation of each nutrient in rice grain requires a complex pathway in which many genes or enzymes are involved. Current advances in protein and fatty acid biosynthesis in other crops and *Arabidopsis* may help understand the pathways in rice.

Phenolics are expected to be an important field because they are proven to benefit human health in many ways (Shao and Bao 2012). Genes for red pericarp formation, *Rc* (brown pericarp) on chromosome 7 and *Rd* (red pericarp) on chromosome 1 have been under‐ stood, but their roles in regulating the flavonoids biosynthesis are unknown. The genes for dark purple pericarp formation, *Pb* and *Pp*, wait for finely mapping and functional characterization. In this field, MAS could be conducted to breed rice accumulating not only anthocyanins (a characteristic of black rice) and proanthocyanidin (a characteristic of red rice). Genetic transformation could be conducted to breed rice with accumulation of the anthocyanins or proanthocyanidin in the endosperm, since these phytochemicals accumu‐ late only in the bran layer (Shao and Bao 2012).

## **5.3. Cooking and eating quality of brown rice**

As concerns about nutritional quality rise, consumption of brown rice will become popular in the near future. Cooking and eating quality of brown rice will be another issue. The knowledge we have established for milled rice may not be applicable to the brown rice. Needless to say the genetic control of the cooking and eating quality of brown rice, what parameters to assess these qualities should be firstly considered. How to make brown rice appeal to consumers through suitable cooking methods should also be considered as well. At last, the question is how to improve the cooking and eating quality of brown rice.

## **5.4. MAS with more genes/QTLs together**

Targeting more traits with more markers, such as *Wx*, *SSIIa*, and *fragrance* (Jin et al. 2010), is increasingly needed in the breeding programs. MAS for quality and yield and resistance traits should be considered together in the future. Strategies for more effective selection should be developed when many markers are used at the same time. *In silica* molecular breeding is coming into the era, with which alleles of different markers are designed in the computer; the phenotypes of new rice could also be designed and displayed in the computer.

## **6. Conclusion**

CoQ10 is a very popular food supplement. Takahashi et al. (2009) produced CoQ10-enriched rice plants by introduction of the gene for decaprenyl diphosphate synthase. In CoQ10 enriched rice plants, seed CoQ10 content per weight was increased to up to 10 times that of wild-type rice, but its level is still insufficient for practical use. Combination of the transgene with giant embryo mutant lines produced giant embryo line-type CoQ10-enriched rice with seed CoQ10 content per weight increased to up to 1.4-1.8 times. It was found that CoQ was

Great progress has been achieved in our understanding of the genetic and molecular basis of grain quality of rice. This is especially true for grain appearance and grain shape, since they are not only linked with grain quality, but also with grain yield, a more important trait. Cooking and eating quality has a strong relation with starch biosynthesis pathway which has been well understood. Markers derived from the starch biosynthesis related genes have been widely applied in MAS. However, there are four major problem areas that challenge research‐

Genetic understanding of milling quality is quite poor since only limited numbers of QTLs have been detected, and no QTL has been finely mapped or cloned. To make in-deep research into the area of milling quality, (1) rapid and accurate analytical tools are needed to measure the trait; (2) finely dissection of QTLs with large effect should be carried out; (3) because no mutants for milling quality have been reported, the mutants such as those induced by T-DNA insertion may provide a good start to characterize the genes responsible for milling quality. For grain chalkiness, two finely mapped QTLs await further characterization, and transcrip‐ tome for chalkiness formation during seed development have been described (Yamakawa et

Nutrition quality of rice will be a new area for further research because people keep increas‐ ingly concern about the health benefit of the food they eat. Nutrition quality covers a wide range of traits, for example, protein, amino acids, fat and phenolics. In this area, naturally occurring variation for protein, amino acids, fat and fatty acid compositions have been under exploration, but only few genes have been characterized. Formation of each nutrient in rice grain requires a complex pathway in which many genes or enzymes are involved. Current advances in protein and fatty acid biosynthesis in other crops and *Arabidopsis* may help

Phenolics are expected to be an important field because they are proven to benefit human health in many ways (Shao and Bao 2012). Genes for red pericarp formation, *Rc* (brown pericarp) on chromosome 7 and *Rd* (red pericarp) on chromosome 1 have been under‐

al. 2007; Liu et al. 2010). It looks optimism to see more progress from this area.

preferentially accumulated in bran and germ of rice seed.

ers working on molecular genetics of grain quality.

**5.1. Functional genes for milling quality and chalkiness**

**5.2. Molecular genetics studies for nutritional quality**

understand the pathways in rice.

**5. Future directions**

262 Rice - Germplasm, Genetics and Improvement

Grain quality of rice as a whole is a complex trait that is comprised of appearance quality, milling quality, eating and cooking quality, and nutritional quality etc. Researches on the genetic control of the quality traits have made a great progress, especially for the appearance quality, cooking and eating quality. More genetic studies are needed for milling quality and nutritional quality.

The progress on the molecular genetics on grain quality has allowed MAS to be conducted more efficiently. However, only MAS for cooking and eating quality and genetic engineering for nutritional quality have made some achievements. More molecular breeding practices are needed for improvement of grain quality.

With social development and improvement of living standards, cooking and eating quality of brown rice will be a new theme that deserves greater attention from researches. Studies including cooking methods, parameters for cooking and eating, genetics, and molecular breeding are among the top priorities.

## **Acknowledgements**

This work was financially supported by Zhejiang Provincial Natural Science Foundation (LZ13C0001) and Special Fund for Agro-scientific Research in the Public Interest (201103007) from the Ministry of Agriculture, China.

## **Author details**

Jinsong Bao\*

Address all correspondence to: jsbao@zju.edu.cn

Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Zhe‐ jiang University, Hangzhou, China

## **References**


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**Acknowledgements**

264 Rice - Germplasm, Genetics and Improvement

**Author details**

Jinsong Bao\*

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## **Chinese Experiences in Breeding Three-Line, Two-Line and Super Hybrid Rice**

Liyong Cao and Xiaodeng Zhan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56821

## **1. Introduction**

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278 Rice - Germplasm, Genetics and Improvement

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Because rice is a staple food for over half of the world's population, it is estimated that the world rice production must be annually increased by approximately 1% to meet the growing demand for food, resulting from population growth and economic development (Rosegrant et al., 1995). Rice is one of the main food crops in China with the second largest planting area, most total yield and highest per unit yield (Table 1), it feeds more than 60% of the population and contributing nearly 40% of total calorie intake in China (Cheng and Li, 2007). China is the largest producer and consumer of rice, and also a pioneer in the utilization of hybrid rice technology in the world. Hybrid rice has resulted in a substantial increase of food production in China over the past 40 years. China average rice yield has risen from 1.89 tons per ha (t/ha) in 1949 to 6.71 t/ha in 2012, which created the highest historical record (http:// futures.xinhua08.com/a/20121018/1042507.shtml). Hybrid rice has played an important role for total grain production to consecutively increase for nine years in China (http:// www.aqzyzx.com/system/2012/10/31/006110920.shtml).


Source: http://datacenter.cngrain.com/IndexProduce.aspx?Flag=1&IsHome=0&TId=74&Str=PP

**Table 1.** Planting area, total production and yield of three main food crops in China in 2011

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Variety improvement plays a leading role in increasing of grain yield characterized as two quantum leaps in rice (Chen et al., 2002). The first one was brought about by the development of semi-dwarf varieties in the late 1950s in China and early 1960s at the International Rice Research Institute (IRRI). In 1956, a dwarf mutant was found in *indica* variety Nantehao in Guangdong Province, China. Since then, Huang et al. (2001) had initiated dwarf rice breeding in Guangdong and southern China, and released semi-dwarf *indica* rice varieties Guangluai 4, Guichao 2, Teqin, etc. subsequently. The semi-dwarf varieties displayed a yield potential up to 7.5 t/ha, which is 20%–30% higher than the traditional tall varieties, owing to the improved tolerance to lodging for standing higher rates of fertilizer. In 1966, IR8 as the first semi-dwarf variety from IRRI was released to tropical irrigated lowlands (Peng et al., 1994; 2008). The second leap arose from commercial use of hybrid rice in 1976 in China (Yuan et al., 1994). Compared with inbred rice, hybrid rice can increase grain yield by approximately 20%. These two major breakthroughs have brought China's average rice yield up to a new level in the mid-1970s and mid-1980s, respectively. Thereafter, with popularization of hybrid rice due to improved hybrid seed production methods, rice yield was further elevated to 6.0 t/ha in early 1990s in China, which was the world level, then. However, the yield ceiling witnessed in various crop species has also been encountered in the rice production in China since 1990. Considering that the annual per capita rice consumption is 150 kg and rice cropping area maintains at 31.57 million hectares in China, it is estimated that rice total production and yield per unit must be increased by 35% and 32%, respectively in 2030 (Cheng et al., 2005). This estimation implies a great challenge to rice community, and the 3rd leap of rice yield is definitely needed for the challenge.

In response to the challenge, Chinese Ministry of Agriculture (CMOA) organized China National Super Hybrid Rice Symposium at Shenyang in 1996, where rice scientists from all over China united to design a national proposal to breed super hybrid rice and develop cultivation methodologies to realize yield potential of the super hybrid. In 2000, because of the leadership of CMOA and the leading role of China National Rice Research Institute, China Super Rice Cooperative Research Group released super hybrid rice cultivars and reached the phaseⅠtarget of 10.5 t/ha. In 2005, the phase II target of 12 t/ha was accomplished, and cultivation of super hybrid rice cultivars developed in the phase I was dramatically extended to a large area nationwide. Till 2012, the grain production has consecutively increased for nine years, and nationally produced grain of more than 500 million tons has maintained for five consecutive years in China. The 500 million tons set a new record of grain production in China, which is the planning level of grain production in 2020. The abundant harvest will play an important role in maintaining economy to develop steadily in China.

Meanwhile, steady rice production in China has to keep dealing with decreasing growth area along with an increasing population, biotic and abiotic stresses, extensive use of chemical fertilizers, and water shortage. Therefore, it seems at present that the most effective and economic way is to develop and extend super inbred rice and hybrid rice cultivars with wide adaptation and super high yielding potential, which is also an alternative solution to China's future food security problem and an important way to maintain social stability (Chen et al., 2007).

## **2. Genetic mechanism of rice heterosis**

Variety improvement plays a leading role in increasing of grain yield characterized as two quantum leaps in rice (Chen et al., 2002). The first one was brought about by the development of semi-dwarf varieties in the late 1950s in China and early 1960s at the International Rice Research Institute (IRRI). In 1956, a dwarf mutant was found in *indica* variety Nantehao in Guangdong Province, China. Since then, Huang et al. (2001) had initiated dwarf rice breeding in Guangdong and southern China, and released semi-dwarf *indica* rice varieties Guangluai 4, Guichao 2, Teqin, etc. subsequently. The semi-dwarf varieties displayed a yield potential up to 7.5 t/ha, which is 20%–30% higher than the traditional tall varieties, owing to the improved tolerance to lodging for standing higher rates of fertilizer. In 1966, IR8 as the first semi-dwarf variety from IRRI was released to tropical irrigated lowlands (Peng et al., 1994; 2008). The second leap arose from commercial use of hybrid rice in 1976 in China (Yuan et al., 1994). Compared with inbred rice, hybrid rice can increase grain yield by approximately 20%. These two major breakthroughs have brought China's average rice yield up to a new level in the mid-1970s and mid-1980s, respectively. Thereafter, with popularization of hybrid rice due to improved hybrid seed production methods, rice yield was further elevated to 6.0 t/ha in early 1990s in China, which was the world level, then. However, the yield ceiling witnessed in various crop species has also been encountered in the rice production in China since 1990. Considering that the annual per capita rice consumption is 150 kg and rice cropping area maintains at 31.57 million hectares in China, it is estimated that rice total production and yield per unit must be increased by 35% and 32%, respectively in 2030 (Cheng et al., 2005). This estimation implies a great challenge to rice community, and the 3rd leap of rice yield is definitely

In response to the challenge, Chinese Ministry of Agriculture (CMOA) organized China National Super Hybrid Rice Symposium at Shenyang in 1996, where rice scientists from all over China united to design a national proposal to breed super hybrid rice and develop cultivation methodologies to realize yield potential of the super hybrid. In 2000, because of the leadership of CMOA and the leading role of China National Rice Research Institute, China Super Rice Cooperative Research Group released super hybrid rice cultivars and reached the phaseⅠtarget of 10.5 t/ha. In 2005, the phase II target of 12 t/ha was accomplished, and cultivation of super hybrid rice cultivars developed in the phase I was dramatically extended to a large area nationwide. Till 2012, the grain production has consecutively increased for nine years, and nationally produced grain of more than 500 million tons has maintained for five consecutive years in China. The 500 million tons set a new record of grain production in China, which is the planning level of grain production in 2020. The abundant harvest will play an

Meanwhile, steady rice production in China has to keep dealing with decreasing growth area along with an increasing population, biotic and abiotic stresses, extensive use of chemical fertilizers, and water shortage. Therefore, it seems at present that the most effective and economic way is to develop and extend super inbred rice and hybrid rice cultivars with wide adaptation and super high yielding potential, which is also an alternative solution to China's future food security problem and an important way to maintain social stability (Chen et al.,

important role in maintaining economy to develop steadily in China.

needed for the challenge.

280 Rice - Germplasm, Genetics and Improvement

2007).

Heterosis, or hybrid vigor, refers to the phenomenon that progeny of diverse inbred varieties is superior over both parents on yield, panicle size, number of spikelets per panicle, number of productive tillers, stress tolerance etc. This phenomenon to be a powerful force in the evolution of plants has been exploited extensively in crop production. Successful development of hybrid maize in 1930 gave great impetus to breeders of other crops including rice to utilize the principle of hybrid production by exploiting heterosis. In fact, the exploitation of heterosis has been the greatest practical achievement of the science of genetics and plant breeding (Alam et al., 2004). The impact of this phenomenon can be judged by the fact that rice dramatically varies on the number of grains per square meter among 1) wild ancestors with only a few hundreds, 2) improved inbred varieties with about 40, 000, and 3) rice hybrids with about 52, 000 (Mir, 2002). Rice heterosis was first reported by Jones (1926) who observed that some F1 hybrids had more culms and greater yield than their parents. Between 1962 and 1967, a number of suggestions came from different places of the world for commercial exploitation of heterosis to become a major component of rice improvement programs at national and international level. For example, rice breeders from Japan, China, United States, India, the former Soviet Union and Philippines started their projects to utilize rice heterosis. However, progress had not been sound because of the difficulty for rice to be a strictly self-pollinated crop unlike corn, which made out crossing absolutely essential for hybrid seed production extremely difficult.

