**3. Discovery of genetic male sterile lines**

*Protecting Rice Grains in the Post-Genomic Era*

**16**

**Figure 2.**

**Figure 3.**

*factor (sterile).*

*either indica (I) or japonica (J) [7].*

CMS-HL, *Rf5* and *Rf6* restores 75% of fertile pollen in the F1. Further analysis by sequencing and cloning concluded that *Rf1* is the same gene as *Rf5* located in chromosome 10 [19]. *Rf3* and *Rf4* restores fertility in CMS-WA and are located in chromosome 1 and 10 respectively with the latter recently cloned [20]. CMS-LD fertility can be restored by *Rf1* or *Rf2* while CMS-CW can only be restored by a single gene *Rf17* [12].

*Schematic diagram of CMS three line system. Rfrf = nuclear gene homozygote recessive, RfRf = nuclear gene homozygote dominant, Rfrf = nuclear gene heterozygous, N = cytoplasmic factor (fertile), s = cytoplasmic* 

*A schematic presentation of the five well-studied rice CMS types. Abbreviations for cytoplasm sources are RWA for wild-abortive Oryza rufipogon, RRA for red-awned O. rufipogon, and RW1 for Chinese wild rice (O. rufipogon) accession W1; IBT and ILD for indica Boro-II type and Lead rice, respectively. Nucleus sources are* 

Diversification of both CMS maintainers and restorer lines are very important

to guarantee the continued progress of finding the best hybrid combinations.

**2.2 Breeding and diversification of CMS sources**

Although the CMS three-line system greatly increased yields in hybrid rice, there are difficulties and limitations on its use. One of the difficulties is the need to simultaneously develop maintainer lines (B lines) by subsequent nucleus substitution of the original CMS line with the B lines through repeated backcrossing. Furthermore, there are also limited choices available for restorer lines (R lines) with only about 5% of the current existing lines can be used that carries the restorer gene [7]. The discovery of genetic male sterility or photoperiod and/or thermosensitive male sterile lines addresses these problems. These lines responds to photoperiod, temperature or a combination of both which cause the plant to be fertile or sterile depending on the critical daylength or temperature [23]. With the two-line system using genetic male sterile, there is no need to develop a maintainer line and any fertile line can be used as a restorer. This greatly reduce the time and resources in making hybrid combination and parental seed production. Moreover, it broadens the available choices of restorers that can generate more combinations which in turn increases the probability of finding the best hybrids [4].

Extensive studies suggest that genetic male sterile lines can be broadly classified into three categories; photoperiod genetic male sterile (PGMS), thermosensitive genetic male sterile (TGMS), and photoperiod and thermosensitive genetic male sterile (PTGMS) [15, 24, 25]. The first reported genetic male sterile came from spontaneous mutant in a japonica cultivar Nongken 58 discovered in Hubei China and were later called as Nongken 58S [2]. Further studies after its discovery revealed that the male sterility is regulated mainly by photoperiod and thus referred to as photoperiod genetic male sterile (PGMS). Nongken 58S showed complete pollen sterility when grown under long day conditions (>14 h), fertility was restored when subjected to <10 h of light under controlled environment [26].

A thermosensitive type of male sterility was discovered in a spontaneous mutant AnnongS-1 (Ans-1) in 1997. The pollen remained sterile at both long and short day when exposed to 33°C and reverts back to fertile when the temperature reached 24°C [27]. Additional lines exhibiting thermosensitivity were also discovered in Zhu1S, Hengnong 1S and Guangzhang 63S where the fertility rates vary at different controlled temperatures regardless of daylength [28–30].

The third classification of genetic male sterility affects both photoperiod and temperature. Pei'ai 64S is a line derived from the original male sterile mutant Nongken 58S with genetic backgrounds such as *indica* and *javanica* [31]. A study conducted on the response of Pei'ai 64S and another line 8902S showed fertility under long daylength (>14.5 h) and low temperature (24°C) or short daylength (10 h) and high temperature (28°C) conditions, but were consistently sterile at long daylength (14 h) and high temperature conditions (28°C) [32].

