Fine Mapping And Candidate Genes Cloning Of Cytoplasmic Male Sterility Restorer Gene Rf2 In Soybean

Soybean(Glycine max(L.)Merr.)originated in China.As one of the world most important food and oil crops,it is widely cultivated and used.The yields of the conventional soybean varieties are increasing slowly,which cannot meet the increasing demands.Therefore,it is an urgent need for means or measures which can improve soybean yield,stress resistance and quality.One of the most effective methods is heterosis utilization.Heterosis utilization is mainly based on the CMS/Rf system,which has the advantage that we can obtained 100%sterile individual plants in the female row in the field.In the CMS/Rf system,the restorer lines are used as the male parents,and the strength of its restoration ability affects the fertility and stability of the hybrids directly.However,the long process and the large time-laborious consuming of the strong restorer lines selection have greatly restricted the selecting process of the strong dominant hybrids.Therefore,mapping and cloning fertility restorer gene and using molecular biological methods to assist breeding process and artificially creating strong restorer lines have become the important parts of efficient screening and breeding of the CMS/Rf system.With the development of molecular biology,genomics and high-throughput sequencing technology,a great number of fertility restorer genes have been mapped in plants.And some of them have been cloned and identified.In soybean,there are five main CMS types:RN-type,ZD-type,M-type,N-type and XXT-type.Most of them are the type of gametophytic sterility.Moreover,the "JiYu" series hybrid soybean varieties which were bred by RN-type CMS/Rf system have the largest number of varieties and are used the most widely.Therefore,mapping and cloning the CMS-RN type fertility restorer gene is of a great significance to clarify the CMS fertility restoration mechanism,and then assist the selecting process and create strong restoration lines which can be applied in production.In this study,we used the RN-type soybean CMS line JLCMS9A and the restorer line JLR500 as parents to construct an F2 isolated population which contained the Rf2 gene.Based on the preliminary bulk segregant RNA sequencing,we obtained the Rf2 gene candidate interval.Then we used the parental resequencing combined with the SSR markers and developed a great number of InDel and SNP markers to fine map the Rf2 gene.Moreover,we further cloned the candidate genes of the Rf2 gene and we run the sequence analysis to testified the cloned gene.This provides the basis and ideas for the application of marker assisted selection of restorer lines and the use of genetically modified methods to create new restorer line sources in the future.Through the study,we mainly obtained the following conclusions:(1)We used the male sterile line JLCMS9A which is the type of gametophytic sterility and the restorer line JLR500 which bred from a near-isogenic line introduced with the restorer gene Rf2,as the parents.And we selected V9 isolated population from 10 isolated populations and we identified the pollen fertility and genetic mapping analysis.Through the fertility identification and genetic pattern analysis,we confirmed that the restorer gene Rf2 was controlled by only one pair of dominant nuclear genes,which was in line with the single dominant gene pattern in the background of gametophytic sterile cytoplasm.(2)Based on the previous BSR-Seq technology,we obtained the chromosome interval of the target restorer gene.Compared with other candidate positions of soybean CMS fertility restorer genes that are already being mapped,the restorer gene was initially judged to be a new one.We temporarily named it as Rf2.We selected 90 pairs of SSR markers which cover this interval and used them to screen the interval and map the gene.Through marker screening and mapping by the JoinMap 4.0 software,we initially mapped the Rf2 gene between BARCSOYSSR-16-0921 and BARCSOYSSR-16-0932.The genetic distances were 0.4 cM and 0.6 cM,respectively.And the physical interval was 29 666 812-29 877 997 bp,within a length about 211 kb.(3)Based on the SSR marker mapping interval and the analysis of parental resequencing data,we developed 10 pairs of SNP markers and 119 pairs of InDel markers for fine map gene within the candidate interval.Through screening and verification,we found BARCSOYSSR-16-0921,InDel-16-110,and CAPS-16-8 closely linked to the Rf2 gene.The genetic distances were 0.4 cM,0.3 cM,and 0.3 cM,respectively.The minimum physical interval was 29 666 812-29 831 111 bp,within a length about 164 kb.(4)According to the physical position of the fine mapping molecular marker and corresponding to the soybean reference genome Williams82.a2.vl,there were 13 genes in this candidate interval.Then we retrieved the information of their sequence and functional annotation information,Glyma.16G139800 is a PPR protein family gene.This was in line with the characteristic of the cloned restorer gene encoding for PPR protein which in other plants.We designed the specific primers which located in the Glyma.16G139800 coding region.After we cloned the target gene in each control material,run the sequence analysis and the comparation,we found there were 2 InDels and 1 SNP in the Glyma.16G139800 coding region in JLR500.These caused prematurely terminate of its translation and formed a new coding sequence.We speculated that Glyma.16G139800-Phe-Ser-Leu+Val+Thr→Asn was the Rf2 gene.According to the characteristics and functions of the cloned PPR restorer genes,the protein which encoded by the Rf2 gene may be guided into the mitochondria through the mitochondrial targeting signal peptide carried by it,which affects the transcription or translation of sterility genes in the mitochondria and achieved fertility restoration of the F1 generation.