## **2.1. Genetic hypotheses for crop heterosis**

Classic quantitative genetic explanations for heterosis center on two concepts, dominance and over dominance (Crow, 1952). With advances on genetic study of quantitative traits and high density molecular linkage maps, many research groups prefer epistasis as a major genetic basis of heterosis (Wright, 1968; Hallauer and Miranda, 1988).

"Dominance" originally means that heterosis is resulted from action and interaction of favorable dominant genes brought together in an F1 hybrid from two parents. This hypothesis assumes that genes that are favorable for vigor and growth are dominant, and the genes contributed by another parent result in more favorable combination of dominant genes in the F1 than either parent. For instance, we have a combination of five dominant genes ABCDE favorable for yields, patent one (P1) has the genotype AAbbCCDDee (possessing three dominant genes ACD) and parent two (P2) has the genotype aaBBccddEE (possessing two dominant genes BE); the F1 hybrid derived from the two parents will have five dominant genes as shown below (Fig. 1).

The F1 hybrid therefore would exhibit higher yield than either of the parents because each parenthas only a part of five dominant genes. According to this hypothesis, inbreeding depression occurs when unfavorable recessive genes hidden in the heterozygous conditions in the F1 generation become homozygous in subsequent generations with inbreeding. Crossing unrelated homozygous lines obscures the deleterious recessives and restores vigor.

$$\begin{array}{ccc} \text{P1 } \text{A} \text{A} \text{b} \text{b} \text{C} \text{C} \text{D} \text{e} \text{e} \times \begin{array}{c} \text{P2 } \text{a} \text{a} \text{B} \text{b} \text{c} \text{d} \text{d} \text{E} \\\\ \bullet \\\\ \text{F1} \qquad \text{A} \text{a} \text{B} \text{b} \text{C} \text{c} \text{D} \text{d} \text{E} \text{e} \text{ (performance of heterosis)} \end{array}$$

parents will have five dominant genes as shown below (Fig. 1) .

and high density molecular linkage maps, many research groups prefer epistasis as a

"Dominance" originally means that heterosis is resulted from action and interaction of favorable dominant genes brought together in an F1 hybrid from two parents. This hypothesis assumes that genes that are favorable for vigor and growth are dominant, and the genes contributed by another parent result in more favorable combination of dominant genes in the F1 than either parent. For instance, we have a combination of five dominant genes ABCDE favorable for yields, patent one (P1) has the genotype AAbbCCDDee (possessing three dominant genes ACD) and parent two (P2) has the genotype aaBBccddEE (possessing two dominant genes BE); the F1 hybrid derived from the two

The F1 hybrid therefore would exhibit higher yield than either of the parents because each parenthas only a part of five dominant genes. According to this hypothesis, inbreeding depression occurs when unfavorable recessive genes hidden in the heterozygous conditions in the F1 generation become homozygous in subsequent generations with inbreeding. Crossing unrelated homozygous lines obscures the deleterious recessives and

major genetic basis of heterosis (Wright, 1968; Hallauer and Miranda, 1988).

**Figure 1.** Illustration of dominance hypothesis

The second historical explanation for heterosis is "over dominance," which refers to allelic interactions in the hybrid, such that the heterozygous class performs better than either homozygous class (Fig. 2). Thus, an individual such as the F1 hybrid with the greatest number of heterozygous alleles will be mostly vigorous compared to two parents. The second historical explanation for heterosis is "over dominance, " which refers to allelic interactions in the hybrid, such that the heterozygous class performs better than either homozygous class (Fig. 2). Thus, an individual such as the F1 hybrid with the greatest number of heterozygous alleles will be mostly vigorous compared to two parents. Because these two explanations for heterosis were developed under the conditions with non-additive effects and supposed all the genes have the same influences to different traits, they have limitations and can't explain the heterosis in molecular level. Therefore, they are of diminished utility for describing the molecular parameters that accompany heterosis. Because these two explanations for heterosis were developed under the conditions with non-additive effects and supposed all the genes have the same influences to different traits, they have limitations and can't explain the heterosis in molecular level. Therefore, they are of diminished utility for describing the molecular parameters that accompany heterosis.

 **Fig. 1 Illustration of dominance hypothesis** 

**Figure 2.** Illustration of over-dominance hypothesis

The hypothesis of over dominance advocating that the hybrids exhibit superiority to the better parent has been agreed by increasing number of studies. However, this hypothesis The hypothesis of over dominance advocating that the hybrids exhibit superiority to the better parent has been agreed by increasing number of studies. However, this hypothesis completely denies the function of dominant genes in heterosis. It is well known that heterosis doesn't perfectly comply with heterozygosity of alleles. For instance, some rice hybrids do not perform better than their homozygous parents at some specific traits.

 **Fig 2 Illustration of over-dominance hypothesis** 

completely denies the function of dominant genes in heterosis. It is well known that heterosis doesn't perfectly comply with heterozygosity of alleles. For instance, some rice hybrids do not perform better than their homozygous parents at some specific traits. The hypothesis of epistasis regards heterosis to be genetically controlled by many genes because a complex character such as yield includes many components. Heterozygosity with gene interaction is the primary genetic basis for explanation of heterosis because the hybrid is heterozygous across all genetic loci that different between the parents. Thus, the degree of heterosis depends on which loci are heterozygous and how within locus alleles and inter-locus alleles interact with each other. Interaction of within locus alleles results in The hypothesis of epistasis regards heterosis to be genetically controlled by many genes because a complex character such as yield includes many components. Heterozygosity with gene interaction is the primary genetic basis for explanation of heterosis because the hybrid is heterozygous across all genetic loci that different between the parents. Thus, the degree of heterosis depends on which loci are heterozygous and how within locus alleles and inter-locus alleles interact with each other. Interaction of within locus alleles results in dominance, partial dominance, or over dominance, with a theoretical range of dominance degree from zero (no dominance) to larger than 1 (over dominance). Interaction of inter-locus alleles results in epistasis. Genetic mapping results have indicated that most QTLs involve in heterosis and other quantitative traits have a dominance effect. Epistasis has been found more frequently and proven to be a common phenomenon in the genetic control of quantitative traits including heterosis (Yu et al., 1997; Luo et al., 2001; Hua et al., 2003). Study of Yu et al. (1997) provided strong evidence for two-loci and multi-loci interactions (epistasis) especially for traits such as grain yield, which are complex in nature. They found that heterosis is not controlled by a single

> dominance, partial dominance, or over dominance, with a theoretical range of dominance degree from zero (no dominance) to larger than 1 (over dominance). Interaction of interlocus alleles results in epistasis. Genetic mapping results have indicated that most QTLs involve in heterosis and other quantitative traits have a dominance effect. Epistasis has been found more frequently and proven to be a common phenomenon in the genetic control of quantitative traits including heterosis (Yu et al., 1997; Luo et al., 2001; Hua et

locus, even the locus behaves in dominant or overdominant patten, linkage and epistasis has a major role. Thus, the effects of dominance, over dominance and epistasis of various forms are not mutually exclusive in the genetic basis of heterosis, as opposed to what was previously debated in favor of different hypothesis. All of these components have a role to play depending on the genetic architecture of the population (Hua et al., 2003), i. e. single-locus heterotic effects (caused by partial, full-and over-dominance), all three forms of digenic interactions (AA/AD/ DA and DD) and probably multi-locus interactions.

Thus, these results may help reconcile the century long debate on the role of dominance, overdominance and epistasis as genetic basis of heterosis. Two different types of allelic interaction, both within-locus and inter-locus, should play an important role in the genetic control of heterosis. A full understanding of heterosis has to wait for breakthrough achievements on cloning and functional analysis of all genes related to heterosis. This process would be very similar to the understanding of disease resistance genes with aid of standard check variety.

## **2.2. Molecular basis of heterosis**

and high density molecular linkage maps, many research groups prefer epistasis as a

"Dominance" originally means that heterosis is resulted from action and interaction of favorable dominant genes brought together in an F1 hybrid from two parents. This hypothesis assumes that genes that are favorable for vigor and growth are dominant, and the genes contributed by another parent result in more favorable combination of dominant genes in the F1 than either parent. For instance, we have a combination of five dominant genes ABCDE favorable for yields, patent one (P1) has the genotype AAbbCCDDee (possessing three dominant genes ACD) and parent two (P2) has the genotype aaBBccddEE (possessing two dominant genes BE); the F1 hybrid derived from the two

The F1 hybrid therefore would exhibit higher yield than either of the parents because each parenthas only a part of five dominant genes. According to this hypothesis, inbreeding depression occurs when unfavorable recessive genes hidden in the heterozygous conditions in the F1 generation become homozygous in subsequent generations with inbreeding. Crossing unrelated homozygous lines obscures the deleterious recessives and

The second historical explanation for heterosis is "over dominance," which refers to allelic interactions in the hybrid, such that the heterozygous class performs better than either homozygous class (Fig. 2). Thus, an individual such as the F1 hybrid with the greatest number of heterozygous alleles will be mostly vigorous compared to two parents.

Because these two explanations for heterosis were developed under the conditions with non-additive effects and supposed all the genes have the same influences to different traits, they have limitations and can't explain the heterosis in molecular level. Therefore, they are of diminished utility for describing the molecular parameters that accompany

major genetic basis of heterosis (Wright, 1968; Hallauer and Miranda, 1988).

parents will have five dominant genes as shown below (Fig. 1) .

P1 AAbbCCDDee × P2 aaBBccddEE

F1 AaBbCcDdEe (performance of heterosis)

 **Fig. 1 Illustration of dominance hypothesis** 

The second historical explanation for heterosis is "over dominance, " which refers to allelic interactions in the hybrid, such that the heterozygous class performs better than either homozygous class (Fig. 2). Thus, an individual such as the F1 hybrid with the greatest number of heterozygous alleles will be mostly vigorous compared to two parents. Because these two explanations for heterosis were developed under the conditions with non-additive effects and supposed all the genes have the same influences to different traits, they have limitations and can't explain the heterosis in molecular level. Therefore, they are of diminished utility for

P a1a1b1b1c1c1d1d1e1e1× a2a2b2b2c2c2d2d2e2e2

F1 a1a2b1b2c1c2d1d2e1e2 (performance of heterosis)

 **Fig 2 Illustration of over-dominance hypothesis** 

The hypothesis of over dominance advocating that the hybrids exhibit superiority to the better parent has been agreed by increasing number of studies. However, this hypothesis completely denies the function of dominant genes in heterosis. It is well known that heterosis doesn't perfectly comply with heterozygosity of alleles. For instance, some rice hybrids do not perform

The hypothesis of epistasis regards heterosis to be genetically controlled by many genes because a complex character such as yield includes many components. Heterozygosity with gene interaction is the primary genetic basis for explanation of heterosis because the hybrid is heterozygous across all genetic loci that different between the parents. Thus, the degree of heterosis depends on which loci are heterozygous and how within locus alleles and inter-locus alleles interact with each other. Interaction of within locus alleles results in dominance, partial dominance, or over dominance, with a theoretical range of dominance degree from zero (no dominance) to larger than 1 (over dominance). Interaction of inter-locus alleles results in epistasis. Genetic mapping results have indicated that most QTLs involve in heterosis and other quantitative traits have a dominance effect. Epistasis has been found more frequently and proven to be a common phenomenon in the genetic control of quantitative traits including heterosis (Yu et al., 1997; Luo et al., 2001; Hua et al., 2003). Study of Yu et al. (1997) provided strong evidence for two-loci and multi-loci interactions (epistasis) especially for traits such as grain yield, which are complex in nature. They found that heterosis is not controlled by a single

The hypothesis of over dominance advocating that the hybrids exhibit superiority to the better parent has been agreed by increasing number of studies. However, this hypothesis completely denies the function of dominant genes in heterosis. It is well known that heterosis doesn't perfectly comply with heterozygosity of alleles. For instance, some rice

hybrids do not perform better than their homozygous parents at some specific traits.

The hypothesis of epistasis regards heterosis to be genetically controlled by many genes because a complex character such as yield includes many components. Heterozygosity with gene interaction is the primary genetic basis for explanation of heterosis because the hybrid is heterozygous across all genetic loci that different between the parents. Thus, the degree of heterosis depends on which loci are heterozygous and how within locus alleles and inter-locus alleles interact with each other. Interaction of within locus alleles results in dominance, partial dominance, or over dominance, with a theoretical range of dominance degree from zero (no dominance) to larger than 1 (over dominance). Interaction of interlocus alleles results in epistasis. Genetic mapping results have indicated that most QTLs involve in heterosis and other quantitative traits have a dominance effect. Epistasis has been found more frequently and proven to be a common phenomenon in the genetic control of quantitative traits including heterosis (Yu et al., 1997; Luo et al., 2001; Hua et

restores vigor.

describing the molecular parameters that accompany heterosis.

better than their homozygous parents at some specific traits.

**Figure 1.** Illustration of dominance hypothesis

282 Rice - Germplasm, Genetics and Improvement

heterosis.

**Figure 2.** Illustration of over-dominance hypothesis

Since heterosis is a phenomenon of superior growth, development, differentiation, and maturation caused by the interaction of genes, metabolism and environment, a simple explanation of heterosis solely based on the nuclear genome heterozygosity appears untenable. Several distinct lines of evidence from biochemical, physiological, ultrastructural and restric‐ tion endonuclease DNA fragment analyses in a variety of organisms are available to show that all three genetic sources of nuclear genome, mitochondrial genome and chloroplast genome, in staed just one of them, are at work during the manifestation of heterosis.