### **3.1 Genetics of male sterility**

Numerous genetic studies concluded that genetic male sterility can be controlled by single, two genes or multiple genes depending on the genetic background and

the environment. The original Nongken 58S when crossed to conventional *indica* and *japonica* lines produced F1's that are all fertile [15]. The F2 reciprocal crosses to Nongken 58S concluded that that male sterility is controlled by a single recessive gene [33]. Further studies showed similar results when Nongken 58S (sterile) and Nongken 58 (fertile) were crossed showing a typical single gene recessive segregation in the F2 population under long day conditions [34, 35]. A single locus segregation of genetic male sterile lines was also reported in several TGMS lines under long day and high temperature field conditions [29, 30, 36, 37]. Previous research also discovered two gene recessive segregation when Nongken 58S was crossed to an *indica* variety. F2 segregation exhibited a ratio of 15 fertile: 1 sterile pollen fertility [2]. Several *indica* derived male sterile lines such as Pei'ai 64S displayed similar genetic ratios [31, 38, 39]. Additional studies detected segregation that followed either a bimodal or a continuous distribution in some populations of genetic male steriles [26, 40].

Studies conducted under US conditions on *indica* male sterile line 2008S originated from China showed that male sterility is controlled by two or three recessive genes depending on the location and year of planting (**Figure 4**). When planted in

### **Figure 4.**

*Male sterile line 2009S grown in the greenhouse (A) and in the field (C). 2008S male sterile line grown in the greenhouse (B) and in the field (D). Both lines exhibited 100% sterility when grown at H. Rouse Caffey Rice Research Station in Crowley, Louisiana USA under high temperature and long day conditions.*

**19**

**Figure 5.**

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice*

only a three-gene model was found for 2008SxCL131 [4].

**3.2 Candidate genes and mechanism of genetic male sterile rice**

Several candidate genes were identified in controlling male sterility in PGMS lines. More recent studies using Nongken 58S discovered that the gene in LOC\_Os12g36030 which was previously mapped as pms3 and is also allelic to p/ tms12-1 located on chromosome 12 regulates photoperiod genetic male sterility. The single nucleotide polymorphism (SNP) mutation located in non-coding region increased the methylation on the promoter which in turn reduced transcription of LOC\_Os12g36030.The reduced transcription caused the pre-programmed cell death

A second study conducted in PTGMS line Pei'ai 64S revealed that the gene LOC\_Os07g12130 previously mapped as pms1(t) encodes a protein containing Myb-like DNA binding domain that affect the transcription of a protein responsible for the photo-thermosensitive response. RT-PCR results showed that mRNA levels of LOC\_Os07g12130 changes at different photoperiod and temperature conditions [31]. However, the gene is yet to be cloned and further study needs to be conducted. De Guzman [4] sequenced both locus in male sterile line 2008S and found both SNPs present. When QTLs were analyzed in two segregating populations using both single marker analysis and interval mapping, each locus and their interaction gives

For lines exhibiting thermosensitivity, candidate genes associated with TGMS lines were mapped on chromosome 2. QTL mapping using bulk segregant analysis approach (BSA) identified the ptgms2-1 locus [30]. Further analysis showed

*Pollen sterility distribution of F2 plants in populations of 2008S/CL131 and 2009S/10HB020 male sterile lines* 

*planted in 2013 at H. Rouse Caffey Rice Research Station, Crowley LA USA.*

**Figure 5** for 2008S and 2009S F2 populations.

in developing anthers causing pollen sterility [42, 43].

significant effects [25].

Stuttgart Arkansas USA, 2008SxCL131 F2 population fit a three-gene model, while the same population planted in Crowley, Louisiana USA segregated in two-gene model during 2013. In 2014, the 2008S/Cypress population fit both 15:1 and 63:1 ratios in Arkansas, whereas 2008S/CL131 only fit a 15:1 segregation ratio. In the same year at Crowley, Louisiana a single 15:1 ratio exhibited in 2008SxCypress, and

Another male sterile line 2009S currently used by the LSU Agcenter for their hybrid rice breeding program shows a single gene recessive inheritance (**Figure 4**). Field trials carried out in Crowley Louisiana over 2 years (2013–2014) in two F2 and four BC1F2 populations showed consistent results both in seed and pollen fertilities [25, 41]. A comparison of the pollen sterility frequency distribution is presented in

*DOI: http://dx.doi.org/10.5772/intechopen.85191*

### *Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice DOI: http://dx.doi.org/10.5772/intechopen.85191*

Stuttgart Arkansas USA, 2008SxCL131 F2 population fit a three-gene model, while the same population planted in Crowley, Louisiana USA segregated in two-gene model during 2013. In 2014, the 2008S/Cypress population fit both 15:1 and 63:1 ratios in Arkansas, whereas 2008S/CL131 only fit a 15:1 segregation ratio. In the same year at Crowley, Louisiana a single 15:1 ratio exhibited in 2008SxCypress, and only a three-gene model was found for 2008SxCL131 [4].

