Fine Mapping And Study Of Sterility Mechanism Of The Interspecific Hybrid Sterility Locus S40 In Rice(Oryza Sativa L.)

Asian cultivated rice(Oryza saliva L.)was domesticated from wild species(Oryza rufipogon)thousands of years ago,and during this process,the genetic diversity within this cultivated rice has been gradually reduced.More importantly,owing to the frequent use of a few adapted progenitors in breeding,the decrease of genetic variation has been amplified.These challenges limit the increases in yield of new varieties and also make rice more susceptible to disease and insect epidemics.Thus,wild rice with the AA genome containing favorable genes(alleles)could be a valuable resource to overcome these obstacles in rice improvement.Oryza longistaminata Chev.is closely related to O.ssativa,as they have a similar AA genome.It is also an excellent gene pool in modern rice breeding because it possesses various outstanding traits,including resistance to bacterial blight disease,weed suppression effects,rhizomes,high biomass production on poor soils and high nitrogen use efficiency.Most importantly,transferring genes from O.longi.staminata to O.saliva through sexual hybridization can significantly enhance the yield of Asian cultivated rice.However,severe reproductive isolation between wild rice(including O.longistaminata)and O.saliva by hybrid sterility and inviability limits extensive utilization of these species and the exploitation of advantageous heterosis effects in related species.Therefore,it is essential to isolate sterility loci and discover wide compatibility varieties containing neutral alleles to overcome reproductive barriers.To dissect the mechanism of interspecific hybrid sterility,we developed a near-isogenic line(NIL)using indica variety RD23 as the recipient parent and O.longistaminata as the donor parent.Using this NIL,we identified a hybrid sterility locus controlling both pollen and spikelet fertility on chromosome 1 and temporarily named it as S40.The main conclusions are as followed:1.Hybrid sterility locus S40 was involved in both male and female development in hybrids between RD23 and O.longistaminata.During the tillering and heading stages,F1 hybrids exhibited no obvious differences from the parents,RD23 and NIL-540.At anthesis,however,the anthers of the F,hybrids demonstrated partial dehiscence.In vivo/in vitro pollen germination tests showed that mature pollen grains from RD23,F1 hybrids and NIL-S40 could germinate efficiently,while aborted pollen grains in F1 hybrids could not germinate.Due to partial anther dehiscence,fewer numbers of pollen grains were poured on the stigma of F,hybrids than those ofRD23 and NIL-S40.When RD23,as a female parent,was pollinated with the pollens of F,hybrids(NIL-S40/RD23),the setting rate of RD23 was approximately 50.3412.11%,but when pollinated with pollens of RD23,the setting rate was higher,at 84.3712.32%.This suggested that partial anther dehiscence in F,hybrids did affect the setting rate.The pollen staining ability of 12-KI revealed that pollen grains of F,hybrids exhibited typical semi-sterility,whereas both RD23 and NIL-S40 were fertile with pollen fertility>90%.Cytological observation indicated that microspores in the F,hybrids were hindered from the first mitosis at the early bicellular pollen stage.Consequently,the proportion of aborted pollen grains with one or no nucleus in the F,hybrids was significantly higher than those in RD23 at mature stage.Despite the normal function of tapetum,the pollen wall structure in F,hybrids was incomplete.In addition,spikelet fertility was much lower in the F,hybrids than in RD23 and NIL-S40.When female Fi hybrids were pollinated with pollens from RD23,the setting rate of F,hybrids was only approximately 35.6±1.7%,which implied that female development in F,hybrids was also impaired.Partial embryo sacs were arrested at the function megaspore formation stage,and subsequent mitosis was thus unable to continue.Above all,partial dehiscence of the anther and semi-sterile embryo sac together led to the lower spikelet fertility of F,hybrids.2.To analyze genetic patterns of the S40 locus,we constructed two backcross populations(RD23//RD23/NIL-S40,RD23/NIL-S40//RD23).NIL-S40 carried a locus genotype S40lS40l(l:longistaminata),while RD23 harbored S40iS4i(i:indica).The segregation ratio of genotypes and phenotypes in progeny was fit to Mendel's law when pollinating RD23 and RD23/NIL-5S40 with RD23/NIL-5S40 and RD23 pollen grains.In these two populations,the number of homozygote S40iS40i was almost equal to the number of heterozygote S40iS40l.Based on the sex-independent TRX)(siTRD)system,the parameters km and kf of the S40i and S40l alleles transmitted efficiently through male and female gametes were 0.51 and 0.49,respectively,which indicated that both male and female gametes carrying the S40i or S40l allele could be transmitted equally.In addition,in the F2 population derived from RD23/NIL-S40,all homozygotes,S40iS40i and S40lS40l;were fertile,while the heterozygotes S40iS40l demonstrated typical pollen and embryo sac semi-sterility,which resulted in a 1:1 segregation ratio of fertile and semi-sterile plants.The genetic mechanism of S40 was consistent with "one-locus sporophytic model".3.A total of 202 plants from the NIL-S40/RD23 F2 population were used to perform the primary mapping of S40.Then,the S40 locus was located to an interval of approximately 1.3 Mb between molecular markers Z116 and HLY43,spanning the short arm of chromosome 1.To further map the S40 locus,the flanking markers Z116 and HLY43 were used to screen a total of 16,600 individuals from the F2 population,and a total of 203 recombinants were obtained.Finally,the S40 locus was finely mapped between ES-2 and ES-60,with one recombination event with marker ES-2 and three with marker ES-60.In addition,one marker,ES-19,was determined to co-segregate with the S40 gene.Based on reference sequences of an indica variety 93-11,the genomic region containing the S40 locus was approximately 80 kb in length.Through sequencing,we determined that the distance between ES-2 and ES-60 was also approximately 80 kb on the O.longistaminata chromosome.F2:3 populations of 8 key recombinants(between markers E45 and ES-60)validated the fine-mapping results.4.Gene prediction of the 80-kb DNA sequence containing S40 using the online Gramene database identified eight putative open reading frames(ORF1-ORF8).Sequencing and expression analysis was performed to determine possible candidate genes.We firstly sequenced the genome of seven ORFs(ORF2,3,4,5,6,7 and 8)that had functional annotation from RD23 and NIL-S40.Because the amino acid sequences of ORF2,ORF6 and ORF7 were the same as those of RD23 and NIL-S40,we excluded them as candidate genes.The amino acid sequences of ORF3,ORF4,ORF5 and ORF8 had differences from those of RD23 and NIL-S40.ORF3 and ORF4 might not be the candidate genes either because they were barely expressed in mature anthers or pistils.ORF5 and ORF8 are the most possible candidate genes for S40.ORF5,encoding an ulpl protease,was highly expressed in pistils from stage 6 to the mature stage.An A742G mutation located in the third exon caused one variant amino acid at amino acid position 189 in NIL-S40,which has asparagine(Asn,N)instead of aspartic acid(Asp,D)in RD23.ORF8,encoding a R2R3 MYB family transcription facto,r was predominantly expressed in anther from stages 9 to 11.Because of a 3 bp deletion in coding region of cDNA in RD23,RD23 has an amino acid deletion(alanine)between amino acids 235 and 236 compared to NIL-S40.