Some molecular studies support the over dominance hypothesis (Stuber et al., 1992, Yu et al., 1997, Li et al., 2001), but another supports the dominance hypothesis (Xiao et al 1995). Yu et al. (1997) reported over dominance at several main-effect quantitative trait loci (QTLs) and a stronger additive epistasis affecting grain yield and its components in F3 progenies from Shan You 63, the most widely grown hybrid in China. Furthermore, Li et al. (2001) concluded that most QTLs associated with inbreeding depression and heterosis in rice appeared to be involved in epistasis, and almost 90% of the QTLs contributing to heterosis appeared to be over dominant. Zhang et al. (2001) assessed the relationship between gene expression and heterosis by assaying the patterns of different genes expression in hybrids related their parents using a diallel cross. The analysis revealed that differentially expressing fragments occurred in only one parent of the cross were positively correlated with heterosis, but the fragments detected only in F1 generation not in the respective parents were negatively correlated with heterosis. Using a total of 384 fragments recovered from gels which hybridized with the mRNAs from seedling and flag-leaf tissues, Zhang et al. (2000) detected an overall elevated level of gene expression in the hybrid compared with the parents, where several fragments showed a higher expression in the high-heterotic hybrid than in the low-heterotic hybrids. Studying the molecular mechanism of differential gene expression between Chinese super-hybrid rice cultivars and their parental lines concluded that many genes were up-regulated in the superhybrid, whereas other genes were down-regulated (Zhang et al, 2006). These findings pointed out a role of enhanced photosynthesis in the heterosis of the super-hybrid combinations. Using different display techniques for a set of diallel cross involved eight elite hybrid rice parents, Xiong et al. (1998) studied the relationship between banding patterns of differentially dis‐ played gene expressions and the level of heterosis, and showed that dominant type of differential gene expression in flag leaf tissue failed to be correlated with heterosis on yield traits, while differential inhibition of gene expression in the hybrids appeared to be signifi‐ cantly correlated with heterosis. Huang et al. (2006) analyzed gene expression profiles of an elite rice hybrid with the parents at three stages of young panicle development, a cDNA microarray consisting of 9198 expressed sequence tags (ESTs) was used for the objective to reveal gene expression patterns that may be associated with heterosis in yield. The results showed that the biochemical and physiological activities took place in the hybrid relatively rather than in the parents. Identification of genes showing expression polymorphisms among different genotypes and heterotic expression in the hybrid may provide new avenues for exploring the biological mechanisms underlying heterosis.

Nonetheless, a lack of a clear understanding of the genetic or molecular basis of heterosis has not prevented plant breeders from exploiting this phenomenon to raise crop yields.

## **3. Methods and strategies in hybrid rice breeding**

## **3.1. The methods in hybrid breeding**

Prof. Yuan proposed the breeding strategy to Chinese scientists for developing hybrid rice in the following phases. Three approaches are for breeding methodology, 1) three-line method or CMS (cytoplasmic male sterility) system, 2) two-line method or PTGMS (photo/temperature sensitive genic male sterility) system and 3) one-line method or apomixis system. Three ways are for increasing the degree of heterosis, 1) inter-varietal hybrids, 2) inter-sub-specific hybrids and 3) inter-specific or intergeneric hybrids (distant hybrids).

*Indica* and *japonica* (both tropical and temperate) are two main subspecies of *Oryza sativa* in Asia. *Javanica* is genetically between *indica* and *japonica*. Among them, *indica* and *temperate japonica* subspecies have the most apparent difference in morphological and agronomic traits. Many studies indicate that the degree of heterosis in different kinds of hybrid rice varieties has the following general trend: *indica/japonica>indica/javanica>japonica/javanica>indi‐ ca/indica>japonica/japonica* (Yuan, 1996). The first three kinds are inter-subspecific hybrid, and the last two are inter-varietal. Currently, hybrid rice technology mainly uses intervarietal heterosis, *indica* × *indica* and *japonica* × *japonica*. The high-yielding inter-subspecific hybrids yield 15% to 20% more than the best inbred varieties when they are grown under similar conditions. But now, it has been quite difficult to make the genetic differ‐ ence between parents great enough for the inter-subspecific hybrids, so that their yields have almost reached a ceiling. In *japonica*/*javanica* hybrids, there are a few fertility prob‐ lems till present. Therefore, using heterosis between *japonica/japonica* and *indica/japonica* would be an effective approach for increasing rice yields. *Indica*/*japonica* hybrids posses the highest yield potential in both sink and source. Their theoretical yield can be 30% more than the best existing inter-varietal hybrid varieties. But inter-subspecific heterosis has not been commercially utilized because of high spikelet sterility and long growth duration.

The discovery of wide compatibility (WC) genes provides a possibility to resolve the problems of seed setting and growth duration in the inter-subspecific hybrids. IRRI, China, and India are making a great effort to develop inter-subspecific hybrids. Recently, Chinese scientists have developed super high-yielding rice hybrids from crosses involving *indica*/ *japonica* derivative parents. Most of the inter-specific crosses in cultivated species are within either *O. sativa* or *O. glaberrima*, and their hybrids are heterotic but not so useful in terms of yield and plant stature. Most inter-specific hybrids from wide hybridization have elevation of genetic variability and bring in desirable genes for resistance to several biotic and abiotic stresses, such as the progeny of *O. sativa* × *O. longistaminata*, *O. sativa* × *O. rufipogon*, and *O. sativa* × *O. perennis*.

## **3.2. The principles for parental selection**

different display techniques for a set of diallel cross involved eight elite hybrid rice parents, Xiong et al. (1998) studied the relationship between banding patterns of differentially dis‐ played gene expressions and the level of heterosis, and showed that dominant type of differential gene expression in flag leaf tissue failed to be correlated with heterosis on yield traits, while differential inhibition of gene expression in the hybrids appeared to be signifi‐ cantly correlated with heterosis. Huang et al. (2006) analyzed gene expression profiles of an elite rice hybrid with the parents at three stages of young panicle development, a cDNA microarray consisting of 9198 expressed sequence tags (ESTs) was used for the objective to reveal gene expression patterns that may be associated with heterosis in yield. The results showed that the biochemical and physiological activities took place in the hybrid relatively rather than in the parents. Identification of genes showing expression polymorphisms among different genotypes and heterotic expression in the hybrid may provide new avenues for

Nonetheless, a lack of a clear understanding of the genetic or molecular basis of heterosis has

Prof. Yuan proposed the breeding strategy to Chinese scientists for developing hybrid rice in the following phases. Three approaches are for breeding methodology, 1) three-line method or CMS (cytoplasmic male sterility) system, 2) two-line method or PTGMS (photo/temperature sensitive genic male sterility) system and 3) one-line method or apomixis system. Three ways are for increasing the degree of heterosis, 1) inter-varietal hybrids, 2) inter-sub-specific hybrids

*Indica* and *japonica* (both tropical and temperate) are two main subspecies of *Oryza sativa* in Asia. *Javanica* is genetically between *indica* and *japonica*. Among them, *indica* and *temperate japonica* subspecies have the most apparent difference in morphological and agronomic traits. Many studies indicate that the degree of heterosis in different kinds of hybrid rice varieties has the following general trend: *indica/japonica>indica/javanica>japonica/javanica>indi‐ ca/indica>japonica/japonica* (Yuan, 1996). The first three kinds are inter-subspecific hybrid, and the last two are inter-varietal. Currently, hybrid rice technology mainly uses intervarietal heterosis, *indica* × *indica* and *japonica* × *japonica*. The high-yielding inter-subspecific hybrids yield 15% to 20% more than the best inbred varieties when they are grown under similar conditions. But now, it has been quite difficult to make the genetic differ‐ ence between parents great enough for the inter-subspecific hybrids, so that their yields have almost reached a ceiling. In *japonica*/*javanica* hybrids, there are a few fertility prob‐ lems till present. Therefore, using heterosis between *japonica/japonica* and *indica/japonica* would be an effective approach for increasing rice yields. *Indica*/*japonica* hybrids posses the highest yield potential in both sink and source. Their theoretical yield can be 30% more

not prevented plant breeders from exploiting this phenomenon to raise crop yields.

exploring the biological mechanisms underlying heterosis.

**3. Methods and strategies in hybrid rice breeding**

and 3) inter-specific or intergeneric hybrids (distant hybrids).

**3.1. The methods in hybrid breeding**

284 Rice - Germplasm, Genetics and Improvement

High-yield, good quality and multiple resistances are the eternal targets in rice breeding. Matching proper parents together is the key and basis to breed excellent hybrid combina‐ tions. Selection of excellent hybrid combinations must base on heterosis with the follow‐ ing specific considerations.

#### *3.2.1. Selection of parents with big variation in genetic basis*

Hereditary diversity is the basis of heterosis, and within a certain range, the genetic diversity decides heterosis. Because rice varieties are different in kinship, geographical origin or ecotype, the heterosis is produced by genetic diversity between both parents. Widely commercialized hybrid rice combinations usually have widely different parents in ecological types and geographic origins, such as the parents for Shanyou63, ShanYou10 and XieYou46. Further‐ more, the parents may differ in *indica*-*japonica* affinity.

#### *3.2.2. Screening the parents with good traits*

Presently, hybrid rice varieties in commercial production have complementary good traits from their parents. Thus, hybrid rice combinations are comprehensively better than the parents, such as Shanyou63, Shanyou10, Liangyou Peijiu, Zhongzhe You 1 and so on. The hybrid combinations gather many good characteristics of parents such as disease-resistance, late mid-maturity, strong tillering ability and high grain weight. Studies on heredity law of essential traits in hybrid rice showed that some traits of hybrids have certain relationships with the average of parents, such as number of grains per panicle, filled grain number per panicle, grain weight, efficient panicles per unit area, growth period and plant height. These traits have highly significant correlations between a hybrid and the average of its parents. According to this relationship, we may choose the combinations with excellent parents to breed new restorer or maintainer lines.

## *3.2.3. Selection of parents with good combining ability*

General combining ability (GCA) refers to the average performance of an offspring from one parent that is crossed with many other parents. GCA is determined by the number of favorable genes and the size of gene function in parents, and often influenced by additive effects. Specific combining ability (SCA) refers to the performance of a unique offspring deviation from a specific pair of parents. SCA is mainly controlled by non-additive effects. Therefore, not only the yield of parents is necessary for, but also the combining ability of parents is very important for hybrid rice breeding.

## **3.3. Strategies for hybrid rice breeding**

In addition to the breeding principles described above, the following strategies are also very important in hybrid rice breeding to result in hybrid cultivars with high yield, good quality and disease resistance.

## *3.3.1. Amplification of genetic diversity in parents*

Through pedigree analysis, most rice cultivars originate from few parents which genetic diversity is small. The main reason for hybrid rice yield to stand still since 1980s maybe relate to short in genetic diversity in China. Since the discovery of semi-dwarfism in *indica*, genetic diversity among cultivars has become increasingly narrow, so that strong hybrid vigour has hardly been achieved by combining *indicas* cultivars. *Japonica* materials are full developed and utilized in temperate zone, resulting in a narrow genetic diversity among cultivars. The narrow diversity makes it very hard to achieve hybrid preponderance in inter varietal crosses. Tropic *japonica* (*Javanica*) and *nuda* types of rice (*japonica*) are not widely utilized in breeding because they are limited in special regions with some particular traits. Recently breeders have paid much attention to wide compatibility germplasm derived from tropic *japonica* and *nuda* rice. After Yuan (1987) proposed the strategy to utilize heterosis between *indica-japonica*, breeders have made a great effort to amplify parents' genetic diversity and breed super high-yield *indica-japonica* hybrid through effective utilization of wide compatibility. *Indica-japonica* combinations from direct crosses hardly have super high-yield because of sterility. Introgress‐ ing *japonica* genes into *indica* restorer lines in the south and introgressing indica genes into *japonica* in the north are proven to be effective for super high-yield hybrid rice breeding in China. Taking this strategy, three new *indica* restorer lines, Zhong413, R9308 and T2070, are bred by China National Rice Research Institute. They all contain about 25% of *japonica* components, and their hybrid combinations have super high-yield.

#### *3.3.2. Increasing the biological outputs*

Ideotype is an ideal plant model which is expected to yield the most for a specific environment. Hybrid rice ideotype (HRI) is a best parental combination to yield the most grains with good quality in a certain ecological environment. HRI does not only contain desire morphological characteristics but also have resistances to biotic and abiotic stresses for the environment. Otherwise, the most grains with good quality are not achievable. For instance, Taoyuan in Yongsheng County, Yunnan Province, China is a perfect ecological environment for rice, where the yield of indica hybrid rice Shanyou63 can be up to 15.27 t/ha with a harvest index up to 0.54 in small field trial. However, under normal conditions in most areas of China, it is not possible for Shanyou63 to yield 15.27 t/ha. This difference may imply the importance of plant biomass for grain yield. Increasing plant biomass with maintaining or improving harvest index may lead to raise grain yield directly. It is well known that high biological-outputs need abundance of sunshine and high levels of nitrogen nutrition. In addition to high plant biomass, high grain yield needs a series of good characteristics such as tough stems, erect leaves and rational operation of photo synthetic products. Otherwise, the population will lodge to flat and the leaves shade each other, favorable for pest and disease infestations. At the end, rice yields will decline instead of increasing.

Increasing the biological outputs is the key to further improve yield potential under a certain harvest index condition for high-yielding dwarf rice. It is no doubt to properly raise plant height beneficial for increasing biological products, but we must enhance the capacity of lodging resistance at the same time and ensure all set grains harvestable. In current highyielding rice fields, when the number of spikelets per square meter is about 40, 000 or slightly less, 50, 000 to 60, 000 and 60, 000 to 70, 000, the corresponding grain yield could be 7.5 t/ha, 11.25 t/ha and 15 t/ha, respectively. To a specific rice variety, the panicle number per unit and grains per panicle varied upon to ecological conditions, thus, we must improve leaf position and leaf quality to increase the number of spikelets per unit and the total output. Meanwhile, strong root activity, no immature stems and leaves, long time of photosynthesis, rough vascular bundles in stems and spikes are all very important for grain production.

## *3.3.3. Improvement of leaf posture and quality*

*3.2.3. Selection of parents with good combining ability*

for hybrid rice breeding.

286 Rice - Germplasm, Genetics and Improvement

and disease resistance.

**3.3. Strategies for hybrid rice breeding**

*3.3.1. Amplification of genetic diversity in parents*

components, and their hybrid combinations have super high-yield.