Another male sterile line 2009S currently used by the LSU Agcenter for their hybrid rice breeding program shows a single gene recessive inheritance (**Figure 4**). Field trials carried out in Crowley Louisiana over 2 years (2013–2014) in two F2 and four BC1F2 populations showed consistent results both in seed and pollen fertilities [25, 41]. A comparison of the pollen sterility frequency distribution is presented in **Figure 5** for 2008S and 2009S F2 populations.

## **3.2 Candidate genes and mechanism of genetic male sterile rice**

Several candidate genes were identified in controlling male sterility in PGMS lines. More recent studies using Nongken 58S discovered that the gene in LOC\_Os12g36030 which was previously mapped as pms3 and is also allelic to p/ tms12-1 located on chromosome 12 regulates photoperiod genetic male sterility. The single nucleotide polymorphism (SNP) mutation located in non-coding region increased the methylation on the promoter which in turn reduced transcription of LOC\_Os12g36030.The reduced transcription caused the pre-programmed cell death in developing anthers causing pollen sterility [42, 43].

A second study conducted in PTGMS line Pei'ai 64S revealed that the gene LOC\_Os07g12130 previously mapped as pms1(t) encodes a protein containing Myb-like DNA binding domain that affect the transcription of a protein responsible for the photo-thermosensitive response. RT-PCR results showed that mRNA levels of LOC\_Os07g12130 changes at different photoperiod and temperature conditions [31]. However, the gene is yet to be cloned and further study needs to be conducted. De Guzman [4] sequenced both locus in male sterile line 2008S and found both SNPs present. When QTLs were analyzed in two segregating populations using both single marker analysis and interval mapping, each locus and their interaction gives significant effects [25].

For lines exhibiting thermosensitivity, candidate genes associated with TGMS lines were mapped on chromosome 2. QTL mapping using bulk segregant analysis approach (BSA) identified the ptgms2-1 locus [30]. Further analysis showed

### **Figure 5.**

*Pollen sterility distribution of F2 plants in populations of 2008S/CL131 and 2009S/10HB020 male sterile lines planted in 2013 at H. Rouse Caffey Rice Research Station, Crowley LA USA.*

*Protecting Rice Grains in the Post-Genomic Era*

the environment. The original Nongken 58S when crossed to conventional *indica* and *japonica* lines produced F1's that are all fertile [15]. The F2 reciprocal crosses to Nongken 58S concluded that that male sterility is controlled by a single recessive gene [33]. Further studies showed similar results when Nongken 58S (sterile) and Nongken 58 (fertile) were crossed showing a typical single gene recessive segregation in the F2 population under long day conditions [34, 35]. A single locus segregation of genetic male sterile lines was also reported in several TGMS lines under long day and high temperature field conditions [29, 30, 36, 37]. Previous research also discovered two gene recessive segregation when Nongken 58S was crossed to an *indica* variety. F2 segregation exhibited a ratio of 15 fertile: 1 sterile pollen fertility [2]. Several *indica* derived male sterile lines such as Pei'ai 64S displayed similar genetic ratios [31, 38, 39]. Additional studies detected segregation that followed either a bimodal or a continuous distribution in some populations of genetic male steriles [26, 40]. Studies conducted under US conditions on *indica* male sterile line 2008S originated from China showed that male sterility is controlled by two or three recessive genes depending on the location and year of planting (**Figure 4**). When planted in

*Male sterile line 2009S grown in the greenhouse (A) and in the field (C). 2008S male sterile line grown in the greenhouse (B) and in the field (D). Both lines exhibited 100% sterility when grown at H. Rouse Caffey Rice* 

*Research Station in Crowley, Louisiana USA under high temperature and long day conditions.*

**18**

**Figure 4.**

LOC\_Os02g12290 encode a ribonuclease Z gene that when the SNP is present, it created a premature stop codon rendering the RNase ZS1 defective. The mechanism described that when the mutant male sterile is exposed under high temperature (28°C), it induced the accumulation of mRNA ribosomal protein UbL40 in microspore mother cell that were not processed by the defective RNase ZS1 enzyme consequently causing pollen degeneration. At lower temperatures (23°C), UBL40 mRNAs levels remained low allowing production of normal pollen. This mutation was reported in TGMS varieties Guangzhang 63S, Ans-1 and Zhu1S [28]. Zhang [44] discovered that the locus tms5, ptgms2-1 and tms9 were allelic and were all mapped to chromosome 2 that contains the similar ribonuclease Z gene. De Guzman [25] sequenced the locus LOC\_Os02g12290 in line 2009S and discovered the same SNP present in TGMS lines. Inheritance studies on F2 and BC1F2 showed similar segregation ratios. SNP marker were developed using CEL1 nuclease to identify association of the marker to the trait and showed that the markers were able to predict 95–100% of male sterile lines in F2 and BC1F2 population [41].

