Genome-wide Association Study For Salt Tolerant Traits Of Alfalfa At Germination Stage Based On RNA-Seq

Alfalfa is known as the“king of pastures”.Salt damage is one of the main non-biological stress factors in alfalfa.The ability of seeds to germinate is an important precondition for the promotion and utilization of saline-alkali land.In this study,119 breeds of alfalfa were used as experimental materials,13 phenotypic traits of alfalfa as the research object,salt-tolerance analysis was conducted,and alfalfa cultivars were sequenced and explored new SNPs locus by high-throughput sequencing technology.The whole genome wide association study of salt tolerance of alfalfa was analyzed by combining phenotype data and molecular data.The results of the study are as follows:?1?Analysis of salt tolerance phenotype of alfalfaTo analyse the variance of 13 identification indicators during the germination,119 alfalfa cultivars were used as experimental materials.All the identified indicators were significant or extremely significant differences between different materials.13 indicators of salt tolerance were distributed under different treatments,All indicators were normal distribution,indicating that the phenotypic data can be used for subsequent GWAS.The correlation of salt tolerance of the 13 identification indexes showed that all the germination traits were significantly positive correlation,and there was a significant positive correlation between the germination traits and the seedling traits.According to the D value of the tested alfalfa germplasm materials,the K-means clustering method was used to cluster analysis of the tested alfalfa germplasm.119 alfalfa germplasm materials could be divided into 5 groups:High salt tolerance?11?,Salt tolerance?26?,Medium?38?,Salt sensitivity?34?and High salt sensitivity?10?.?2?Transcriptome data analysis and marker developmentThe total sequence length of the full-length transcriptome sequencing for three-generation was 148458528bp,and their sequence lengths ranged from 139bp to 6943bp,focused on 1500 to 5500 bp,the number of sequence was 52005,and the length of sequences about 5000 bp was the largest.The longest transcript length is 6943bp,the shortest is 139bp,the median is 2725bp,the N50 length is 3102bp,the average length is 2854bp,and the GC content is 38.15%.After the second generation transcriptome sequencing,a total of 937597transcripts were obtained with a total length of 449080794 bp,of which the longest transcript was 16671 bp in length,the shortest was 201 bp,N50 was 536 bp in length,the median was322 bp,the average length was 478 bp,GC content was 36.74%.The second and third generation sequencing results of the transcriptome were spliced and assembled,and the reference transcriptome of alfalfa was established.The number of transcripts was 989620 and the total length of the sequences was 597539277 bp.The longest transcript length was 16671bp and the shortest was 139 bp.The N50 length was 868 bp,median was 332 bp,average length was 603 bp,GC content was 37.09%.Genome comparison analysis showed that the reference genome match rate was an average of 90.43%and could be used for the next GWAS analysis.SNP markers were used to the correlation analysis of the salt tolerance traits.A total of243 single association sites were detected.Among them,47 and 196 sites were detected in distilled water and salt stress,respectively,and the number of merging sites was 36.The range of the phenotypic variation rate?R²?of these 36 marker locis was 17.671%?Root Fresh Weight loci of Snp_?7728₁470?to 64.986%?Total Dry Weight loci of Snp_?15807₂89?.The most important trait of correlation markers was shoot dry weight?57?,the least trait of correlation markers was shoot high?1?.The most associated traits marked was Snp_?7728₁470?10 traits?,followed by Snp_?21498₁449?9 traits?and Snp_?159874₁44?9 traits??...