Mapping,Cloning And Development Of Functional Molecular Markers Of Nuclear Sterility Gene Ms6 In Soybean

Soybean（Glycine max（L.）Merr.）is native to China,but in recent years,the yield of soybean in China has been lower than the average level of the world.Therefore,it is imperative to improve soybean yield in China.The utilization of heterosis could improve crop yield rapidly and effectively,but soybean is a self pollinated crop,which has small floral organs and is difficult to cross.The discovery and utilization of soybean male sterile lines,as well as the development of insect vector cross pollination technology,greatly promoted the process of hybrid soybean breeding.In soybean male sterile lines,the discovery and utilization of nuclear male sterility is relatively early,and it is widely used in recurrent selection of breeding,but it is rarely used in hybrid breeding due to low utilization rate of seed production and sensitive environmental conditions.With the rapid development of the third generation crop hybrid breeding technology and the improvement of the intelligent genic male sterile（GMS）system,as an important functional element,the genic male sterile genes have been widely cloned and applied in maize,rice and other crops.In the early years,researchers found 14 genic male sterile genes in soybean and mapped them with molecular markers.However,in recent years,only 2 genes have been successfully cloned,and the research on the molecular mechanism of soybean genic male sterility is relatively scarce.In view of the important role of GMS gene,if we can localize GMS gene with molecular markers,further clone GMS gene and clarify the genetic mechanism of GMS gene,it will provide material basis and theoretical support for further using GMS gene to construct soybean third generation intelligent GMS system and develop new methods of soybean heterosis utilization.In this study,the F₂ segregating population containing recessive genic male sterile gene ms6 in soybean was used to carry out related experiments.According to the high-throughput sequencing technology with the method of bulked segregation analysis（BSA）,the candidate interval was selected and combined with simple sequence repeat（SSR）molecular markers to locate the sterile gene.The ms6 candidate gene was successfully cloned,and the single nucleotide polymorphism（SNP）site where the mutation occurred was clarified.Based on this,the derived cleaved amplified polymorphic sequence（d CAPS）molecular markers were developed for rapid and accurate identification of soybean ms6 genotypes,which provided important theoretical basis and technical support for soybean cross breeding using ms6 gene.The main conclusions are as follows:（1）Based on the field investigation and pollen fertility identification of four segregation populations,M92 population with the same growth and more stable segregation ratio was selected for the mapping of ms6 gene.There were 225 fertile plants and 76 sterile plants in the segregation population.The segregation ratio of 3:1 was consistent with the chi square test.Therefore,it was clear that ms6 gene was only controlled by a pair of recessive nuclear genes.（2）The results showed that the ms6 was located on chromosome 13 of soybean by BSA sequencing and polymorphism SSR molecular marker screening.It was located between the SSR markers BARCSOYSSR-13-0259 and BARCSOYSSR-13-0275.The genetic distances were 0.4 c M and 3.2 c M,respectively.The physical interval was 16428596 bp～16683664 bp,and the interval length was about 255 Kb.（3）According to the physical distance between SSR molecular markers,it was mapped to the soybean reference genome Williams 82.a2.v1,in which there were 24 known genes.There Only Glyma.13G066600 was found in the cross region.The sequence alignment revealed that it was a homologous gene encoding the transcription factor TDF1 of tapetum and pollen wall in Arabidopsis thaliana.Therefore,Glyma.13G066600 gene was identified as a candidate gene for soybean MS6.（4）The specific primers were designed for the coding region of Glyma.13G066600 gene.Sequencing and alignment showed that the 227th base of CDS in the sterile offspring changed from T to A,the corresponding amino acid changed from Leu to His,which changed the function of the original gene.Therefore,Glyma.13G066600^76Leu→Hisis supposed to be the recessive genic male sterile gene ms6 in soybean.（5）There 12 d CAPS markers were developed according to the mutation site of ms6 gene.3d CAPS markers,ms6-d CAPS-1,ms6-d CAPS-2 and ms6-d CAPS-3,showed obvious polymorphic bands after digestion.Further verification showed that ms6-d CAPS-3 marker had high stability and good repeatability.The restriction enzyme Hind III could accurately distinguish homozygous fertile,heterozygous fertile and homozygous sterile plants in ms6progeny segregation population,which was suitable for molecular marker assisted selection of ms6 genotype materials.