Fine Mapping And Candidate Gene Analysis Of Soybean CMS Restorer-of-fertility Gene GmRf1

The hybrid breeding in soybean(Glycine max)mainly relies on the three-lines system derived from cytoplasmic male sterility.Identification of optimal restorer lines is therefore of importance.Since checking the fertility of F1 hybrids derived from the tested restorer lines crossing with sterile lines is time-consumption and laborious,genetic identification of the strong restorer-of-fertility(Rf)gene(s)and its use for marker-assisted selection(MAS)will greatly improve the efficiency of breeding.Inthis study,the F2 segregation population derived from the CMS-RN type sterile line JLCMS204A(female parent)and the restorer line JLR230(male parent,containing the unknown Rf gene)was investigated.The inheritance pattern of restorer-of-fertility gene contained in the restorer line was clarified.Based on the bulked segregant analysis(BSA)of both parents and two pools(fertile and semi-sterile),and the location interval of the gene locus was predicted and named GmRf1.The simple sequence repeat(SSR)molecular marker was used for polymorphism analysis to preliminarily locate the GmRf1 gene.Then combined with parental resequencing to further develop single nucleotide polymorphism(SNP)markers,insertion-deletion(InDel)markers and sequence tag site(STS)markers for GmRf1 gene identification Fine mapping,and cloning,sequence alignment analysis and expression analysis of the predicted candidate genes within the positioning interval to preliminarily confirm the GmRf1 gene.The main conclusions of this study are as follows:(1)According to the microscopic examination results observed under the microscope,the populations were separated and 119 were found to be fertile and 124 to be semi-sterile.After the chi-square test,the separation ratio of the two is in line with the Mendelian 1:1 separation ratio,indicated that the restorer-of-fertility gene contained in the restorer line was controlled by a pair of dominant single genes,which conformed to the single gene gametophytic inheritance pattern.(2)Based on the BSA of both parents and two pools(fertile and semi-sterile),the genetic locus was allocated on chromosome 16.Using SSR molecular markers for polymorphism analysis,the gene was initially located between the markers BARCSOYSSR＿16＿1069 and BARCSOYSSR＿16＿1076,the genetic distances were 0.4 cM and 0.3 cM,and the physical distances were between 32 703 286 bp and 32 932 950 bp,the genetic locus was named GmRf1.(3)Combined with the mapping interval of SSR markers and parental resequencing data,further develop dCAPS,InDel and STS markers for fine mapping of GmRf1 gene.According to the polymorphism screening results of individual plants in the F2 population based on dCAPS,InDel and STS markers.GmRf1 was finally delimited between the marker dCAPS-1 and BARCSOYSSR＿16＿1076,in which the genetic distance were 0.1 cM and 0.3 cM,respectively.On the basis of the ZH13 v2.0 reference genome,GmRf1 was located between 32 708 896 bp and 32 932 950 bp.The interval length is about 224.1 kb.The physical distance of the restoration gene GmRf1 was converted from ZH13 v2.0 to the Williams82.a2.v2 reference genome.The physical distance was between 32 094 021 bp and 32 309 902 bp,the interval length becomes 215.9 kb.(4)Reference genome with Williams82.a2.v1,there are 27 candidate genes in this interval searched through the soybean genome database.The 27 genes were functionally annotated,and7 PPR protein family genes with typical characteristics of restoration genes were found,and cloned and sequenced were analyzed,and combined with subcellular localization prediction,it was found that Glyma.16G161900,Glyma.16G162000 and Glyma.16G163100 may contain the GmRf1 characteristic of restorer-of-fertility gene,presumably to restorer-of-fertility gene GmRf1.(5)qRT-PCR was used to detect the relative expression levels of seven PPR genes in the male restorer line JLR230 and the female sterile line JLCMS204 A.It was found that the expression level of Glyma.16G161900 was the highest and the expression level of this gene in the leaves of the restorer male parent JLR230 and the sterile line female parent JLCMS204 A was significantly different.(6)Through the comparison of the Glyma.16G161900 gene sequencing between the parents,it was found that because the Glyma.16G161900 gene had 3 single nucleotide mutations in the coding region of the father,resulting in a change in the encoded amino acid.The base was mutated from C to T at 256 bp in the CDS region,the base was mutated from A to G at 760 bp,and the base was also mutated from C to T at 826 bp.Therefore,it is speculated that Glyma.16G161900 has a functional mutation in this gene.The CDS length of the resulting new gene was changed to 1731 bp,encoding 576 amino acids.It was speculated that it was the target restorer-of-fertility gene GmRf1.(7)According to the three mutation sites on the Glyma.16G161900 gene,two functional markers,dCAPS-2 and dCAPS-3,were developed,which can distinguish different genotypes containing GmRf1 sites,it provides technical support for the identification and assisted breeding of GmRf1-containing restorer lines using the above molecular markers.