Association Analysis Of Restoration Of Fertility Genes For Wild-Abortive And Hong-Lian Cytoplasmic Male Sterility And Linkage Mapping Of Quantitative Trait Loci For Stigma Exsertion Rate In Rice

Hybrid rice has made great contribution to the food security of our country and even the world.A hybrid combination consists of two members,the male sterile line and the restorer line.The three-line hybrid rice is a key component of hybrid rice system,of which the theoretical basis is the interaction between the gene conferring cytoplasmic male sterility（CMS）in the mitochondrial genome and the gene for fertility restoration（Rf）in the nuclear genome.Nowadays,Wild-abortive CMS（WA-CMS）and Hong-Lian CMS（HL-CMS）have been employed in the thee-line hybrid rice expanded in our country.Both the fertility restoration of WA-CMS and that of HL-CMS are controlled by major genes and minor genes,according to previous studies.In addition,stigma exsertion rate is a key factor in determining the outcrossing rate of male sterile lines,thus significantly associated with the yield of hybrid rice production.Previous studies showed that stigma exsertion rate in rice is a typical quantitative trait and controlled by many genes.In this study,in order to investigate the genetic basis of fertility restoration of WA-CMS and HL-CMS,we constructed hybrid combination populations derived from a indica core germplasm population from worldwide and sterile lines with WA-CMS and HL-CMS,respectively,and performed genome wide association study（GWAS）.Haplotype analysis was performed for the repeatedly detected major loci.In order to dissect the genetic basis of stigma exsertion rate,a QTL analysis was performed with a recombinant inbred lines（RILs）population.The effect of two QTLs was validated with backcross population,and one QTL was further fine mapped.The main results are as follows:1.F₁ hybrid combination populations with the background of WA-CMS and HL-CMS were constructed respectively with a indica core germplasm population consisting of 337 rice cultivars from worldwide being the paternal parent and the WA-CMS line Hua1517A and the HL-CMS line Yuetai A being the maternal parent.GWAS was performed for pollen fertility,seed-setting rate on bagged panicles（BSS）and seed-setting rate on natural panicles（NSS）of the two F₁ populations in year 2013 and 2014.A total of 13significant loci were detected in the whole population for WA-CMS,and the locus around the 18.8Mb on chromosome 10 was repeatedly detected in the whole population and the population with cultivars from the indica II subgroup being the paternal parent,and responsible for pollen fertility,BSS and NSS,which is co-located with Rf4,the cloned major gene for fertility restoration of WA-CMS.A total of 6 significant loci were detected in the whole population for HL-CMS,and the locus around the 18.8Mb on chromosome 10 was repeatedly detected in the whole population and the population with cultivars from the indica I subgroup being the paternal parent,and responsible for BSS and NSS,which is co-located with Rf5,the cloned major gene for fertility restoration of HL-CMS.2.Four main haplotypes of Rf4 are existed in the core germplasm population,among which the H1 type is carried by the restorer line Minghui 63,while the H4 type is carried by the sterile lines Hua1517Aand Zhenshan 97A.Of the four types,H1 and H2 are functional types,and mainly distributed in the indica II subgroup.H3 and H4 are loss-of-function types.H3 is mainly distributed in the aus subgroup,while H4 is mainly in the indica I subgroup.3.Two main haplotypes of Rf5 are existed in the core germplasm population,which are the H1 functional type carried by the restorer line 9311,and the H2 loss-of-function type carried by the sterile line Yuetai A.236 cultivars carry the H1 type,while only 34 carry the H2 type.4.The genetic basis of stigma exsertion rate was investigated in the RIL population derived from a cross between Zhongguo Xiangdao（ZX）and Chuanxiang 29B（CX29B）.A total of 18QTLs were detected in year 2010 and 2011,among which 8 conferred dual stigma exsertion rate（DSE）and 10 conferred single stigma exsertion rate（SSE）.Two QTL clusters,located between marker RM10105 and RM10142 on chromosome 1 and between marker RM253 and L41 on chromosome 6,were stablely detected for both DSE and SSE,and were named qSe1and qSe6.5.The backcross populations of qSe1 and qSe6 were developed under the background of CX29B,respectively.In the BC₄F₂ random population of qSe1,the values of DSE and SSE of the lines carrying homozygous qSe1 regions from ZX were 17.17%and 15.82%higher than that from CX29B,respectively.In the BC₃F₂ random population of qSe6,the values of DSE and SSE of the lines carrying homozygous qSe1 regions from ZX were 9.25%and11.80%lower than that from CX29B,respectively.6.The genetic basis of stigma exsertion rate was investigated in two F₂ populations derived from a cross between Hua1971B and CX29B.A total of 12 QTLs on chromosome 6 were detected in year 2013 and 2014,among which 6 conferred DSE and 6 conferred SSE.Progeny test of seven F₂ lines showed that the region conferring stigma exsertion rate was located between maker RM314 and L41,which was co-located with qSe6 from the RIL population of ZX and CX29B.7.The BC₇F₂ random population of qSe1 was developed under the background of CX29B and named as the NIL（near-isogenic line）population of qSe1.The value of stigma exsertion rate of NIL-qSe1^ZX was 12.53%higher than that of NIL-qSe1^CX29B.In addition,the values of grain width and kilo-grain weight of NIL-qSe1^ZX were significantly higher than that of NIL-qSe1^CX29B.8.Progeny test and marker analysis were performed for the 51 recombinant plants from the NIL population of qSe1,which was subdivided into two QTLs,qSe1.1 and qSe1.2.New markers were developed and used to analyze recombinant plants in the qSe1.1 region,and qSe1.1 was narrowed down to a region of about 60 kb,which contained seven annotated genes in the reference genome of Nipponbare,two encoding retrotransposon proteins,three encoding expressed proteins,one encoding a transcription factor and one encoding a cation efflux family protein.Relative expression analysis at three stages of young panicle and comparative sequencing analysis were performed for annotated genes,and finally,two genes were treated as candidate genes for qSe1.