### **3.3 Breeding and production of genetic male sterile rice**

Since PTGMS and TGMS have different responses to temperature and photoperiod, methods in seed multiplication and breeding varies including selection of specific location and time of planting. On PTGMS lines, weak and strong photoperiodic responses were reported [45]. In strong photoperiodic response such as in long daylength, the critical sterility inducing temperature (CST) is low (21°C) and at short daylength high (25–26°C).

For a PTGMS lines with weak photoperiodism, the (CST) is about 22°C under short daylength. In China, Chen [45] suggested that seed production of this type should be bred during autumn in Guangdong and Guangxi, and in winter in Hainan province. Cold water irrigation treatment has been used extensively in lines with both weak and strong photoperiod. This solved the problem of low yields in multiplication of PTGMS lines such as Pei'-ai 64 s with low CST [46].

For TGMS lines, there is no weak or strong photoperiodism, thus timing and selection of location is important for seed production of male steriles and hybrids. On most TGMS lines, the ideal CST is about 22.5°C. Seed production are also treated with continuous cold water irrigation to increase seed yield during the winter [46].

In the US, RiceTec of Alvin Texas successfully used both ptgms and tgms lines however, seed production locations are unknown and specific methods of seed multiplications of male sterile lines are undisclosed.

In Louisiana USA, the LSU AgCenter initiated the hybrid rice breeding program in 2009 using ptgms and cytoplasmic male sterile lines obtained via a Material Transfer Agreement with the Guangxi Academy of Agricultural Sciences, Nanning, China. Test crosses made from these lines showed equal or superior grain and head rice yield compared to the current RiceTec commercial varieties. However, high chalk, lodging, and late maturities were observed that warrant the development of male sterile with improved agronomic traits and are suitable to the southern US conditions [47]. The breeding cycle starts with crosses of adapted lines to male sterile lines to produce F1's. Seeds from hybrids then harvested and spaced planted. Single plant selections of male sterile lines were performed by looking at sterile pollen stained with 1% I2KI under the microscope during early heading. Plants suitable for generation advance were selected with the following characteristics: 98–100% pollen sterile, 60–80 cm in height, short flag leaf, intermediate tillering and with compact growth habit. Selected plants were uprooted, placed in one gallon pots and transferred in the greenhouse. The plants were ratooned by cutting ~9–10 cm from the soil line. The ratoons are allowed to grow up to early booting stage where the

**21**

November [48, 49].

**Figure 6.**

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice*

majority of the shoots have the measurement distance of about of 1–3 cm between the flag leaf collar to the leaf node or at about the early booting stage (meiotic division of pollen mother cell). Plants are then subsequently treated in a growth chamber with temperatures 22°C during the night and 28°C during the day at 11 h daylength for 10 d. After treatment, plants are then transferred to the greenhouse for 30–35 days for the seed to mature (**Figure 6**). Seed multiplication were carried out in Puerto Rico agricultural experiment station planted during October to

*Methods of selection and production of male sterile lines in LSU Agcenter hybrid rice breeding program.*

Introgression of disease resistance traits in hybrid rice becomes a necessity largely due to the narrow genetic diversity of both CMS and genetic male sterile sources. A study showed leading hybrids from China that was introduced in Africa were out yielded by inbred checks due to non-adaptability and susceptibility to diseases and pests [50]. There were also reported insect and disease incidence in China that are more frequent in hybrid rice than on inbred varieties [5]. Research institutes such as the International Rice Research Institute (IRRI) was aware of these issues and has continued to develop new CMS, maintainer, restorers and genetic male steriles in diverse background. Current improvement on hybrid rice focuses on incorporation of resistance gene identified from inbred and wild sources. For instance, blast resistance genes have been introgressed in maintainer, restorer and S lines through hybridization, backcross and marker assisted selection (MAS) [6]. In the early 2000, varieties with multiple bacterial panicle blight resistance were released in Indonesia and China. These varieties were developed using MAS

and produced significant yield gain demonstrated in farmer's field [51].