*3.3.2. Increasing the biological outputs*

General combining ability (GCA) refers to the average performance of an offspring from one parent that is crossed with many other parents. GCA is determined by the number of favorable genes and the size of gene function in parents, and often influenced by additive effects. Specific combining ability (SCA) refers to the performance of a unique offspring deviation from a specific pair of parents. SCA is mainly controlled by non-additive effects. Therefore, not only the yield of parents is necessary for, but also the combining ability of parents is very important

In addition to the breeding principles described above, the following strategies are also very important in hybrid rice breeding to result in hybrid cultivars with high yield, good quality

Through pedigree analysis, most rice cultivars originate from few parents which genetic diversity is small. The main reason for hybrid rice yield to stand still since 1980s maybe relate to short in genetic diversity in China. Since the discovery of semi-dwarfism in *indica*, genetic diversity among cultivars has become increasingly narrow, so that strong hybrid vigour has hardly been achieved by combining *indicas* cultivars. *Japonica* materials are full developed and utilized in temperate zone, resulting in a narrow genetic diversity among cultivars. The narrow diversity makes it very hard to achieve hybrid preponderance in inter varietal crosses. Tropic *japonica* (*Javanica*) and *nuda* types of rice (*japonica*) are not widely utilized in breeding because they are limited in special regions with some particular traits. Recently breeders have paid much attention to wide compatibility germplasm derived from tropic *japonica* and *nuda* rice. After Yuan (1987) proposed the strategy to utilize heterosis between *indica-japonica*, breeders have made a great effort to amplify parents' genetic diversity and breed super high-yield *indica-japonica* hybrid through effective utilization of wide compatibility. *Indica-japonica* combinations from direct crosses hardly have super high-yield because of sterility. Introgress‐ ing *japonica* genes into *indica* restorer lines in the south and introgressing indica genes into *japonica* in the north are proven to be effective for super high-yield hybrid rice breeding in China. Taking this strategy, three new *indica* restorer lines, Zhong413, R9308 and T2070, are bred by China National Rice Research Institute. They all contain about 25% of *japonica*

Ideotype is an ideal plant model which is expected to yield the most for a specific environment. Hybrid rice ideotype (HRI) is a best parental combination to yield the most grains with good quality in a certain ecological environment. HRI does not only contain desire morphological characteristics but also have resistances to biotic and abiotic stresses for the environment. Otherwise, the most grains with good quality are not achievable. For instance, Taoyuan in Plant growing, tillering and expanding ability of tillers at early stage, stand upright, and enough green leaves at later stage are all extremely important for rice to use solar energy highly efficiently. Thickness of leaves and maximum leaf nitrogen content should be exploited under intensive cultivation conditions because their advantages for improving photosynthetic capacity. Besides maintaining the photosynthetic ability of leaves, maintaining the flag leaves upright is also very important. Studies have indicated that the thicker the leaves are, the more mesophyll cells per unit area and the larger intercellular spaces there are (Zhu et al., 2001; Fu et al, 2012). Therefore, it is also very important to select thicker leaves in hybrid breeding because they are beneficial for diffusing carbon dioxide and maintaining high level of chlor‐ ophyll and nitrogen content inside the leaves.

Curling degree of leaf is another important index for leaf posture in super-rice breeding due to its benefit for photopermeability by increasing the under surface. Zhu et al. (2001) studied the relationships between curl degree and photopermeability, and classified the leaf curl degrees into high, intermediate and low with 44o -47o , 15o -16o and 10o -11o , respectively. Testing the photosynthesis rate between under and upper surface of leaves resulted in 1.19-1.32 in the high, 0.90-1.02 in the intermediate and 0.82-0.85 in the low curl degree hybrid combinations, respectively. Compared with the low curl degree combinations, the high and intermediate curl degree combinations had smaller leaf angle, higher leaf straightness and lower extinction efficient. So, we should select the lines with high or middle leaf curl degree in breeding.

#### *3.3.4. Increasing the ability of root system*

Long time ago, researchers were attracted by the influence of root system on rice associated to grain yield. Nagai proposed the concept of root type for the first time in 1957. Ling et al (1989) studied the close relationship between the direction of root stretching and leaf angle, and proposed that cultivating root type was favorable for ideo-type as well as rice high-yield cultivation. Root vigour, especial during the filling stage, guarantees a super high-yield of rice, undoubtedly. A radical reason for bad grain plumpness in hybrid rice is root senescence. However, improving root system properties has not been embodied in rice breeding plan till now, so we should do further and more research in root vigour at various aspects such as methodology study for root characteristic, root physiological characteristic, relationship with ground part, diversity among breeding materials and genetic utilization to construct ideal root type.

## **4. Three-line system hybrid rice**

## **4.1. Identification and utilization of cytoplasm male sterility**

The role of cytoplasm on causing male sterility of rice was first reported in 1954 (Sampath and Mohanty 1954). In 1965, Sasahara and Katsuo studied cytoplasmic differences among rice varieties and developed, for the first time, a male sterile line by transferring the nuclear genotype of rice cultivar Fujisaka 5. However, this cytoplasm male sterility (CMS) line could not be used for breeding rice hybrids because of its instability, poor plant type and photoperiod sensitivity. In 1964, Yuan Long Ping put forward the idea to utilize the heterosis in rice and initiated the research on hybrid rice in China for the first time. In November 1970, a pollen abortive wild rice plant (shortly called wild abortive, i.e., WA) was discovered among the plants of common wild rice (*Oryza rufipogon Griff*. L.) at Nanhong Farm of Ya County, Hainan Island which is the south most province of China. After the discovery of WA, a nationwide cooperative program was immediately established to extensively testcross with the WA and screen for its maintainers and restorers. Soon in 1972, the first group of CMS lines such as Erjiunan 1A, Zhenshan 97A and V20A were developed all using WA as the donor of male sterile genes and all using successive backcrossing method. In 1973, the first group of restorer lines such as Taiyin 1, IR24 and IR661 were screened out through direct test crossing method. In 1974, the hybrids with strong heterosis such as Nanyou 2 and Nanyou 3 were released. In another word, the discovery of WA resulted in the subsequent and successful breakthrough in hybrid rice development, so that the three-line hybrid rice system was established. There‐ fore, China became the first country to commercialize hybrid rice for food production in the world.

Three-line hybrid system includes the CMS line (A), maintainer line (B) and restorer line (R) for a commercial production of rice hybrids. The A line cannot produce viable pollen due to the interaction between cytoplasmic and nuclear genes, so called cytoplasmic male sterile, which anthers are pale or white and shriveled. The A line is used as a female parent for hybrid seed production, so it is commonly called the CMS line and the seed parent as well. Because the CMS line is male sterile, it cannot be self-reproduced and has to have a maintainer. The B line is the maintainer line, which morphology is highly similar to its corresponding CMS line except its reproductive function. The B line has viable pollen grains and normal seed setting, so can pollinate the A line and the F1 plants from this pollination are male sterile, again. In this way, the male sterility of the A line is maintained, and the A line is reproduced for further use or commercial use in a large scale. Similarly, the R line has viable pollen grains and normal seed setting and can pollinate the A line. Differently from the pollination with B line, the F1 plants from the pollination with R line are highly fertile, or the male sterility of the A line is restored into fertility by R line in their progeny. Therefore, the R line also called the pollen parent, male parent, and/or restoring line.

#### **4.2. Diversification of CMS sources**

degree combinations had smaller leaf angle, higher leaf straightness and lower extinction efficient. So, we should select the lines with high or middle leaf curl degree in breeding.

Long time ago, researchers were attracted by the influence of root system on rice associated to grain yield. Nagai proposed the concept of root type for the first time in 1957. Ling et al (1989) studied the close relationship between the direction of root stretching and leaf angle, and proposed that cultivating root type was favorable for ideo-type as well as rice high-yield cultivation. Root vigour, especial during the filling stage, guarantees a super high-yield of rice, undoubtedly. A radical reason for bad grain plumpness in hybrid rice is root senescence. However, improving root system properties has not been embodied in rice breeding plan till now, so we should do further and more research in root vigour at various aspects such as methodology study for root characteristic, root physiological characteristic, relationship with ground part, diversity among breeding materials and genetic utilization to construct ideal root

The role of cytoplasm on causing male sterility of rice was first reported in 1954 (Sampath and Mohanty 1954). In 1965, Sasahara and Katsuo studied cytoplasmic differences among rice varieties and developed, for the first time, a male sterile line by transferring the nuclear genotype of rice cultivar Fujisaka 5. However, this cytoplasm male sterility (CMS) line could not be used for breeding rice hybrids because of its instability, poor plant type and photoperiod sensitivity. In 1964, Yuan Long Ping put forward the idea to utilize the heterosis in rice and initiated the research on hybrid rice in China for the first time. In November 1970, a pollen abortive wild rice plant (shortly called wild abortive, i.e., WA) was discovered among the plants of common wild rice (*Oryza rufipogon Griff*. L.) at Nanhong Farm of Ya County, Hainan Island which is the south most province of China. After the discovery of WA, a nationwide cooperative program was immediately established to extensively testcross with the WA and screen for its maintainers and restorers. Soon in 1972, the first group of CMS lines such as Erjiunan 1A, Zhenshan 97A and V20A were developed all using WA as the donor of male sterile genes and all using successive backcrossing method. In 1973, the first group of restorer lines such as Taiyin 1, IR24 and IR661 were screened out through direct test crossing method. In 1974, the hybrids with strong heterosis such as Nanyou 2 and Nanyou 3 were released. In another word, the discovery of WA resulted in the subsequent and successful breakthrough in hybrid rice development, so that the three-line hybrid rice system was established. There‐ fore, China became the first country to commercialize hybrid rice for food production in the

Three-line hybrid system includes the CMS line (A), maintainer line (B) and restorer line (R) for a commercial production of rice hybrids. The A line cannot produce viable pollen due to

*3.3.4. Increasing the ability of root system*

288 Rice - Germplasm, Genetics and Improvement

**4. Three-line system hybrid rice**

**4.1. Identification and utilization of cytoplasm male sterility**

type.

world.

Chinese rice breeders designated various CMS sources arbitrarily without following any systematic nomenclature. Principally, the CMS sources are designated according to the cultivar name from which the male sterile cytoplasme is derived. In some cases, different symbols are assigned by different researchers for the same material. For example, Shinjyo designated the male sterile cytoplasm of Chinsurah Boro II as [CMS-boro] or [CMS-bo], but the Chinese workers designated it as BT (B for Chinsurah Boro Ⅰ and T for Taichung 65 which is the nuclear donor cultivar). The first series of released WA-type CMS lines include Zhenshan 97A, V20A, Erjiu Ai 4A, Erjiu Nan 1A and V41A (Mao, 1993). In order to diversify the genetic background of hybrid rice, other CMS types besides WA-type are developed for three-line hybrid rice varieties in China, including Dwarf Abortive (DA) type, Gambiaka and Dissi (G and D) type, Indonesla 6 (ID) type, K type and Hong Lian (HL) type. DA-type CMS lines are derived from the male sterile dwarf wild rice, including Xieqingzao A. G and D type CMS lines are developed from geographically distant crosses, where West Africa *indica* cultivars Gambiaka Kokoum and Dissi D52/37 are crossed with Chinese *indica* cultivar Aijonante, respectively to yield G46A and D62A, major representative of G and D CMS lines (Li 1997). ID type CMS lines are derived from an abortive plant in an Indonesia rice cultivar Indonesia 6. Zhong 9 A and II-32A are two representative CMS lines of ID type. K type CMS lines are derived from the cross of K52 and Luhongzao 1 with representative K qing A and K 17A. HL Type CMS lines are derived from the cross between red awn wild rice (*O.sativa spontanea* L.) and an indica rice cultivar Lian‐ tangzao with representative Yuetai A (Zeng et al, 2000). Virmani and Wan (1988) listed some of the CMS sources identified in and outside China, where the CMS sources are designated in principle according to the cultivar name from which the male sterile cytoplasme is derived, as well.

Outside China, IRRI used CMS sources from V20A, Kaliya 1, ARC and Gambiaka to develop CMS lines of IR58025A, IR68275A, 68281A, IR68273A, IR68888A, IR68891A and IR68893A. Also, IRRI developed CMS lines with male sterile cytoplasme sources of *Oryza perennis* (e.g. IR66707A) and *O. rufipogon* (e.g. OMS1) (Virmani, 1996). Therefore, the genetic backgrund among three-line hybrid rice varieties are greatly broadened or diversified.

## **4.3. Genetic model of CMS line**

As a self-pollinating crop, rice must use an effective male sterility system to develop and produce F1 hybrid cultivars. The male sterility in CMS system is controlled by an interac‐ tion of cytoplasmic and nuclear genes. The presence of homozygous recessive nuclear genes for fertility restoration combining with cytoplasmic genes for sterility makes a plant male sterile. The cytoplasmic genes for sterility exist in mitochondrial DNA. The nucleocytoplasmic inter-reaction hypothesis explains genetics of three-line hybrids. In this hypothesis, a CMS or A line has sterility cytoplasm but no dominant restorer genes in nucleus, so sterile. A maintainer or B line also has no dominant restorer genes in nu‐ cleus, but has fertile cytoplasm, so fertile.

When B line pollinates A line, the progeny is male sterile because it has a complete sterility cytoplasm of A line with half nucleus from A line and another half nucleus from B line, and both A and B nuclei have no dominant restorer genes. A restoring or R line has dominant restorer genes in the nucleus. Regardless of sterile or fertile cytoplasm in R line, the progeny from crossing A with R line becomes fertile solely because of dominant restorer genes in the nucleus of R line. Accordingly, genetic constitutions can be expressed as *S* (sterile cytoplasm) with *rfrf* (sterile nucleus) in CMS line, *N (*fertile cytoplasm) wit *rfrf* in maintainer line, S/N with *RfRf* (fertile nucleus) in restorer line and S with *Rfrf* in hybrid rice. When the CMS line is crossed with corresponding maintainer, the sterility is main‐ tained and seeds of the CMS line are multiplied. When the CMS line is crossed with the restorer line, the fertility is restored in F1 generation, namely commercial hybrid seed production.

Zhang (1981) made the following conclusions on male sterility and cytoplasmic regulation of gene reaction in rice:


maintaining or normal fertility for restoring. Therefore, the cytoplasmic differences between two male-sterile lines derived from two CMS sources can be ascertained through the reaction of maintainer and restorer by crossing with a set of cultivars.

