Evaluation Of Salt Tolerance And Transcriptome Analysis Of Alfalfa In The Germination Stage

There are large areas of saline land and increased year by year in the world.Soil salinization is an important abiotic stress factors that limit the growth of crop.Medicago sativa.is one of the major leguminous forages with certain ability of soil salinity tolerance.To obtain the transcriptome analysis of germination stage under salt tolerance in alfalfa,we evaluate the salt tolerance varieties and use the transcriptome sequencing technology.The transcriptome analysis of germination stage provided novel insights into the salt stress tolerance response mechanisms in alfalfa.This study includes the evaluation of salt tolerance and the transcriptome analysis in germination stage.The study of the evaluation of salt tolerance contains screening for suitable salt concentration and salt-tolerant varieties(lines),stress relief experiment.The transcriptome analysis is RNA sequencing with reference genome.1.Evaluation of salt tolerance in germination stage:(1)Screening for suitable salt concentration.0?100?150?200?250?300 mM NaCl solution were used to treat Algonquin.When the concentration of NaCl is 150 mM,the germination rate was 29%,52% less than the control treatment.When the concentration of NaCl was 200,250,300 mM,the germination rate was 12%,9% and 2% respectively.It indicated that the germination is seriously inhibited.So 150 mM was selected for the subsequent experiment.(2)Evaluation of salt tolerance.To select the varieties for transcriptome analysis,we screen 40 local varieties and 40 foreign varieties under 150 mM NaCl solution.The performance of salt resistant in most characters showed very significant positive correlation in the correlation analysis,it illustrated that the stress treatment played a inhibited role in plant.According to the germination rate of salt tolerance,80 varieties were divided into 5 levels,high tolerance(accounted for 17.5%),tolerance(accounted for 35.0%),middle tolerance(accounted for 30.0%),Susceptibility(accounted for 15.0%),High susceptibility(accounted for 2.5%).Salt-tolerant varieties Wudi alfalfa and salt sensitive varieties Golden empress were selected for the following experiment.(3)Stress relief experiment.The seeds of Wudi alfalfa and Golden empress with no germination were used to do the stress relief exeperiment after 5-days treatment under 150 mM NaCl solution.The germination rate was not affected when the seed in the contrast treatment transfered into the stress treatment.After stress relief,the germination rate of Wudi alfalfa was20% increased while Golden empresswas 6%.The germination was not affected when the already germinated seeds transfered into the sodium chloride solution.It indicated that the early germination is an important node for seed germination.2.Transcriptome analysis: Wudi alfalfa and Golden empress were chosen for re-water and transcriptome analysis.Correlation analysis showed that most of the salt tolerance performance of all traits had a very significant positive correlation.(1)Summary the quality of sequencing data.A total of 87.062 G raw data and 86.513 G clean data were received from 12 cDNA libraries,145713211 reads in wudi alfalfa and 142662828 reads in golden empress.(2)Analysis of sequence alignment.HISAT was used to align sequences to genome of Medicago truncatula.The compare rate of golden empress is 47.73% and wudi alfalfa is 47.67% under water treat while golden empress is 47.37% and wudi alfalfa is 47.37% under salt treatment.(3)Analysis of differentially expressed genes.Using StringTie and Ballgown,25 DEGs were selected in genes and 42 in transcripts.Using DESeq,583 DEGs were selected in wudi alfalfa and 287 were selected in golden empress under salt stress.(4)GO annotation of differentially expressed genes under salt stress.Function analysis of 100 DEGs related to salt stress by GO annotation.The most representative section in the biological process is metabolic process,cellar process,single-organism process,biological regulation and response to stimulus.The most representative section in the molecular function is catalytic activity,binding,transporter activity,molecular function regulator and nucleic acid binding.The most representative section in the cellular component are membrane,cell part,cell,membrane part and organelle.