**4.2 Trait improvement in parental lines and hybrids**

Hybrid rice has been one of the innovations that led the quantum jump in rice productivity last century. However, the challenge of meeting the increasing demand for rice and making hybrid more sustainable under impeding climatic changes, trait development in parental lines for ideal plant type with substantial yield, grain quality, and resistance/tolerance to multiple biotic and abiotic stresses is necessary. In this context, conventional breeding is more cumbersome, time taking and less précised. The advancement in molecular breeding techniques makes it convenient to improve the parents and hybrids for desirable traits with great precision. Markerassisted selection/MABB has provided strong utensils for indirect selection/trace the trait of interest at any plant growth stage. The bacterial blight and blast are the

**37**

*Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

utilizing genome editing tool "CRISPR/Cas9" [34].

**4.3 Screening of Rf genes in parents**

two-major destructive diseases affecting rice plant at different growth stages and caused substantial yield loss. Resistant genes for BB diseases have been deployed successfully in popular hybrids like Rajalaxmi, Ajay [26], BS 6444G, PRH 10 [29], Shanyou 63, Guangzhan63-4S; seed parent of CR Dhan 701; restorers Minghui 63 and Mianhui 725 [5, 26], Zhonghui 8006 and Zhonghui 218, etc. The popular CMS line Rongfeng A, Pusa 6A female parent of popular basmati hybrids PRH 10, RGD-7S, and RGD-8S [30] were successfully stacked with blast and BB resistant gene(s). Besides, CRMS 31A and CRMS 32A were deployed with submergence and salinity tolerance QTLs (NRRI newsletter 2015). Grain and eating quality in hybrids are concerns which are addressed by stacking QTLs/genes for quality traits in parents. Zhenshan 97A seed parent of several hybrids in China has been stacked with QTLs of AC, GC and GT [31]. Efforts were made toward quality improvement of both the parental lines of popular indica hybrids, viz., Xieyou57, using markerassisted selection for *Wx* locus [32]. Yield-enhancing QTLs, *yld1.1* and *yld2.1*, from *O. rufipogon* to restorer "Ce64" [33] are successfully stacked. Hybrid sterility in inter-subspecific (*indica/japonica*) hybrids is reported to be effectively addressed by

Limited availability of fertility restorer system in rice makes three-line system

very selective and less heterotic. Rice genotypes have fertility restorer ability can only be utilized as pollen parent in three-line hybrid breeding. Identification of genetically compatible, well combining restorers is tedious process, involve laborious test cross generation and evaluation steps. However, prior information on fertility restorer genes in the pollen parent excludes test cross steps thus make it convenient for saving time of hybrid development. Plenty of co-segregating molecular markers (tightly linked or functional markers) for fertility restorer gene(s) having functional specificity to diverse CMS systems are available (**Table 2**). The genic/functional markers, RM6100 and DRRM Rf3–10 of restorer gene(s) *Rf4* and *Rf3*, respectively, are widely utilized for screening the fertility restoration efficacy

of unknown pollen parents for WA and lineage CMS well in advance [15].

Hybrid sterility is common nuisance menacing breeder to exploiting heterosis in inter-subspecific (5–10% more heterosis) hybrids. Generally, *indica* × *japonica* hybrids are sterile due to lack of wide compatibility (*WC*) between parents. It is reported that hybrid sterility in inter-subspecific crosses is mainly affected by the genes at *Sb*, *Sc*, *Sd*, and *Se* [35] loci causes male sterility in F1and the gene at *S5* locus cause female sterility in F1. Presence of these genic regions in at least one parent ensures complete fertility in resulting hybrids. These gene(s) can be assessed in advance by utilizing co-segregating

PSM8, PSM12, and PSM180 (linked SSR); IND19 and ID5 (indel markers) to *Sb*, *Sc*, *Sd*, and *Se*, loci). Thus, it helps breeder in selection of *WC-*positive parent in more predictable way which circumvents laborious test-cross and their evaluations steps.

Genetic distance and level of genetic gain/breeding value in parents are major determinants of extent of heterosis in the resulting hybrid. Molecular markers help in assessing the genetic diversity among parents and breeding values in progenies

) [36] and G02–14827 (genic marker)

**4.4 Screening of parental lines for wide compatibility genes**

markers (S5-InDel, functional marker to S5n

**4.5 Prediction of heterosis**

*Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

*Recent Advances in Rice Research*

genetic purity testing of hybrids.

**4.1 DNA fingerprinting and genetic purity testing**

**4.2 Trait improvement in parental lines and hybrids**

the seed parents CRMS 31A and CRMS 32A. To enhance the seed producibility in seed parents, introgression of stigma exsertion trait from O. longistaminata into CRMS 31A and CRMS 32A, are under progress. To excavate the genetic region responding heterosis in rice, transcriptomic analysis of hybrids Rajalaxmi and Ajay are completed and interpreted. Availability of restorers for WA-CMS lines is very stumpy in nature, only 15% of total rice genotypes having the ability to restore complete fertility in WA-CMS-based hybrid rice [15]. Hence, good combiner genotypes having partial fertility restorers Mahalaxmi and Gayatri were improved by introgressing fertility restorer gene(s) *Rf3* and *Rf4* through MABB approach. Further, to make clear cut identity and ensure pure seed of parents/ hybrids to the stack-holder, 12 signature markers that unambiguously distinguish 32 rice hybrids were developed, which can be utilized for DNA fingerprinting and

**4. Potential application of OMICS approaches in hybrid rice breeding**

Recent advancement in molecular biology has offered tremendous opportunities to the breeder and breeding *per se* in enhancement in their efficacy and speed up the varietal development process. It has diverse applications like mapping, tagging, amplification-based cloning, gene pyramiding, marker-assisted selection (MAS/ MARS), fingerprinting applications, including varietal identification, ensuring seed purity, phylogeny and evolution studies, diversity analysis, and elimination of germplasm duplication. The progress in research related to application of DNA marker technology in hybrid rice improvement may be valuable in following way.