## **4.4. Achievements on three-line hybrid rice**

IR66707A) and *O. rufipogon* (e.g. OMS1) (Virmani, 1996). Therefore, the genetic backgrund

As a self-pollinating crop, rice must use an effective male sterility system to develop and produce F1 hybrid cultivars. The male sterility in CMS system is controlled by an interac‐ tion of cytoplasmic and nuclear genes. The presence of homozygous recessive nuclear genes for fertility restoration combining with cytoplasmic genes for sterility makes a plant male sterile. The cytoplasmic genes for sterility exist in mitochondrial DNA. The nucleocytoplasmic inter-reaction hypothesis explains genetics of three-line hybrids. In this hypothesis, a CMS or A line has sterility cytoplasm but no dominant restorer genes in nucleus, so sterile. A maintainer or B line also has no dominant restorer genes in nu‐

When B line pollinates A line, the progeny is male sterile because it has a complete sterility cytoplasm of A line with half nucleus from A line and another half nucleus from B line, and both A and B nuclei have no dominant restorer genes. A restoring or R line has dominant restorer genes in the nucleus. Regardless of sterile or fertile cytoplasm in R line, the progeny from crossing A with R line becomes fertile solely because of dominant restorer genes in the nucleus of R line. Accordingly, genetic constitutions can be expressed as *S* (sterile cytoplasm) with *rfrf* (sterile nucleus) in CMS line, *N (*fertile cytoplasm) wit *rfrf* in maintainer line, S/N with *RfRf* (fertile nucleus) in restorer line and S with *Rfrf* in hybrid rice. When the CMS line is crossed with corresponding maintainer, the sterility is main‐ tained and seeds of the CMS line are multiplied. When the CMS line is crossed with the restorer line, the fertility is restored in F1 generation, namely commercial hybrid seed

Zhang (1981) made the following conclusions on male sterility and cytoplasmic regulation of

**1.** The occurrence of male sterility depends on "affinity" between the cytoplasm and nucleus. The greater the genetic distance between cytoplasm donor and nucleus donor cultivars is, the easier for their offsping to be male sterile and to breed male sterile line. If we assume that the evolutionary order of cultivated rice is wild, *indica* and *japonica,* the genetic distance between wild and *japonica* should be greater than it between wild and *indica*. Then, the cytoplasm of wild rice has less "affinity" with *japonica* than it with *indica*.

**2.** The cytoplasm and nucleus jointly decide pollen abortion. Pollen abortion is observed from uninucleate stage before first pollen mitosis, until binucleate stage just before anthesis. The earlier the abortion stage is, the more morphologically discernible pollen

**3.** A genotype can function as either a maintainer to one MS cytoplasm or a restorer to another MS cytoplasm, depending upon the ability of either complete sterility for

However, the definition of "affinity" in genetic terms remains unexplained.

among three-line hybrid rice varieties are greatly broadened or diversified.

**4.3. Genetic model of CMS line**

290 Rice - Germplasm, Genetics and Improvement

cleus, but has fertile cytoplasm, so fertile.

production.

gene reaction in rice:

sterility there is.

In order to reduce the potential threats from diseases due to MS cytoplasme uniformity, a variety of cytoplasmic male sterile sources have been utilized by Chinese rice breeders and a number of three-line hybrid CMS lines have been bred. WA CMS source used to be overwhelming for a long time. Other cytoplasm sources named Wild and D type, ID type etc. are gradually increasing lately in commercial extension of hybrid rice. For example, the monopoly of WA CMS source hybrids was broken by D-type CMS source hybrids with heavy panicles that are successully bred in Sichuan on commercial scale.

In recent years, great progresses have been made by Chinese rice breeders to improve grain quality and out-crossing rate of male sterile lines. ID type CMS line Zhong 9A (http:// www.ricedata.cn/variety/varis/601141.htm), developed by China National Rice Research Institute, combines high quality and high outcrossing rate up to 80%. The grain quality of Yixiang 1A cultivated by Yibin Institute of Agricultural Sciences in Sichuan province (Jiang et al, 2008), and Yuefeng A cultivated by Guangdong Academy of Agricultural Sciences has reached international standards of first level high quality rice (Li, 2001). The breed‐ ing success of these CMS lines improved the quality of hybrid rice, especially for the significant improvement on milled rice rate, chalkiness and amylose content.

Rice statistics shows that three-line hybrids are still dominant in rice production in china (Table 2). From 2009 to 2011, the planting areas of top ten three-line hybrids ranged from 110, 700 ha for Jin you 207 in 2011 to 260, 000 ha for Yue you 9113 in 2009. Many threeline hybrids are elite, such as Yue you 9113, Gang you 188, Q you 6, Gang you 725, Tian you 998 and Zhongzhe you 1. Among them, Yue you 9113 is outstanding with the most total planting areas of 724, 7000 ha in the three years because it has good characteristics of high yield and premium quality, resistance to diseases and suitable maturity. Because threeline hybrid combinations have very significant yield increasing ability, it has proven that hybrid rice has a yield advantage of more than 20% over conventional rice. In recent years, hybrid rice covers about 15.5 million ha annually, accounts for 50% of the total rice area, and produces 60% of the total rice produced in China. From 1976 to 2011, the accumulat‐ ed planting area of hybrid rice is 500 million ha, from which 500 million tons of paddy rice has increased over conventional rice. Up to now, three-line hybrids include Indica, Japonica and Indica/Japonica types with different maturities. Thus, hybrid rice produc‐ tion distributes to the entire China, from Hainan in the south to Liaoning in the north, and from Shanghai in the east to Yunnan in the west. Hybrid rice shows not only a highyielding ability but also a wide adaptability. Chinese demonstration has also encouraged IRRI and National rice improvement programs of countries like India, Vietnam, Philip‐ pines, USA, Bangladesh and Indonesia to start hybrid rice breeding programs for utiliza‐ tion of heterosis.


**Table 2.** Planting areas of top 10 three-line hybrid varieties during 2009~2011 (104 ha)

## **5. Breeding and application of two-line hybrid rice**

## **5.1. Photoperiod Sensitive and Thermo-sensitive Genetic Male Sterility**

In 1973, Chinese scientist Shi Mingsun discovered a natural male sterile plant in the field of Nongken 58, a *japonican* late maturing variety, at Shahu Farm of Mianyang County, Hubei Province, China. After eight years of in-house study for confirmation, he announced his discovery as a dual-purpose rice line Nongken 58S in 1981, and proposed a new strategy to utilize heterosis in rice, namely two-line system based on his research results (Shi, 1981). Further studies indicate that the critical stage for fertility transformation must be before the 1st or 2nd of September in Wuhan 30-31 N. When Nongken 58S heads during August 5th ~ 1st or 2nd of September, it is male-sterile (99.5-100%). However, its pollen sterility is reduced to 20% and seed setting rate varies between 10-40% when Nongken 58S heads after 1st or 2nd of September. This sterility-fertility change regulated by heading is true in many other regions. Pollen sterility during the sterile stage in summer is stable, but the fertility in autumn is unstable and varies over locations and years. The original sterile plant Nongken 58S becomes the first dual-purpose line in rice and possesses the characteristics of fertility alteration, i.e., completely sterile under long day period and high temperature conditions, and partially fertile under short day period and low temperature conditions.

Japanese rice scientists (Maruyama et a1., 1991a) reported thermo-sensitive genetic male sterility (TGMS) as a mutant from Japanese rice cultivar Remei treated by 20 kr gamma rays for the first time. This male-sterile mutant, designated as H89-1, sets no seeds under 31/24℃, some seeds under 28/21℃ and full seeds under 25/15℃. Pollen sterility in this mutant does not change along with change of day length period (viz.15, 13.5, 12 h). Behavior of this TGMS mutant is confirmed at IRRI. Like PGMS, TGMS can also be employed to develop rice hybrids rather than three lines. While PGMS can be used in the countries having large territory with striking differences in latitude, TGMS can be used in the area close to the equator where low temperature areas are on top of the hills. Thus, TGMS can be utilized in tropical and subtropical areas.

## **5.2. Breeding methods for two-line hybrid rice**

**Rank**

292 Rice - Germplasm, Genetics and Improvement

**2009 2010 2011 Variety name Area Variety name Area Variety name Area**

1 Yueyou 9113 26.00 Zhongzhe you 1 24.53 Yue you 9113 22.80 2 Tian you 998 25.53 Gangyou 188 23.87 Gang you 188 22.60 3 Gang you 188 24.73 Yueyou 9113 23.67 Q you 6 17.00 4 Jin you 207 21.93 Q you 6 22.80 Gang you 725 14.40 5 Jin you 402 20.33 Gangyou 725 17.93 Tian you 998 13.47 6 Q you 6 19.27 Jin you 207 14.73 Zhongzhe you 1 12.80 7 Zhongzhe you 1 17.33 Tian you 998 14.33 Wu you 308 12.67 8 Gangyou 725 16.60 Ganxin 688 12.80 Tianyou huazhan 12.60 9 II you 838 15.53 Jin you 402 12.47 Fengyuan you 299 11.73 10 Jin you 463 11.53 II you 838 12.13 Jin you 207 11.07

**Table 2.** Planting areas of top 10 three-line hybrid varieties during 2009~2011 (104 ha)

**5. Breeding and application of two-line hybrid rice**

under short day period and low temperature conditions.

**5.1. Photoperiod Sensitive and Thermo-sensitive Genetic Male Sterility**

In 1973, Chinese scientist Shi Mingsun discovered a natural male sterile plant in the field of Nongken 58, a *japonican* late maturing variety, at Shahu Farm of Mianyang County, Hubei Province, China. After eight years of in-house study for confirmation, he announced his discovery as a dual-purpose rice line Nongken 58S in 1981, and proposed a new strategy to utilize heterosis in rice, namely two-line system based on his research results (Shi, 1981). Further studies indicate that the critical stage for fertility transformation must be before the 1st or 2nd of September in Wuhan 30-31 N. When Nongken 58S heads during August 5th ~ 1st or 2nd of September, it is male-sterile (99.5-100%). However, its pollen sterility is reduced to 20% and seed setting rate varies between 10-40% when Nongken 58S heads after 1st or 2nd of September. This sterility-fertility change regulated by heading is true in many other regions. Pollen sterility during the sterile stage in summer is stable, but the fertility in autumn is unstable and varies over locations and years. The original sterile plant Nongken 58S becomes the first dual-purpose line in rice and possesses the characteristics of fertility alteration, i.e., completely sterile under long day period and high temperature conditions, and partially fertile

Japanese rice scientists (Maruyama et a1., 1991a) reported thermo-sensitive genetic male sterility (TGMS) as a mutant from Japanese rice cultivar Remei treated by 20 kr gamma rays for the first time. This male-sterile mutant, designated as H89-1, sets no seeds under 31/24℃, some seeds under 28/21℃ and full seeds under 25/15℃. Pollen sterility in this mutant does not change along with change of day length period (viz.15, 13.5, 12 h). Behavior of this TGMS

Two-line hybrid rice research originates in China and successfully reached to production scale in 1995. The male sterile lines in which sterility expression is controlled by temperature are called thermo-sensitive male sterile (TGMS) lines and those in which expression is controlled by day-length period are called photoperiod-sensitive male sterile (PGMS) lines. The PGMS trait has been transferred to several *Indica* and *Japonica* rice cultivars in China by backcrossing. Rice hybrids developed by this male sterility system are being evaluated in multi-location trials in China. Two-line hybrid rice has similar level of heterosis with three-line hybrid rice, but different in technique process. Unlike three-line hybrids, the male parent of two-line hybrid is not restricted by restorer genes, so we can use not only the good restorer lines with high combining ability as the male parent, but also the good conventional varieties without restorer genes as male parent. The non-restriction of restorer genes brings about greater opportunity to breed elite hybrids.

The developed PTGMS lines such as PA64S, GZ63S, Zhun S, etc. have many advantages for hybrid combinations, such as larger freedom for crossing, higher yielding, better quality and resistance to diseases than CMS lines. Commonly, the yield of improved two-line hybrid rice combinations is higher than it of three-line hybrids as checks. Meanwhile, the techniques of seed production and cultivation for two-line hybrids have been sophisticated enough for production application. Breeding of elite restorer lines is the key for matching heterotic combinations. The following three ways are usually used in breeding restorers and new combinations for two-line hybrids.

## *5.2.1. Testing and screening strong combinations using conventional rice cultivars*

China has a very long history for rice cultivation. Thousands of cultivars have been used for production and all these cultivars can be used as a restorer for testing new two-line combina‐ tions. Especially for some conventional cultivars bred in recent years, they have many advantages such as high-yield, good resistance and excellent quality, thus are easy to be used in two-line hybrid breeding. At present, two-line hybrid rice combinations applied in large production areas are mostly bred using conventional cultivars. For example, Teqing, Shagnq‐ ing11, Yuhong, and 9311 (Yangdao6) are the male parent for elite two-line hybrids Liangyou‐ teqing, Peizashanqing, Peiliangyouyuhong and Liangyoupeijiu, respectively.

## *5.2.2. Testing and screening strong combinations using cytoplasmic male sterile restorers*

Three-line hybrid rice breeding technologies in China are regarded the first class in the world. For the pasted four decades, a large number of restorer lines with strong general combining ability, good resistance to diseases and high quality have been bred. All these restorer lines can be used as male parent for testing two-line hybrid rice combinations. For example, many two-line hybrid rice combinations which are widely used in production such as Peiliangyou 288, 70 You 9 (Wandao20), Liangyou 2163 and Fuliangyou 63 are configured by three-line restorer R288, Wanhui9, Minghui63, respectively.

## *5.2.3. Breed new restorers from crossing*

Although two-line hybrid rice is superior to three-line hybrid rice in quality traits, resistance and yielding, we also need to expand genetic differences of parents, make crosses and breed new two-line restorers, and overcome the shortcomings of parents using complementary effects. For example, researchers in Yahua Seed Industry in Hunan Academy of Agricultural Sciences have bred a restorer line ZR02 which combines good quality, stable growth period, strong resistance and good out-crossing ability together. We can use this restorer line to cross with other lines to improve the traits such as poor quality, late maturity and poor out-crossing ability. Using this restorer line, a new combination Zhuliangyou 02 is bred as a double-crop early maturing hybrid rice with stable and high yield and good quality. As a result, Zhulian‐ gyou 02 has good prospects in Yangtze River.

