Gene Mapping And Functional Investigation Of SNPP1 Controlling Seed Number Per Pod In Mung Bean

Mung bean（Vigna radiata L.,2n=22）is warm-season grain legume with short duration and adapts to relatively harsh conditions such as drought stress and barren soil.It is used as important nutritional dry grain,food legume pulse.In addition,the popular mungbean vegetable sprouts are rich in vitamins as well as amino acids.Seed number per pod is an important yield trait of mung bean.As a quantitative trait,it is controlled by internal genetic loci and the external environment.However,the molecular mechanism controlling seed number per pod of mung bean is not yet clear.In the previous study,from large-scale mutagenesis,7 mutants with increased seed number per pod were identified.Genetic analysis showed that the mutation was controlled by a semi-dominant gene.In this study,two allelic mutants of snppl,M5311 and D008,were selected as research materials to investigate their agronomic traits.Backcrossing progenies from M5311 and Sulu were used to construct 2 extreme bulks and to perform whole genome resequencing.QTL-seq analysis was applied to rapidly map the gene SNPP1（Seed Number Per Pod 1）.In addition,this study also combined the results of RNA sequencing of mung bean samples at the early pod-setting satge to analyze differentially expressed genes.Furthermore,phylogenetic analysis and expression pattern studies on candidate genes were conducted.Main results are as following:1.Phenotype observation of snppl mutantsBy observing the mutants for several generations and seasons,the phenotype was obvious:pods became longer,and this phenotype has already been observed in the early stage of pod development;the number of seeds per pod increased whileas the length,width,height and 100-grain weight of seeds in mutants were significantly reduced,compared to that of the wild-type plants.2.QTL-seq based mapping of gene controling seed number per pod in mung beanThrough Bulk Segregant Analysis,the mutant pool and the wild-type pool were constructed to perform whole genome resequencing.The association interval was obtained by QTL-seq method:SNPP1 was initially located in the 3.49 Mb（92,640-3,584,001 bp）region on chromosome 5 of mung bean,and several molecular markers were developed based on the SNP and InDel provided by sequencing.3.RNA-seq and functional gene mining of mutantsRNA-seq of mung bean samples at early pod-setting satge was carried out.144 differentially expressed genes and several significant enrichment pathways were identified.The results showed that differentially expressed genes are involved in multiple pathways related to catalytic reactions,transcription level regulation,and anabolism of multiple substances,indicating that the regulation of the number of seeds per pod involves multiple biological processes.Combined with the results of whole genome resequencing,the gene Loc106760202 was locked as a candidate gene.4.Phylogenetic analysis and expression patterns of candidate geneThe gene Loc106760178 in mung bean showed high similarity to Loc106760202.Phylogenetic analysis revealed that Loc106760202 encoded the 6mA adenine demethylase of AlkB family.Loc106760202 gene was expressed in root,stem,leaf,and shoot of mung bean,with higher level in the root and the shoot,and very low level in the stem and the leaf.Compared to that of the wild-type plants,Loc106760202 displayed lower expression levels in all of organs in mutants.