Evolutionary Analysis In Indica-japonica Hybrid Sterility Locus S5 And Its Application In Wide-compatibility Breeding

The rice is a main food crop in the world.Asian cultivated rice consists of two subspecies,indica and japonica.The seed-set rate of the hybrids between the subspecies is low,which results from the reproductive isolation.Breeding indica?japonica hybrid rice is supposed to be one of the most effective ways to accelerate the rice yield,by utilizing the strong heterosis expressed in hybrids between the two subspecific cultivator indica and japonica.However,hybrid sterility is a major obstacle in utilization of inter-subspecific heterosis.A special group of rice germplasm,known as wide-compatibility varieties(WCVs),is able to break the reproductive barrier between indica and japonica subspecies,and becomes the important germplasm for the utilization of indica-japonica heterosis.The S5 locus was identified as one of the major loci regulating hybrid sterility.Previous studies have found that a killer-protector system of reproductive isolation at S5 was build up,by the genetic interaction of three tightly linked genes of ORF3,ORF4,and(designed as ORFs3-5),which controlled the indica-japonica hybrid sterility and wide-compatibility by allele interaction.In this study,we investigated the allelic combinations,nucleotide polymorphism,and haplotype networks at the S5 locus from 606 rice accessions including two cultivated rice species and eleven wild rice species.The result provided efficient information in exploring wide-compatibility germplasm harboring neutral allele at S5 locus,which would facilitate rice genetic breeding.In addition,the origin and evolutionary mechanism were systematically analyzed by use of population genetics and molecular evolutionary method.On the other hand,we have incorporated three alleles of Sa-Du,S5-n and f5-n from the broad-spectrum wide-compatible variety 'Dular' into an elite indica.type restorer line 9311,using molecular marker assisted backcross strategy.An efficient wide-compatible molecular breeding system was established,which would provide theoretical foundation and breeding materials for utilizing strong indica-japonica heterosis in the future.The main results in the present studies are as follows:1.The allele frequencies were calculated based on the genotype data of 606 accessions in rice.The frequencies of ORF3+,ORF4+,and ORF5+decreased in O.sativa compared to the wild rice,in which the high frequencies of ORF3+,ORF4+,and ORF5+were observed.However,the frequencies of ORF3-,ORF4-,and ORF5-/n alleles went up significantly in the cultivated rice.These results support the inference that ORF3+,ORF4+,and ORF5+ may be the ancestral alleles.2.There were 12 combinations in theory,which were formed of different allelic groups of the three genes.However,three combinations,ORF3-ORF4-ORF5-,ORF3-ORF4-ORF5+,and ORF3-ORF4+ORF5+,were not observed in the sample investigated.The allelic combination of ORF3+ORF4+ORF5+was the most frequent type in the wild rice species,which indicated that the allelic combination of ORF3+ORF4+ORF5+may be primitive at the S5 locus.The allelic combinations of ORF3+ORF4-ORF5+and ORF3-ORF4+ORF5-,which represented the typical indica-like and japonica-like types,were the most common ones in Asian cultivated rice.These two allelic combinations were also observed in the wild rice with low frequencies.These result suggested that indica-like and japonica-like allelic combinations at the S5 locus already existed before the divergence of the wild and cultivated rice.3.Based on sequence analysis in coding region,the combination of ORFs3-5 in 606 accessions was classified into 93 haplotypes(H1-H93).The haplotype network clearly showed the evolution history of S5 system.The two haplotypes of H31 and H3 3 from ORF3+ORF4+ORF5+ have produced the japonica type ORF3-ORF4+ORF5-and the indica type ORF3+ORF4-ORF5+ respectively.Subsequently,the WC type of ORF3+ORF4-ORF5n emerged from the indica type ORF3+ORF4-ORF5+H26,with deletion of 136 bp in ORF5+.Moreover,the haplotypes H33 had mutated from H31.Our result confirmed the independent origins of indica and japonica haplotypes at S5 locus during the evolutionary process in rice.One mutation event led to the origin of indica haplotype,whereas the japonica haplotype had undergone multiple mutation events.The WC type was generated from the loss-of-function mutation in indica haplotype.4.A total of 104 accessions harboring S5-n were identified in this study.In addition,26 of 112 accessions carried the broad-WCG S5-n,which was named based on the widest compatibility to all the possible allelic combinations at S5 locus.Thus,these broad-WCG resources would benefit the cross-breeding and the utilization of indica-japonica heterosis in the future.5.Through integrated backcross breeding including,positive selection on the target loci based on their linkage and functional markers,negative selection on both sides of the target loci,whole-genome background selection,and phenotypic selection,we incorporated three alleles from the WCV 'Dular' at S5 locus regulating embryo-sac,Sa and f5 loci regulating pollen fertility,into an elite indica restorer line 9311,respectively.Finally four improved lines were developed,eomprised of three single-gene lines NIL-(55-n),NIL-(Sa-Du),NIL-(f5-n),and a double-gene line PL-(S5-n+f5-n).The two near isogenic lines NIL-(S5-n)and NIL-(f5-n)harboring either S5-n or f5-n and a pyramiding line carrying S5-n plus f5-n were obtained,with the recurrent parent genome recovery of 99.95%,99.49%,and 99.44%,respectively.Then,the four lines were evaluated for the wide-compatibility ability.The three lines NIL-(S5-n),NIL-(f5-n)and PL-(S5-n+f5-n)showed normal fertility when crossed to typical indica testers.When testcrossed to five typical japonica varieties,the three lines allowed significant increase of compatibility with constant agronomic performance.The introgressed S5-n could significantly improve 14.7%to 32.9%embryo-sac fertility in indica-japonica hybrids.In addition,with the presence of f5-n fragment,S5-n would increase the spikelet fertility from 9.5%to 21.8%.The introgressed f5-n fragment greatly improved anther dehiscence,embryo-sac and pollen fertility in indica-japonica hybrids,thus leading to improvement of spikelet fertility from 20.4%to 30.9%.Moreover,the pyramiding line showed 33.6%to 46.7%increase of spikelet fertility,suggesting cumulative effect of S5-n and f5-n fragment in seed-set improvement of inter-subspecific hybrids.However,Sa-Du did not exhibit effectiveness in the indica?japonica hybrids.This study focuses on evolutionary analysis in indica-japonica hybrid sterility locus and its application in wide-compatibility breeding.It is useful in understanding the reproductive isolation between indica and japonica,and faciliate improving the fertility of the indica-japonica hybrids by marker-assisted breeding.Therefore,the present study provides theoretical basis for the cross-breeding and the utilization of indica-japonica heterosis.