Evaluation Of Alkali Tolerance Of 130 Alfalfa Resources And Transcriptome Analysis Of Alkali-tolerant Alfalfa

There is a large area of saline-alkali land in the world,and the area of salinization is increasing year by year.Soil salinization is an important ecological and agricultural problem,which seriously inhibits plant growth and limits crop yield and quality.Alfalfa is a perennial legume forage with strong saline-alkali tolerance.Planting alfalfa can significantly reduce the p H of saline-alkali land.With the growth of alfalfa,it can increase vegetation coverage,thereby reducing the evaporation of water in the soil and reducing the rate of salt accumulation in the soil.The salt in the soil can also be used by alfalfa growth,thereby reducing the salt content in the soil and achieving the effect of reducing salt and alkali in the soil.Therefore,cultivating alkali-tolerant alfalfa varieties is an important measure to improve saline-alkali landThe purpose of this study is to evaluate the alkali tolerance of alfalfa resources by measuring the response of seed germination and seedling physiological index under alkali stress,and to use high-throughput sequencing technology to mine the key genes of alkali-tolerant alfalfa.This study is divided into three parts:alkali tolerance evaluation at germination stage,physiological index determination at seedling stage and transcriptome analysis.The main findings are as follows:1.The average relative germination rates of 130 alfalfa species under Na₂CO₃and Na HCO₃stress were 68.54%and 79.17%,respectively.The relative germination potential was 57.3%and 65.78%,respectively.The relative root length was 23.7%and17.61%,respectively.The relative bud length was 53.21%and 46.37%,respectively.The membership function analysis showed that the seeds of LJ112,LJ73,LJ66,LJ33,5010,DARA,Nongjing No.1 and Juneng salt-tolerant varieties had the best germination trend under two alkali stresses.Six alfalfa varieties,LJ33,LJ57,LJ112,alfalfa grade 2,Xinjiang Daye and Nongjing 1,had the best root and shoot growth under two alkali stresses.By calculating the comprehensive membership function values of 130 alfalfa resources under two alkali stresses,it can be seen that Nongjing 1 and LJ33 had the best germination and growth under two alkali stresses.2.Through the determination and analysis of physiological indexes of alkali-sensitive variety Canadian alfalfa and alkali-tolerant variety Nongjing No.1 under two kinds of alkali stress,it was concluded that under two kinds of alkali stress,the superoxide dismutase,peroxidase,catalase,malondialdehyde and soluble sugar of the two alfalfa increased,and the increase was greater under Na₂CO₃stress.The increase of malondialdehyde content in alfalfa leaves reflected the intensity of alkali stress inhibition on the growth and development of alfalfa,and the parameters such as proline gradually increased to improve the adaptability to alkali stress.Compared with the two alfalfa varieties,the physiological parameters of Canadian alfalfa increased less than Nongjing No.1.3.The alkali-tolerant variety Nongjing No.1 was selected for transcriptome analysis,and the following results were obtained.（1）The number of Raw Reads obtained by assembly was 4.0×10⁷to 5.8×10⁷,the average GC content was 43.17%,and the Q30（%）was greater than 92%.Comparative analysis of gene expression levels between different samples.It was found that there were differences in the expression of genes in the three treatments.A total of 16,695genes were co-expressed in all samples,and only 589,588 and 538 genes were expressed in the control group,Na₂CO₃treatment group and Na HCO₃treatment group,respectively.（2）The number of differentially expressed genes in the CK vs Na₂CO₃comparison group was 635,of which 400 genes were up-regulated and 235 genes were down-regulated.There were 739 differentially expressed genes in the CK vs Na HCO₃group,including 379 up-regulated genes and 360 down-regulated genes.For the differentially expressed genes,compared with the control group,the expression levels of 190 and 235 genes were changed after Na₂CO₃and Na HCO₃treatment,and the up-regulated expressions were 58 and 109,respectively.These differentially expressed genes may be involved in the adaptive regulation of alfalfa under alkaline environment.（3）GO functional annotation of differentially expressed genes related to alkali stress:Compared with the control,DEGs after Na₂CO₃treatment were mainly annotated in the redox process,response to biological stimulation,ribosome,extracellular region,oxidoreductase,heme binding and iron ion binding.DEGs after Na HCO₃treatment were mainly annotated in translation,polysaccharide metabolism,ribosome,cell wall,ribosome structure,transferase activity,transfer of hexose and iron ion binding.（4）KEGG annotation of differentialy expressed genes related to alkali stress:The first five metabolic and signal transduction pathways of DEGs enrichment after Na₂CO₃treatment were ribosome,plant MAPK signaling pathway,glycerophospholipid metabolism,cell senescence,and carbon fixation in photosynthetic organisms;after Na HCO₃treatment,the top five metabolic and signal transduction pathways of DEGs enrichment were ribosome,photosynthesis,pyrimidine metabolism,carbon fixation in photosynthetic organisms,and glycerophospholipid metabolism.