## **5.3. Advantages of two-line hybrids**

In the two-line system, only two lines are involved in hybrid rice seed production. One is the male sterile line in which male sterility is genetically controlled by recessive genes, which expression is influenced by environment (temperature, photoperiod, or both). Another is male parent or pollinator line that can be any inbred variety with dominant gene (s) for male sterile locus. There are no constraints for the restoration-maintenance relationship because the male sterility of PGMS and TGMS is controlled by only one or two pairs of recessive genes. There is no need for special R genes to restore fertility, so the choice of parents in developing heterotic hybrids is greatly broadened. Developing hybrid rice varieties with these systems has the following advantages over the classical three-line or CMS system:


**3. Negative effects from the sterile cytoplasm are avoided.** Therefore, the vulnerability to destructive diseases or insects due to uniform resource of male sterile cytoplasme may be eliminated.

Apparently, it is easier to develop rice hybrids that possess higher yield, earlier maturity, better grain quality and improved pest resistance by two-line system than by three-line system. The research findings and production experiences have also proven that two-line hybrid rice outyields three-line hybrid rice by 5%-10%. Furthermore, it is promising to develop elite hybrids for the early cropping rice with both high yield and early maturity, and to develop heterotic *japonica* hybrids by two-line method, which would very likely break the deadlock of stagnant yield and area in hybrid rice. For example, Xiangliangyou 68, an early-cropping two-line hybrid rice combination with high yield, fine grain quality and early maturity, was successfully released to commercial production in 1998. It shows a very promising prospect in overcoming the great difficult long existing in developing high-yield, good-quality and early-maturity hybrid rice in China.

However, it should be pointed out that two-line hybrid rice also faces the risk of seed purity in case of lower temperature occurred in thermo-sensitive stage of TGMS lines in hybrid rice seed production. Because the fertility alteration of TGMS lines is conditioned by temperature, even in hot season like summer, low temperature may occur in rainy days and last for a few days. Therefore, to guarantee the seed purity in two-line hybrid seed production is essentially important for developing practical TGMS lines with their Critical Sterility Inducing Temper‐ ature (CSIT) low enough, generally 23 C for temperate zones and 24 C for subtropical zone.

## **5.4. Achievements in two-line hybrid rice breeding**

ability, good resistance to diseases and high quality have been bred. All these restorer lines can be used as male parent for testing two-line hybrid rice combinations. For example, many two-line hybrid rice combinations which are widely used in production such as Peiliangyou 288, 70 You 9 (Wandao20), Liangyou 2163 and Fuliangyou 63 are configured by three-line

Although two-line hybrid rice is superior to three-line hybrid rice in quality traits, resistance and yielding, we also need to expand genetic differences of parents, make crosses and breed new two-line restorers, and overcome the shortcomings of parents using complementary effects. For example, researchers in Yahua Seed Industry in Hunan Academy of Agricultural Sciences have bred a restorer line ZR02 which combines good quality, stable growth period, strong resistance and good out-crossing ability together. We can use this restorer line to cross with other lines to improve the traits such as poor quality, late maturity and poor out-crossing ability. Using this restorer line, a new combination Zhuliangyou 02 is bred as a double-crop early maturing hybrid rice with stable and high yield and good quality. As a result, Zhulian‐

In the two-line system, only two lines are involved in hybrid rice seed production. One is the male sterile line in which male sterility is genetically controlled by recessive genes, which expression is influenced by environment (temperature, photoperiod, or both). Another is male parent or pollinator line that can be any inbred variety with dominant gene (s) for male sterile locus. There are no constraints for the restoration-maintenance relationship because the male sterility of PGMS and TGMS is controlled by only one or two pairs of recessive genes. There is no need for special R genes to restore fertility, so the choice of parents in developing heterotic hybrids is greatly broadened. Developing hybrid rice varieties with these systems has the

**1. Maintainer lines are not needed.** The PGMS lines (under long day-length) and the TGMS lines (under high temperature) show complete pollen sterility and can thus be used for hybrid seed production. Under short day-length or low temperate conditions, they show almost normal fertility and can multiply themselves by selfing. Therefore, in the PGMS/ TGMS system, no maintainer line is needed in seed multiplication of male sterile lines, thus the cost to produce hybrid seed is cut down because of the simplified production

**2. The parental choice for developing heterotic hybrids is greatly broadened.** Studies showed that more than 97% of tested varieties (within subspecies) could restore fertility of MS lines, indicating no need to identify restoring abilities. Thus, our choice of parents in developing heterotic hybrids is broadened in comparison with the CMS system. In addition, PGMS and TGMS genes can be easily transferred to almost any rice lines with

restorer R288, Wanhui9, Minghui63, respectively.

gyou 02 has good prospects in Yangtze River.

following advantages over the classical three-line or CMS system:

**5.3. Advantages of two-line hybrids**

procedure.

desirable characteristics.

*5.2.3. Breed new restorers from crossing*

294 Rice - Germplasm, Genetics and Improvement

Because the PTGMS lines can be used to produce hybrid seeds in the sterile period and to multiply themselves in the fertile period, a nationwide research was organized to study the mechanism of PTGMS and its application after the discovery of Nongken 58S. Soon after, many *japonica* and *indica* PTGMS lines have been released using male sterile genes in the original Nongken 58S. Furthermore, some other germplasms with fertility alteration such as Annong S-1, 5460 S and Hengnong S-1 are also identified. Up to now, tens of practical PTGMS lines in rice which possess the characteristics of low CSIT to secure hybrid seed production have been technically identified and approved. At present, the PTGMS lines used in rice production mainly derive from either PGMS Nongken 58S or TGMS Annong S. More attention should be paid to the following areas in order to improve screening and utilizing efficiency of photothermo sensitive male sterility (Lu and Zou, 2000).

**1.** Seed production safety: Because the fertility of photo-thermo sensitive male sterile lines is regulated by light and temperature, safely producing hybrid seed in the target region must be taken into account during the selection of sterile lines. The window for sterility to stably occur must be more than 30 days. The stable length of sterility period mainly depends on the critical temperature and critical photo-period length for fertility transition in the selected PTGMS lines. In South China, we generally select temperature-sensitive sterile lines because inter-annual light changes are minor and inter-annual temperature changes are major, where the suitable critical temperature for fertility transition is 24-25°C in this region. In Central China, PGMS, TGMS and P-TGMS are all applicable, but the suitable critical temperature of fertility transition should be 23°C ~ 24°C, and the day length of critical light should be around 13.5 h. We should select the sterile lines with relatively stronger photo-sensitivity but weaker temperature-sensitivity for fertility transition in North and Northeast rice region, because there are longer day length and relatively lower temperature there.


According to above principles, the breeders have bred many elite two-line hybrid variet‐ ies with good quality, high yield and good resistance, such as Fengliangyou Xiang1, Zhunliangyou 527, Yangliangyou 6, Liangyou 288, Zhuliangyou 02, Zhuliangyou 120, et al. After more than twenty years for nationwide collaborative studies, important progress has been made in two-line hybrid rice in both theoretical mechanism and practical applica‐ tion, which has resulted in a yield advantage of 10 percent over three-line hybrids. Along with improvements of techniques on daily bases, two-line hybrid rice is becoming more and more popular in large-scale application. The area planted to two-line hybrid rice increases year after year from 4, 300 ha in 1991 to 704, 400 ha in 1999 in China. In 2000, the growing area of two-line hybrid rice in China reached 1.5 million ha, and total yield reached 109.3 million tons with average yield of 7287kg/ha which was 4.16% more than that of three-line hybrid rice. Currently, the main combinations in commercial production are Liangyou Peijiu, PZS7, Peiliangyou 288, Xiangliangyou 288 and Fengliangyou 1. In 2002, a two-line hybrid rice Liangyou Peijiu took up the first place of planting areas from Shanyou 63, a three-line hybrid rice that had maintained the leading place for more than ten year in China.

In recent years, more and more two-line hybrid rice varieties are released. In 2011, six and 51 two-line hybrid combinations were released from national and provincial institutions, respectively (Fig. 3). The planting area of two-line hybrid rice reached 2.7 million ha, about 9.0% of total rice cultivation area and 18.6% of hybrid rice planting area. In 2010, the top three hybrid varieties with the most planting area were all two-line combinations. The cultivation area of two-line hybrid rice will further expend with the progress of research on seed production.

**Figure 3.** Two-line hybrid rice varieties released in China (2001-2011).

## **6. Super hybrid rice breeding**

changes are major, where the suitable critical temperature for fertility transition is 24-25°C in this region. In Central China, PGMS, TGMS and P-TGMS are all applicable, but the suitable critical temperature of fertility transition should be 23°C ~ 24°C, and the day length of critical light should be around 13.5 h. We should select the sterile lines with relatively stronger photo-sensitivity but weaker temperature-sensitivity for fertility transition in North and Northeast rice region, because there are longer day length and

**2.** Easy to multiply: Because the TGMS lines are fertile when the temperature is below the critical temperature, photo-thermo sensitive male sterile lines must have strong cold tolerance, which makes them survive from the cold water irrigation to have high grain

**3.** High combining ability: A successful hybrid with high vigor is directly determined by the combining ability level of sterile lines. Combining ability (CA) includes specific CA (SCA) and general CA (GCA), the former is for crossing with a specific parent and the latter is for crossing with many parents. The GCA is usually highly related with its comprehensive characters, advantages and disadvantages, so the sterile lines with high GCA have high

According to above principles, the breeders have bred many elite two-line hybrid variet‐ ies with good quality, high yield and good resistance, such as Fengliangyou Xiang1, Zhunliangyou 527, Yangliangyou 6, Liangyou 288, Zhuliangyou 02, Zhuliangyou 120, et al. After more than twenty years for nationwide collaborative studies, important progress has been made in two-line hybrid rice in both theoretical mechanism and practical applica‐ tion, which has resulted in a yield advantage of 10 percent over three-line hybrids. Along with improvements of techniques on daily bases, two-line hybrid rice is becoming more and more popular in large-scale application. The area planted to two-line hybrid rice increases year after year from 4, 300 ha in 1991 to 704, 400 ha in 1999 in China. In 2000, the growing area of two-line hybrid rice in China reached 1.5 million ha, and total yield reached 109.3 million tons with average yield of 7287kg/ha which was 4.16% more than that of three-line hybrid rice. Currently, the main combinations in commercial production are Liangyou Peijiu, PZS7, Peiliangyou 288, Xiangliangyou 288 and Fengliangyou 1. In 2002, a two-line hybrid rice Liangyou Peijiu took up the first place of planting areas from Shanyou 63, a three-line hybrid rice that had maintained the leading place for more than ten year

In recent years, more and more two-line hybrid rice varieties are released. In 2011, six and 51 two-line hybrid combinations were released from national and provincial institutions, respectively (Fig. 3). The planting area of two-line hybrid rice reached 2.7 million ha, about 9.0% of total rice cultivation area and 18.6% of hybrid rice planting area. In 2010, the top three hybrid varieties with the most planting area were all two-line combinations. The cultivation area of two-line hybrid rice will further expend with the progress of research

relatively lower temperature there.

chances to produce high yielding combinations.

output in multiplication.

296 Rice - Germplasm, Genetics and Improvement

in China.

on seed production.

In order to achieve another leap of rice yield and secure food supply in China, after summa‐ rizing the experiences and lessons at home and abroad, Chinese scientists put forward a national program to breed super rice in 1996. A primary goal of this program is to combine ideal plant type with heterosis of *indica*/*japonica*, and improve rice yield, quality and resistance. Through joint research, a series of new super rice varieties have been approved for release from national and provincial institutions. The super rice varieties have demonstrated the yield of 12 t/ha in a scale of 100 mu or 6.7 ha model trial. From 1998 to 2004, the accumulative demonstration and extension areas of super hybrid rice have 10 million ha. Practices show that developing super hybrid rice is a necessary choice to increase rice yield, stabilize total production of rice, improve the efficiency of the rice planting, and ensure food security in China.

## **6.1. Model of super hybrid rice**

Backgrounds are confirmed to be unique based on the results of RFLP variations among *indica* hybrid varieties and their parents. The yield ceiling has remained in hybrid rice for nearly 10 years because of insufficient genetic diversity. Optimal combination means that the hybrid rice combination has a reasonable genetic difference between its parents, such as 1) lowland rice with upland rice varieties, 2) geographically different varieties, 3) ecologically different varieties, 4) dominantly different varieties, and 5) *indica* and *japonica* subspecies (Chen et al., 2007). However, we can only exploit part of the heterosis between *indica* and *japonica* subspe‐ cies, but not the heterosis between typical *indica* and *japonica* rice or excessive *indica* and *japonica* ingredients. Cheng et al. (2007) indicated that when *indica* or indicalinous cytoplasmic male sterile (CMS) lines are crossed with restorer lines having different *indica* and *japonica* genetic backgrounds, the hybrids from indicalinous or japonicalinous restorer lines (*indica*-*japonica* differentiation index 11-15) have the highest yield. Therefore, breeding high yielding hybrids by crossing *indica* with *japonica* with aid of wide compatibility gene has been paid great attentions. In the *indica* rice growing regions, breeders strategically introgress *japonica* consanguinity into *indica* rice, and in the *japonica* rice growing regions, introgress *indica* consanguinity into *japonica* rice, instead. So far, a set of indicalinous or japonicalinous germ‐ plasms for super rice breeding have been intentionally developed. Some of such germplasms have been successfully used in breeding of super inbred and hybrid rice (Table 3). For instance, Shennong 89366 is one of the core parents for IRRI to develop new plant type super rice. ed by Shennong 89366 is bred by Shenyang Agricultural University, China and has served as a donor for short sturdy stems and long-big panicles (Chen et al., 2003). R9308, an indica restorer line from a cross of C57//No. 300/IR26, has been successfully used in the breeding program for super hybrid rice by the China National Rice Research Institute (CNRRI). Xieyou 9308 (Xieqingzao A/R9308), a hybrid rice combination with super high yielding, multi-resistance to diseases and good grain quality, was registered in Zhejiang Province, China in 1999. It is estimated that there are 25% *japonica* and 75% *indica* genetic components in R9308. The hybrid Xieyou 9308 has super high yielding potential with harmonious plant type (Cheng et al., 2005). Another example is Liangyou peijiu, a two-line super *indica* hybrid developed by the Jiangsu Academy of Agricultural Sciences collaboratively with the Hunan Hybrid Rice Research Center, China (Lu et al., 2000). Its female parent is Pei'ai 64S, a thermo-sensitive male sterile line with tropic japonica in its pedigree.