**4. Introgression of disease resistance in hybrid rice**

*DOI: http://dx.doi.org/10.5772/intechopen.85191*

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice DOI: http://dx.doi.org/10.5772/intechopen.85191*

**Figure 6.**

*Protecting Rice Grains in the Post-Genomic Era*

of male sterile lines in F2 and BC1F2 population [41].

at short daylength high (25–26°C).

**3.3 Breeding and production of genetic male sterile rice**

plication of PTGMS lines such as Pei'-ai 64 s with low CST [46].

multiplications of male sterile lines are undisclosed.

LOC\_Os02g12290 encode a ribonuclease Z gene that when the SNP is present, it created a premature stop codon rendering the RNase ZS1 defective. The mechanism described that when the mutant male sterile is exposed under high temperature (28°C), it induced the accumulation of mRNA ribosomal protein UbL40 in microspore mother cell that were not processed by the defective RNase ZS1 enzyme consequently causing pollen degeneration. At lower temperatures (23°C), UBL40 mRNAs levels remained low allowing production of normal pollen. This mutation was reported in TGMS varieties Guangzhang 63S, Ans-1 and Zhu1S [28]. Zhang [44] discovered that the locus tms5, ptgms2-1 and tms9 were allelic and were all mapped to chromosome 2 that contains the similar ribonuclease Z gene. De Guzman [25] sequenced the locus LOC\_Os02g12290 in line 2009S and discovered the same SNP present in TGMS lines. Inheritance studies on F2 and BC1F2 showed similar segregation ratios. SNP marker were developed using CEL1 nuclease to identify association of the marker to the trait and showed that the markers were able to predict 95–100%

Since PTGMS and TGMS have different responses to temperature and photoperiod, methods in seed multiplication and breeding varies including selection of specific location and time of planting. On PTGMS lines, weak and strong photoperiodic responses were reported [45]. In strong photoperiodic response such as in long daylength, the critical sterility inducing temperature (CST) is low (21°C) and

For a PTGMS lines with weak photoperiodism, the (CST) is about 22°C under short daylength. In China, Chen [45] suggested that seed production of this type should be bred during autumn in Guangdong and Guangxi, and in winter in Hainan province. Cold water irrigation treatment has been used extensively in lines with both weak and strong photoperiod. This solved the problem of low yields in multi-

For TGMS lines, there is no weak or strong photoperiodism, thus timing and selection of location is important for seed production of male steriles and hybrids. On most TGMS lines, the ideal CST is about 22.5°C. Seed production are also treated with continuous cold water irrigation to increase seed yield during the winter [46]. In the US, RiceTec of Alvin Texas successfully used both ptgms and tgms lines however, seed production locations are unknown and specific methods of seed

In Louisiana USA, the LSU AgCenter initiated the hybrid rice breeding program

in 2009 using ptgms and cytoplasmic male sterile lines obtained via a Material Transfer Agreement with the Guangxi Academy of Agricultural Sciences, Nanning, China. Test crosses made from these lines showed equal or superior grain and head rice yield compared to the current RiceTec commercial varieties. However, high chalk, lodging, and late maturities were observed that warrant the development of male sterile with improved agronomic traits and are suitable to the southern US conditions [47]. The breeding cycle starts with crosses of adapted lines to male sterile lines to produce F1's. Seeds from hybrids then harvested and spaced planted. Single plant selections of male sterile lines were performed by looking at sterile pollen stained with 1% I2KI under the microscope during early heading. Plants suitable for generation advance were selected with the following characteristics: 98–100% pollen sterile, 60–80 cm in height, short flag leaf, intermediate tillering and with compact growth habit. Selected plants were uprooted, placed in one gallon pots and transferred in the greenhouse. The plants were ratooned by cutting ~9–10 cm from the soil line. The ratoons are allowed to grow up to early booting stage where the

**20**

*Methods of selection and production of male sterile lines in LSU Agcenter hybrid rice breeding program.*

majority of the shoots have the measurement distance of about of 1–3 cm between the flag leaf collar to the leaf node or at about the early booting stage (meiotic division of pollen mother cell). Plants are then subsequently treated in a growth chamber with temperatures 22°C during the night and 28°C during the day at 11 h daylength for 10 d. After treatment, plants are then transferred to the greenhouse for 30–35 days for the seed to mature (**Figure 6**). Seed multiplication were carried out in Puerto Rico agricultural experiment station planted during October to November [48, 49].
