Gene Cloning And Functional Identification Of Seed Germination Based On Genome-wide Association Analysis

Seed germination is the premise of emergence.In practice of modern agricultural cultivation,fast and systematic seed germination is a key to maximize crop yield.Therefore,it is of great significance to explore the gene loci or genes related to seed germination.With the progress of genome sequencing technologies and the reduction of sequencing cost,it is now possible to use the genome wide association study(GWAS)to explore the genes in cotton.In this study,479 upland cotton genotypes were used and data for seed germination related six phenotype parameters,such as seed coat breakthrough rate,endosperm breakthrough rate,radicle elongation,24 hours and 48 hours in 2 time periods,were investigated in combination with 338736 SNP markers to conduct GWAS.We also performed gene cloning and functional analysis of candidate genes.The results are as follows:1.We identified 294 loci that were significantly related to seed germination.Haplotype block structure analysis predicted 27 candidate genes for seed germination based on the most significant site of each single peak associated with seed germination traits,According to the expression level of candidate genes in different plant tissues,homologous gene annotation in Arabidopsis of candidate genes,significance analysis of SNP and amino acid changes caused by SNP,three genes,GhMIEL1,GhDRM1 and GhGlcD1,were selected from 27 candidate genes.The three genes were related to seed germination experimentally verified through gene cloning and functional identification.The identified loci or genes can provide a basis for rapid and systematic germination of cotton seeds,thereby enhancing the development of cotton industry.2.Analysis of the spatiotemporal expression and specific expression of GhMIEL1.The spatiotemporal expression of GhMIEL1 gene as a whole increased first and then decreased.GhMIEL1 is highly expressed in radicle and relatively low in endosperm and cotyledon;GhMIEL1 subcellular localization analysis showed that GhMIEL1 protein is on the cell membrane and nucleus;GhMIEL1 over-expressing transgenic Arabidopsis materials were obtained and subjected to standard germination tests.The results showed that GhMIEL1 functionally acquired transgenic Arabidopsis thaliana showed a reduced seed germination rate.3.Analysis of the spatiotemporal expression and specific expression of GhDRM1 showed that the overall spatiotemporal expression of GhDRM1 gene first increased and then decreased.GhDRM1 was highly expressed in the radicle and cotyledons and rarely expressed in the endosperm;Subcellular localization analysis of GhDRM1 showed that GhDRM1 protein was on the cell membrane and nucleus;GhDRM1 over-expressing transgenic Arabidopsis materials were obtained and subjected to standard germination tests;The results showed that GhDRM1 functionally-acquired transgenic Arabidopsis showed faster seed germination.4.Analysis of the spatiotemporal expression and specific expression of GhGlcD1 showed that the overall spatiotemporal expression of GhGlcD1 gene first increased and then decreased.GhGlcD1 was specifically expressed in endosperm and hardly expressed in radicle and cotyledon;Subcellular localization analysis of GhGlcD1 showed that GhGlcD1 protein was on the cell membrane;GhGlcD1 over-expressing transgenic Arabidopsis materials were obtained and subjected to standard germination tests.The results showed that GhGlcD1 functionally acquired transgenic Arabidopsis thaliana showed faster seed germination rate.