**Table 3.** Some super rice hybrids derived from gene introgression of *indica* (*i*), *japonica* (*j*) and medium (m) type

## **6.2. Strategies to breed super hybrid rice**

2007). However, we can only exploit part of the heterosis between *indica* and *japonica* subspe‐ cies, but not the heterosis between typical *indica* and *japonica* rice or excessive *indica* and *japonica* ingredients. Cheng et al. (2007) indicated that when *indica* or indicalinous cytoplasmic male sterile (CMS) lines are crossed with restorer lines having different *indica* and *japonica* genetic backgrounds, the hybrids from indicalinous or japonicalinous restorer lines (*indica*-*japonica* differentiation index 11-15) have the highest yield. Therefore, breeding high yielding hybrids by crossing *indica* with *japonica* with aid of wide compatibility gene has been paid great attentions. In the *indica* rice growing regions, breeders strategically introgress *japonica* consanguinity into *indica* rice, and in the *japonica* rice growing regions, introgress *indica* consanguinity into *japonica* rice, instead. So far, a set of indicalinous or japonicalinous germ‐ plasms for super rice breeding have been intentionally developed. Some of such germplasms have been successfully used in breeding of super inbred and hybrid rice (Table 3). For instance, Shennong 89366 is one of the core parents for IRRI to develop new plant type super rice. ed by Shennong 89366 is bred by Shenyang Agricultural University, China and has served as a donor for short sturdy stems and long-big panicles (Chen et al., 2003). R9308, an indica restorer line from a cross of C57//No. 300/IR26, has been successfully used in the breeding program for super hybrid rice by the China National Rice Research Institute (CNRRI). Xieyou 9308 (Xieqingzao A/R9308), a hybrid rice combination with super high yielding, multi-resistance to diseases and good grain quality, was registered in Zhejiang Province, China in 1999. It is estimated that there are 25% *japonica* and 75% *indica* genetic components in R9308. The hybrid Xieyou 9308 has super high yielding potential with harmonious plant type (Cheng et al., 2005). Another example is Liangyou peijiu, a two-line super *indica* hybrid developed by the Jiangsu Academy of Agricultural Sciences collaboratively with the Hunan Hybrid Rice Research Center, China (Lu et al., 2000). Its female parent is Pei'ai 64S, a thermo-sensitive male

sterile line with tropic japonica in its pedigree.

II you 7954 II-32A/Zhehui 7954

298 Rice - Germplasm, Genetics and Improvement

Guodao 1 Zhong 9A/R8006

Guodao 3 Zhong 8A/R8006 Guodao 6 Neixiang 2A/R8006

**Combination name Parental cross Pedigree of major parent**

Liangyou Peijiu Pei'ai 64 S/R9311 Pei'ai 64S: Nongken 58S (*j*) /Peiai 64 (*i*) // Peiai 64 (*i*) /

Zhehui 7954: R9516/M105 R9516: Peiai'64S/Teqing

M105: Miyang 46 (*i*) /Lunhui 422 (*m*)

T2070: WL1312 (*j*) /Lunhui 422 (*m*) /Minghui 63 (*i*)

R8006: IRBB60//T2070/Duoxi 1

**Table 3.** Some super rice hybrids derived from gene introgression of *indica* (*i*), *japonica* (*j*) and medium (m) type

Xieyou 9308 Xieqingzao A/R9308 R9308: C57 (*j*) //No.300 (*j*) /IR26 (*i*)

Liaoyou 5218 Liao 5216A/C418 C418: Lunhui422 (*m*) /Miyang 23 (*i*)

II you 602 II-32A/Luhui 602 Luhui602: 02428 (*j*) /Gui 630 (*i*) //IR24 (*i*)

#### *6.2.1. Construction of harmonious plant type based on substantial biomass production*

Evolutionary change of rice variety in China indicates that increasing yield through dwarfing is due to the increase of harvest index, whereas through hybrid rice is due to the increase of biological production or biomass. In the experiment under the special ecological conditions at Yongsheng county, Yunnan Province, China, the plot grain yield, biological yield and harvest index of an *indica* hybrid Shanyou 63 were 15.27 t/ha, 28.29 t/ha and 0.54, respectively. Its harvest index under normal ecological conditions is almost the same (about 0.5) (Yang et al., 2006). We think that the key to further increase grain yield is the increase of biomass with a stable harvest index. Undoubtedly, proper increase of plant height is beneficial for increasing biomass, but the lodging resistance should be increased as well. Currently, leaf area index (LAI) in some high yielding varieties is 8-10, which seems to be the maximum. In order to increase the spikelet number and filled grain number per panicle, an indirect strategy is to properly raise plant height, ameliorate leaf stature and leaf quality, and strengthen root system vigor. It is known that the erect and slight rolling uppermost three leaves favor the full utilization of light energy after heading, by promoting CO2 diffusion, increasing photosyn‐ thetic rate on the back face of leaves, accelerating the increase in biological yield, mitigating the conflict between panicle number and size, and improving the lodging resistance of rice plants (Cheng et al., 2007). Now, a set of super hybrids with slight rolling leaves have been developed and used in production. They generally have more than 12 t/ha of yield potential in combination with erect and late senescent leaves and lodging resistant culms.

#### *6.2.2. Utilization of intersubspecific heterosis*

It is known that the heterosis of inter-subspecific hybrids is much stronger than that of intervarietal hybrids. Therefore, utilization of inter-subspecific hybrids is the most feasible approach for realizing super high yield. At present efforts have been focused on using Pei'ai 64S as a major female parent in the selection of super high-yielding combinations. Because Pei'ai 64S is an intermediate type between *indica* and *japonica*, it has a very wide compatibility. To exploit the heterosis of inter-subspecific hybrids and improve the efficiency of super highyielding hybrid breeding, the emphasis is on the development of various widely compatible lines, especially those that have a broad spectrum of compatibility, including restorer lines and male sterile lines of *indica* type, *japonica* type and the intermediate type with different growth durations. This emphasis will create abundant parental lines for breeding various super highyielding hybrids to well adapt different ecological environments in China.

#### *6.2.3. Improvement of important agronomic traits by molecular breeding techniques*

Genetic engineering techniques, such as anther culture, marker-assisted selection and gene transformation, offer reliable opportunities to accelerate breeding progress, increase selection efficiency and overcome genetic barriers to transfer genes across species. These techniques have played important roles in breeding super hybrid rice as well. For example, dramatic progress has been made in the development of transgenic rice plants with a high level of resistance to insects (stem borer, brown planthopper), diseases (tungro virus, rice yellow stunt virus, blast, bacterial blight), herbicide (glufosate), and abiotic stresses (salinity and drought), as well as better nutritional value (e.g. glutelin, vitamin A) and higher yield. Some transgenic rice plants have already been subjected to evaluation under field conditions. In 1995, based on molecular analysis and field experiments carried out as part of a cooperative research program with Cornell University, China National Hybrid Rice Research and Development Center (CNHRRDC) identified two favourable quantitative trait loci (QTLs) (yld1 and yld2) from wild rice (*O. rufipogon* L.). Each of the QTL genes contributed a yield advantage of 18% over the high-yielding hybrid V64 (one of the most elite hybrids in China, with a yield potential of 80 kg/ha per day). By means of molecular marker-assisted backcrossing and selection, the development of near-isogenic lines carrying these two QTL genes is under way (Xiao et al., 1996). If biotechnology can be used to transfer apomixis to rice from grass species, hybrid rice production will be revolutionized and reach even higher levels.

## **6.3. Achievements of super rice breeding in China**

In recent years, super rice breeding and extension for heterosis demonstration in China have made outstanding progress. On the bases to actively use conventional hybrid rice technology for new variety breeding, more and more attention is paid to strengthen the breeding tech‐ nology innovation by combining molecular breeding technology with the conventional breeding technology. Bacterial blight broad-spectrum resistance gene *Xa21* has been intro‐ gressed into restorer lines by marker-assisted selection technology to develop restorer line R8006 with disease-resistance, good quality and high combining ability. As a result, R8006 has produced a series of successful combinations such as Guodao 1, Guodao 3, II you 8006 and Guodao 6 in China National Rice Research Institute. Among them, Guodao 6 has tall and erect plant type as obvious high-yield characteristics, so that the record in southern area was broken by it. In 2004, Guodao 6 had the average yield of 12.08 t/ha in a 100 mu or 6.7 ha model trial. It was released by National Crop Variety Approval Committee in 2006. Similarly, bacterial blight broad-spectrum resistance gene *Xa4* and *Xa21* are introgressed into restorer lines by marker-assisted selection technology in Rice Research Institute of Sichuan Agricultural University. This introgression has resulted in developing restorer line Shuhui527 with diseaseresistance and high combining ability, from which a series of combinations such as superior two-line hybrid rice combination Zhunliangyou 527 and three-line hybrid rice Dyou 527, Gangyou 527, Xieyou 527, Guyou 527 have been bred. Some of the 527 hybrids reached record of high yield frequently. A comprehensive review paper "Super Hybrid Rice Molecular Breeding Research" published on China Rice Science has been downloaded more than 10, 000 times from China rice information network during last two years.

Over last 16 years, super rice research in China has gained significant advances in the aspects of breeding methodology, creation of breeding materials and selection and promotion of elite rice varieties. New pathway is proposed to utilize inter-subspecies heterosis between *indica* and *japonica* and harmonious plant type construction. Under the guidance of breeding methods, the super rice breeding program has been successively conducted and a series of new super rice varieties have been commercially released, such as the three-line super hybrid rice combinations Xieyou 9308, II youming 86, II youhang 1, II you 162, D you 527, Zhong 9 you 8012 and II you 602; the two-line super hybrid rice combinations Liangyoupeijiu, Fen‐ gliangyou 1, Xinliangyou 6 and Zhunliangyou 527; and super inbred rice varieties Jijing 88, Shennong 265 and Shennong 606. Hitherto, a total of 101 new inbred rice varieties or hybrid rice combinations have been identified and nominated as super rice by the Chinese Ministry of Agriculture (CMOA), about half of which are the three-line hybrid rice combinations from South China. The demonstration and promotion of super rice have resulted in an increase of rice yield. According to the CMOA statistics, the accumulative planting area of super rice has increased to 23.85 million hectares (Fig. 4). The average yield of super rice is over 9 t/ha, 0.75 t/ha higher than that of traditional rice varieties. Totally, super rice yield has increased by 17.7 million tons since 1998. These super rice varieties cover rice regions in the Yangzte River Valley, South China and Northeast of China. In 2011, the yield of super hybrid rice Y Liangyou 2 was up to 13.9 t/ha in 6.6 ha demonstration area in Hunan province, which passed, for the first time, the yield target of the third phase in National Super RiceProgram.

**Figure 4.** Planting area of super hybrid rice from 2005 to 2009 (the Ministry of Agriculture, P. R. China)

## **6.4. Outstanding elite hybrid rice varieties in China**

resistance to insects (stem borer, brown planthopper), diseases (tungro virus, rice yellow stunt virus, blast, bacterial blight), herbicide (glufosate), and abiotic stresses (salinity and drought), as well as better nutritional value (e.g. glutelin, vitamin A) and higher yield. Some transgenic rice plants have already been subjected to evaluation under field conditions. In 1995, based on molecular analysis and field experiments carried out as part of a cooperative research program with Cornell University, China National Hybrid Rice Research and Development Center (CNHRRDC) identified two favourable quantitative trait loci (QTLs) (yld1 and yld2) from wild rice (*O. rufipogon* L.). Each of the QTL genes contributed a yield advantage of 18% over the high-yielding hybrid V64 (one of the most elite hybrids in China, with a yield potential of 80 kg/ha per day). By means of molecular marker-assisted backcrossing and selection, the development of near-isogenic lines carrying these two QTL genes is under way (Xiao et al., 1996). If biotechnology can be used to transfer apomixis to rice from grass species, hybrid rice

In recent years, super rice breeding and extension for heterosis demonstration in China have made outstanding progress. On the bases to actively use conventional hybrid rice technology for new variety breeding, more and more attention is paid to strengthen the breeding tech‐ nology innovation by combining molecular breeding technology with the conventional breeding technology. Bacterial blight broad-spectrum resistance gene *Xa21* has been intro‐ gressed into restorer lines by marker-assisted selection technology to develop restorer line R8006 with disease-resistance, good quality and high combining ability. As a result, R8006 has produced a series of successful combinations such as Guodao 1, Guodao 3, II you 8006 and Guodao 6 in China National Rice Research Institute. Among them, Guodao 6 has tall and erect plant type as obvious high-yield characteristics, so that the record in southern area was broken by it. In 2004, Guodao 6 had the average yield of 12.08 t/ha in a 100 mu or 6.7 ha model trial. It was released by National Crop Variety Approval Committee in 2006. Similarly, bacterial blight broad-spectrum resistance gene *Xa4* and *Xa21* are introgressed into restorer lines by marker-assisted selection technology in Rice Research Institute of Sichuan Agricultural University. This introgression has resulted in developing restorer line Shuhui527 with diseaseresistance and high combining ability, from which a series of combinations such as superior two-line hybrid rice combination Zhunliangyou 527 and three-line hybrid rice Dyou 527, Gangyou 527, Xieyou 527, Guyou 527 have been bred. Some of the 527 hybrids reached record of high yield frequently. A comprehensive review paper "Super Hybrid Rice Molecular Breeding Research" published on China Rice Science has been downloaded more than 10, 000

Over last 16 years, super rice research in China has gained significant advances in the aspects of breeding methodology, creation of breeding materials and selection and promotion of elite rice varieties. New pathway is proposed to utilize inter-subspecies heterosis between *indica* and *japonica* and harmonious plant type construction. Under the guidance of breeding methods, the super rice breeding program has been successively conducted and a series of new super rice varieties have been commercially released, such as the three-line super hybrid

production will be revolutionized and reach even higher levels.

times from China rice information network during last two years.