Varietal identity of hybrids and parents is imperative to assure the ownership (IPR issue) and pure seeds to the stakeholders. The genetic purity testing of hybrid seed is done by conducting Grow-Out-Test (GOT) which is time taking (needs one full growing season), tedious and very expensive. Molecular markers in this context found to be a suitable alternative, provide an unbiased means of identifying crop varieties. Among available DNA-based markers, sequence-tagged microsatellites (STMSs), which are co-dominant in nature, are widely used for speedy genetic purity assessment of the hybrids and parental lines [27, 28]. Besides, ICAR-NRRI has developed another set of nine signature markers which can distinguish parents CRMS 31A, CRMS 32A; and hybrids Ajay, Rajalaxmi and CR Dhan 701,

Hybrid rice has been one of the innovations that led the quantum jump in rice productivity last century. However, the challenge of meeting the increasing demand for rice and making hybrid more sustainable under impeding climatic changes, trait development in parental lines for ideal plant type with substantial yield, grain quality, and resistance/tolerance to multiple biotic and abiotic stresses is necessary. In this context, conventional breeding is more cumbersome, time taking and less précised. The advancement in molecular breeding techniques makes it convenient to improve the parents and hybrids for desirable traits with great precision. Markerassisted selection/MABB has provided strong utensils for indirect selection/trace the trait of interest at any plant growth stage. The bacterial blight and blast are the

**36**

unambiguously.

two-major destructive diseases affecting rice plant at different growth stages and caused substantial yield loss. Resistant genes for BB diseases have been deployed successfully in popular hybrids like Rajalaxmi, Ajay [26], BS 6444G, PRH 10 [29], Shanyou 63, Guangzhan63-4S; seed parent of CR Dhan 701; restorers Minghui 63 and Mianhui 725 [5, 26], Zhonghui 8006 and Zhonghui 218, etc. The popular CMS line Rongfeng A, Pusa 6A female parent of popular basmati hybrids PRH 10, RGD-7S, and RGD-8S [30] were successfully stacked with blast and BB resistant gene(s). Besides, CRMS 31A and CRMS 32A were deployed with submergence and salinity tolerance QTLs (NRRI newsletter 2015). Grain and eating quality in hybrids are concerns which are addressed by stacking QTLs/genes for quality traits in parents. Zhenshan 97A seed parent of several hybrids in China has been stacked with QTLs of AC, GC and GT [31]. Efforts were made toward quality improvement of both the parental lines of popular indica hybrids, viz., Xieyou57, using markerassisted selection for *Wx* locus [32]. Yield-enhancing QTLs, *yld1.1* and *yld2.1*, from *O. rufipogon* to restorer "Ce64" [33] are successfully stacked. Hybrid sterility in inter-subspecific (*indica/japonica*) hybrids is reported to be effectively addressed by utilizing genome editing tool "CRISPR/Cas9" [34].

#### **4.3 Screening of Rf genes in parents**

Limited availability of fertility restorer system in rice makes three-line system very selective and less heterotic. Rice genotypes have fertility restorer ability can only be utilized as pollen parent in three-line hybrid breeding. Identification of genetically compatible, well combining restorers is tedious process, involve laborious test cross generation and evaluation steps. However, prior information on fertility restorer genes in the pollen parent excludes test cross steps thus make it convenient for saving time of hybrid development. Plenty of co-segregating molecular markers (tightly linked or functional markers) for fertility restorer gene(s) having functional specificity to diverse CMS systems are available (**Table 2**). The genic/functional markers, RM6100 and DRRM Rf3–10 of restorer gene(s) *Rf4* and *Rf3*, respectively, are widely utilized for screening the fertility restoration efficacy of unknown pollen parents for WA and lineage CMS well in advance [15].

#### **4.4 Screening of parental lines for wide compatibility genes**

Hybrid sterility is common nuisance menacing breeder to exploiting heterosis in inter-subspecific (5–10% more heterosis) hybrids. Generally, *indica* × *japonica* hybrids are sterile due to lack of wide compatibility (*WC*) between parents. It is reported that hybrid sterility in inter-subspecific crosses is mainly affected by the genes at *Sb*, *Sc*, *Sd*, and *Se* [35] loci causes male sterility in F1and the gene at *S5* locus cause female sterility in F1. Presence of these genic regions in at least one parent ensures complete fertility in resulting hybrids. These gene(s) can be assessed in advance by utilizing co-segregating markers (S5-InDel, functional marker to S5n ) [36] and G02–14827 (genic marker) PSM8, PSM12, and PSM180 (linked SSR); IND19 and ID5 (indel markers) to *Sb*, *Sc*, *Sd*, and *Se*, loci). Thus, it helps breeder in selection of *WC-*positive parent in more predictable way which circumvents laborious test-cross and their evaluations steps.

#### **4.5 Prediction of heterosis**

Genetic distance and level of genetic gain/breeding value in parents are major determinants of extent of heterosis in the resulting hybrid. Molecular markers help in assessing the genetic diversity among parents and breeding values in progenies

(through genomic selection, high-density SNP genotyping) with great convenient. There are abundant STMS and SNP markers available which can be utilized for assessment of genetic diversity/genetic distance between parents and genomic selection in progenies easily [37]. Hence, this is helpful in the selection of diverse parents with maximum breeding values in turn higher heterosis or genetic gain in hybrids.
