Location And Cloning Of A Yellow-leaf Controlling Gene GmYLD1 In Soybean

The mutant is a good material for cloning and studying of gene functions.However,there are relatively few studies on isolating genes by the use of soybean mutants.Photosynthesis is closely related to crop yield and leaf chlorosis causes photosynthetic efficiency and yield decrease.Isolating function genes with leaf-color mutants and deeply analyzing the genetic laws and Molecular mechanism of leaf color regulation can help providing marker genes and references for breeding of high light-efficiency.In this study,Hedou 12,a high yield and quality variety of Shandong Province,and Williams 82 were treated by EMS mutagenesis.And a batch of heritable mutants were obtained.One of these mutants names gmyld1(yellow leafchlorophyll deficient1)was studied,and the results are as follows:Through EMS mutagenesis,we totally screened out 64 genetic M2/3-generation soybean mutants,28 of which are leaf shapes and leaf color mutants,8 are sterile lines and 28 are about the size and color mutation of seeds.Then,15 of these mutant lines were hybridized with Williams 82,and their F2 generation popμ Lations were constructed.The phenotype analysis of the gmyld1 mutant showed that its young leaves are yellow and exhibits delayed greening.Its Chlorophyll content decreased 88.41%and photosynthetic efficiency also decreased significantly.Chlorophyll synthetic key product content analysis found that,the content of Mg-Proto,Pchilde,and Childe in the mutant is decreased,while the Urogen content is increased significantly.It suggests that the mutant’s chlorophyll synthesis was blocked at the biochemical process of Urogen to Mg-Proto.In this pathway,the expression level of genes which encode UROD(uroporphyrinogen decarboxylase)and Magnesium chelatase was significantly decreased.TEM shows that the grana stacking and the chloroplast development in gmyldl were in disordered.Genetic analysis showed that the phenotype of yellow leaf in the gmyld1 mutant is controlled by a single recessive nuclear locus.Genome sequence alignment with Hedou 12 and Williams 82,we obtained 610,515 SNPs and 12,515 Indels respectively.Then we developed 84 indel markers distributed on chromosome 11-20 and screened as anchor Molecular marker.And then,we constructed the physical map.BSA-seq analysis was performed on wild and mutant materials isolated from gmyldl and Hedou 12 backcross F2 popμLation(BC1F2).The Mutmap and SNP association analysis revealed that Chr20:8,012,091...10,281,887 was the candidate region,including 112 genes and 3candidate SNPs.With the32 F2 yellow-leaf mutants,the mutation site was initially mapped to a 21.31 Mb interval of chromosome 20,which contains the BSA-seq localization interval.Then,with an expanding F2 popμLation,the mutation site was further mapped to the region of Chr:8,438,195...8,822,768,with a length of 383,5 Kb and containing 16 genes.Further analysis showed one of the 16 gene was the same as that of candidate gene(Glyma.20G046200)found in BSA-seq.Futher sequencing of the sequencing genes,we found only one gene sequence has changed,and it was just Glyma.20G046200.So,we set Glyma.20G046200 as a candidate gene.In Gmyld1,CDS of candidate gene in+1629 site base G was substitute by base A,and the corresponding amino acid changed from Phe to Ser.We then named the candidate gene as GmYLD1(Yellow Leaf chlorophyll Deficient1).RNA-seq analysis of gmyld1 found that 511 genes were up-regulated and 265 genes were down-regulated in gmyld1compared with the CK(Hedou 12 wild type).Among them,the expression level of photosynthesis and chlorophyll synthesis genes were significantly changed,the expression of PsbA(photosystem Ⅰ 1 related gene),PsbA(photosystem Ⅱ related genes),Pet B(cytochrome b6/f complex related genes)and ATP synthase related genes were significantly up-regulated,while PsbK(photosystem Ⅱ related genes),the expression of Lhca2,Lhcb1 and Lhcb6 gene was significantly down-regulated.Gene expression pattern analysis showed that the candidate gene GmYLD1 was expressed in roots,stems and leaves of soybean,and the expression quantity was root>leaf>stem.