**6.3. Achievements of super rice breeding in China**

300 Rice - Germplasm, Genetics and Improvement

Guodao 6 was released in 2007, which elite traits include high yielding and good quality with a yield potential of 12.5 t/ha and planting area of 95, 000 ha in 2010 (Table 4). Y Liangyou 1 was released in 2006, which elite traits include high yielding, good quality, and wide adaptability with a yield potential of 12.50 t/ha and planting area of 353, 000 ha in 2010. Xin Liangyou 6 was released in 2005, which elite traits include good quality and high yielding with a yield potential of 12.5 t/ha and planting area of 271, 000 ha in 2010. Zhongzheyou 1 was released in 2004, which elite traits include high yielding, good quality, and ideotype with a yield potential of 12.3 t/ha and planting area of 245, 000 ha in 2010.


**Table 4.** Outstanding elite hybrid rice varieties.

## **6.5. Future directions for super rice breeding**

Although great achievements have been resulted from past 13 years in super rice breeding and the yields of some hybrids have approached to the designed target, Chinese scientists are consistently making their efforts to further increase grain yield of rice, regardless of more difficulties and more constraints than even before.

## **6.6. Strengthen the exploitation and utilization of favorable genes**

The core of super rice breeding is an effective use of germplasms and favorable genes. Because the genetic diversity in rice variety gene pool is limited, we must extensively utilize exogenous genes for variety improvements. We can exploit valuable genes from not only the cultivated rice, but also wild rice species, and even other crops to increase yield, quality and resistance to diseases and insects, and tolerance to adverse circumstances. The molecular marker-assisted selection techniques should be effectively used for transformation of high-yielding genes and other important agronomic trait genes from various sources into current variety genetic background. Especially for some complicated traits that lack in rice such as high photosynthetic efficiency gene in maize, stem borer resistant gene (*Bt*) and herbicide-resistant gene in microorganism, the transgenic technology is the best choice to improve rice in the future.

## **6.7. Strengthen the evaluation on root system (including physiological traits)**

In recent years of super hybrid rice breeding practices, a conflict of large panicle with prema‐ ture senescence becomes more and more troublesome to rice breeders and scientists. The large panicle needs longer time for the gains to fully fill and the premature senescence closes the sink before the completion of filling process. Therefore, studying root system to delay senescence of rice plant has become a hot subject in rice community (Wu and Cheng, 2005). Rice root system is not only a vital organ to stand the plant, absorb water and minerals, but also an important place where bioactive substances (hormones, amino acids, etc.) are synthe‐ sized. Root senescence at the late developmental stage directly affects the life span of functional leaves, grain filling and root vigor, especially during the grain-filling period. Obviously, the root vigor is the guarantee for high-yielding of super rice. So far, the genetic research on the root-related morphological traits such as root length, thickness, number, dry weight, density, volume, penetration depth, and root/shoot ratio, as well as other physiological traits such as absorption ability to N, P and K, and root vigor, etc. has achieved great progress (Wu, 2006). However, these studies are still at preliminary stage, and further systematical study is required to propose the key indicator and the appraisal methods for rice breeding, and to explore the gene regulation of the root system and the relationship with the plant organs above ground.

## **6.8. Strengthen the seed production with high security and efficiency**

As we know, super hybrid rice could not be commercially and successfully utilized if hybrid seed production costs too much, seed yield is too low and seed purity is not high enough. In the future, we should strengthen the research on the characteristics of flowering time, stigma exsertion and out-crossing rate of the female parents for super hybrid rice, and establish a new system for super high-yielding seed reproduction. The efficiency of seed production will bring seed price down. The security of seed production will not only yield good quantity and quality of hybrid seed, but also reduce production risk. Therefore, improving seed production will promote the rapid and stable extension of super hybrid rice.

#### **6.9. Strengthen the combination of super rice with suitable cultivation management**

China plays a leading role in global rice production, and the key from Chinese experience is the integration of superior varieties with suitable cultivation management. Research and promotion of the cultivation technology have played an important role in the two break‐ throughs of rice yield in China. Besides high yielding, super rice should have high grain quality, and high efficiency to utilize resources with low environmental pollution. The superiority of varieties and suitability of cultivation practices jointly determine the production scale for a super hybrid rice to be promoted in commercialization.

## **7. Challenges and prospects**

## **7.1. Challenges**

**Aariety name**

302 Rice - Germplasm, Genetics and Improvement

**Year of release**

Y liangyou 1 <sup>2006</sup> high yielding, good quality, wide

Zhongzhe you 1 <sup>2004</sup> high yielding, good quality,

**6.5. Future directions for super rice breeding**

difficulties and more constraints than even before.

**Table 4.** Outstanding elite hybrid rice varieties.

adaptability

ideotype

**6.6. Strengthen the exploitation and utilization of favorable genes**

**6.7. Strengthen the evaluation on root system (including physiological traits)**

**Elite traits Yield potential**

Guodao 6 2007 high yielding, good quality 12.50 95, 000

Xin liangyou 6 2005 good quality, high yielding 12.50 271, 000

Although great achievements have been resulted from past 13 years in super rice breeding and the yields of some hybrids have approached to the designed target, Chinese scientists are consistently making their efforts to further increase grain yield of rice, regardless of more

The core of super rice breeding is an effective use of germplasms and favorable genes. Because the genetic diversity in rice variety gene pool is limited, we must extensively utilize exogenous genes for variety improvements. We can exploit valuable genes from not only the cultivated rice, but also wild rice species, and even other crops to increase yield, quality and resistance to diseases and insects, and tolerance to adverse circumstances. The molecular marker-assisted selection techniques should be effectively used for transformation of high-yielding genes and other important agronomic trait genes from various sources into current variety genetic background. Especially for some complicated traits that lack in rice such as high photosynthetic efficiency gene in maize, stem borer resistant gene (*Bt*) and herbicide-resistant gene in microorganism, the transgenic technology is the best choice to improve rice in the future.

In recent years of super hybrid rice breeding practices, a conflict of large panicle with prema‐ ture senescence becomes more and more troublesome to rice breeders and scientists. The large panicle needs longer time for the gains to fully fill and the premature senescence closes the sink before the completion of filling process. Therefore, studying root system to delay senescence of rice plant has become a hot subject in rice community (Wu and Cheng, 2005). Rice root system is not only a vital organ to stand the plant, absorb water and minerals, but also an important place where bioactive substances (hormones, amino acids, etc.) are synthe‐ sized. Root senescence at the late developmental stage directly affects the life span of functional leaves, grain filling and root vigor, especially during the grain-filling period. Obviously, the root vigor is the guarantee for high-yielding of super rice. So far, the genetic research on the root-related morphological traits such as root length, thickness, number, dry weight, density,

**(t/ha)**

12.50 353, 000

12.30 245, 000

**2010 planting area (ha)**

> Although tremendous achievements have been made in hybrid rice breeding, there also are some constraints and challenges in its development. To sum up, the major problems are as follows.

#### *7.1.1. Planting area has not been at a standstill for years*

In 1991, the acreage of hybrid rice reached its peak at 17.6 million ha, but after that the acreage decreased and remained at about 14.2 million ha in 2011. The main reasons are considered to be the cease and even decrease in the acreage of double cropping early hybrid rice and japonica hybrid rice. Recently, only 20% of early cropping rice area in South China are covered by hybrid rice, while over 90% of late cropping rice area are under hybrid rice in the same region. The availability of early cropping hybrid rice varieties is very limited to growers because it is very difficult to integrate short growth duration and acceptable grain quality into elite high yielding combinations.

## *7.1.2. Grain quality of hybrid rice needs improving*

With the increase of living standards for rice consumers in China, grain quality of rice is required to be improved. In comparison with conventional rice, hybrid rice usually has poorer grain quality measured mainly by the traits of head rice recovery and chalkiness. How to develop rice hybrids with both high yield and good grain quality is still a challenge for breeders.

## *7.1.3. Limited sources of male sterile cytoplasm to develop better CMS lines*

Currently, more than 75% of the CMS lines used in commercial production belong to WA types. This dominant cytoplasm creates a great uniformity of WA cytoplasm, and genetic uniformity has been responsible for an epidemic of a destructive pest. Therefore, more efforts should be paid to diversify male sterile cytoplasms.

#### **7.2. Prospcects**

Conventional breeding has played an essential role in rice cultivar innovation for decades. Large-area application of three-line hybrid rice has showed that hybrid rice technology brings rice yield up to its potential level of physiological yield. With advanced root system and heterosis of seedling and nutrition in early stage, the application of hybrid rice to not only irrigated areas, but also low-lying fields, rain-fed fields and upland fields should be equally important. To commercialize the hybrid rice worldwide, studies on mechanical operation of hybrid seed production and male sterile line regeneration will also be an important subject in hybrid rice research.

In the past dozen years, we have made great progress in rice genome researches, such as establishing a dense molecular linkage map, locating a large amount of major and minor genes underlying important traits including resistance to bacterial blight and rice blast, plant height, reproductive period and tillers, and fully sequencing both *indica* and *japonica* DNA and subsequent functional genomic studies. With research advancement on rice genome, molec‐ ular breeding technology has become a new breeding technology to screen and breed new cultivars according to both phenotype and genotype, thus has been applied to rice breeding. Marker-assisted selection (MAS), quantitative trait locus (QTL) analysis and genetic transfor‐ mation techniques are the most useful tools for rice molecular breeding, and have been used to identify new germplasms and elite rice cultivars. Chinese rice geneticists and breeders have made great progress in identifying QTLs responsible for important agronomic traits such as grain yield and quality, growth and development, disease and pest resistance and abiotic tolerance (Wang et al., 2005). MAS is a method to use molecular markers closely linked to a target gene as a molecular tag, so that the target gene can be quickly identified from breeding populations in the lab. In China, MAS is widely used to pyramid functional genes into popular hybrid rice cultivars to improve important agronomic traits of hybrid rice, such as resistance and grain quality.

In summary, hybrid rice has made a great contribution to safeguarding the food supply in China and is still a major source of elite rice cultivars. However, hybrid rice production is rather time-consuming and the limited available genetic resources leave little room for the continued improvement of rice. With the completion of rice genome sequence, scientists are better equipped to unravel rice gene functions on a genome-wide scale, providing breeders with abundant genetic resources for continued generation of elite rice varieties to maintain a sustainable food supply in China. We expect that the successful implementation of a combi‐ natorial approach using hybrid rice technology will play a crucial role in our effort to improve rice cultivars in China. The immediate goal is to breed varieties with a further improved yield potential, enhanced stress resistance and good grain quality by using molecular and genomic information to break the rice yield plateau in the future.

## **Author details**

difficult to integrate short growth duration and acceptable grain quality into elite high yielding

With the increase of living standards for rice consumers in China, grain quality of rice is required to be improved. In comparison with conventional rice, hybrid rice usually has poorer grain quality measured mainly by the traits of head rice recovery and chalkiness. How to develop rice hybrids with both high yield and good grain quality is still a challenge for

Currently, more than 75% of the CMS lines used in commercial production belong to WA types. This dominant cytoplasm creates a great uniformity of WA cytoplasm, and genetic uniformity has been responsible for an epidemic of a destructive pest. Therefore, more efforts should be

Conventional breeding has played an essential role in rice cultivar innovation for decades. Large-area application of three-line hybrid rice has showed that hybrid rice technology brings rice yield up to its potential level of physiological yield. With advanced root system and heterosis of seedling and nutrition in early stage, the application of hybrid rice to not only irrigated areas, but also low-lying fields, rain-fed fields and upland fields should be equally important. To commercialize the hybrid rice worldwide, studies on mechanical operation of hybrid seed production and male sterile line regeneration will also be an important subject in

In the past dozen years, we have made great progress in rice genome researches, such as establishing a dense molecular linkage map, locating a large amount of major and minor genes underlying important traits including resistance to bacterial blight and rice blast, plant height, reproductive period and tillers, and fully sequencing both *indica* and *japonica* DNA and subsequent functional genomic studies. With research advancement on rice genome, molec‐ ular breeding technology has become a new breeding technology to screen and breed new cultivars according to both phenotype and genotype, thus has been applied to rice breeding. Marker-assisted selection (MAS), quantitative trait locus (QTL) analysis and genetic transfor‐ mation techniques are the most useful tools for rice molecular breeding, and have been used to identify new germplasms and elite rice cultivars. Chinese rice geneticists and breeders have made great progress in identifying QTLs responsible for important agronomic traits such as grain yield and quality, growth and development, disease and pest resistance and abiotic tolerance (Wang et al., 2005). MAS is a method to use molecular markers closely linked to a target gene as a molecular tag, so that the target gene can be quickly identified from breeding populations in the lab. In China, MAS is widely used to pyramid functional genes into popular

*7.1.3. Limited sources of male sterile cytoplasm to develop better CMS lines*

combinations.

304 Rice - Germplasm, Genetics and Improvement

breeders.

**7.2. Prospcects**

hybrid rice research.

*7.1.2. Grain quality of hybrid rice needs improving*

paid to diversify male sterile cytoplasms.

Liyong Cao\* and Xiaodeng Zhan

\*Address all correspondence to: caolycgf@mail.hz.zj.cn

China National Center for Rice Improvement, China National Rice Research Institute, Zhe‐ jiang,, China

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## *Edited by Wengui Yan and Jinsong Bao*

Rice is a staple food for half of the worlds population mostly in Asia. Productivity of rice has largely been improved since the Green Revolution in 1960s. Further improvement of rice yield is necessary to keep pace with population growth, which is a challenging task for breeders. This book, Rice - Germplasm, Genetics and Improvement, as its name implies, comprehensively reviews current knowledge in germplasm exploration, genetic basis of complex traits, and molecular breeding strategies in rice. In the germplasm part, we highlight the application of wild rice in rice breeding. In the genetics part, most of the complex traits related with yield, disease, quality have been covered. In the improvement part, Chinese experiences in hybrid rice breeding have been summarized together with many molecular breeding practices scattering in different chapters.

Rice - Germplasm, Genetics and Improvement

Rice

Germplasm, Genetics and Improvement

*Edited by Wengui Yan and Jinsong Bao*