Map-based Cloning And Functional Analysis Of Genes Controlling Seed Coat Color In Soybean(Glycine Max(L.)Merr.)

Seed coat color is an important agronomic trait in soybean for morphology marker,biochemical function,and domestication.It is closely related to the appearance quality and commodity quality of seeds.Moreover,anthocyanins,the main substance formed by seed color,have health care function to human body.Therefore,the research on soybean seed coat color not only has important theoretical significance,but also has practical value.In this study,the genes/loci controlling soybean seed color were identified by association analysis and BSA linkage analysis,and the target genes were fine mapped and the gene controlling green seed coat color was cloned by map-based cloning strategy using biparental segregating population derived from cultivated soybean and wild soybean.The main results are as follows:1.A total of 14 loci,including nine novel and five previously reported ones,were identified by GWAS method.Four of these loci were confirmed and further mapped using a biparental population.By genotyping entire F₂ population using flanking markers located in fine-mapping regions,the genetic basis of seed coat color was fully dissected and these four loci could explain all variations of seed colors in this population.2.The candidate gene GmSG for qSC1 was identified by map-based cloning.The variation of A-G at the end of the eighth intron of candidate gene,lead to the early termination of the gene.Analysis of genetic variation in soybean accession indicated that GmSG underwent artificial selection during soybean domestication and people intended to select modern soybean cultivars with yellow seed coat and early maturity.3.The qSC1 NIL appeared stay green during seed maturation and leaf senescence.The seed coat of NIL-G remained green,and chlorophyll degraded slowly during seed development,while the seed coat of NIL-Y turned yellow at maturity and chlorophyll degraded rapidly at R7 stage.Meanwhile,dark treatment experiments showed that NIL-G leaves remained green after 7 and 14 days,chlorophyll degradation was slow,and chlorophyll b remained basically unchanged.4.Overexpression of GmSG resulted in stay green seed coat and leave.In order to verify the function of GmSG,the coding sequence of two GmSG alleles driven by its own promoter and CaMV35S promoter were introduced into soybean genotype Jack using Agrobacterium-mediated transformation system.Transgenic soybean showed green seed coat,slow degradation of chlorophyll in dark leaves,and delayed senescence of the whole plant.Transgenic soybeans had significant effect on 100-seed weight and maturity of T₂ and T₃ plants.5.GmSG may play an important role in regulating chlorophyll degradation during leaf senescence and seed maturation by interacting with soybean CCEs.The different expression genes were identified by transcriptome analysis at different stages of dark treatment in leaves of transgenic soybean and Jack control.The correlation analysis of gene expression showed that chlorophyll degradation pathway genes were positively correlated with the target gene,which might be the key enzymes.Co-expression trend analysis revealed that 563 different expression genes co-expressed with target genes,including plant hormone signal transduction,porphyrin and chlorophyll metabolism,carotenoid biosynthesis,may critial genes.Moreover,eleven genes encoding transcription factors were also identified from different expressing genes co-expressing with the target gene,four of which were ethylene responsive transcription factors,indicating that ethylene pathway may be affected by the target gene.Ethylene treatment of leaves from transgenic soybeans also proveed that overexpression of GmSG could inhibit the effect of ethylene on promoting senescence,indicating that this gene may interact with ethylene pathway to regulate plant senescence.6.Two loci of stay green cotyledon were located on chromosome 1 and chromosome 11 by BSA sequencing,and the candidate genes were identified as homologous genes D1 and D2 of stay green gene SGR by fine mapping.