Screening Of Salt-Tolerant Germplasm And Cloning Of Salt-Tolerance Related Genes In Brassica Napus L.

As one of the major oil crop in China,rapeseed is crucial for ensuring the country’s oil security.In recent years,the planting area of rapeseed in China’s main production areas has shown a downward trend,seriously affecting the safety of edible oil in China.Therefore,there is an urgent need to increase the planting area of new rapeseed.However,the current problem of soil salinization limits the development of rapeseed industry in China.Studying the salt tolerance of rapeseed can not only help with the effective development and utilization of saline-alkali land,but also ensure the oil supply of China.In this study,the salt tolerance of Brassica napus L.at the bud and seedling stages was identified,and salt-tolerant rapeseed germplasms were screened;molecular markers were developed using the main effect loci obtained from genome-wide association analysis of salt tolerance-related traits by previous researchers,and verified in the above-mentioned identification accessions;salt tolerance related genes were cloned and analyzed.The following research results were obtained:1.Screening of salt-tolerant germplasm in B.napus L.（1）We identified the salt tolerance of 123 rapeseed lines during the bud stage under simulated salt stress with 200 mmol/L and 230 mmol/L Na Cl.The germination rate,bud length,root length and other indicators were measured.Combining principal component analysis,membership function and cluster analysis,we screened 35 salt-tolerant rapeseed germplasms.Among them,there were three highly salt-tolerant germplasms,namely P4,P64and P79;four moderately salt-tolerant germplasms,including P7,P9,P15 and P92;seven generally salt-tolerant germplasms,including P2,P10,P11 and so on;and 21 were lowly salt-tolerant.（2）In order to screen for B.napus L.germplasm that is salt-tolerant during both the germination and seedling stages,10 salt-tolerant materials and 10 salt-sensitive materials screened at the bud stage were identified for salt tolerance at the seedling stage.Using the method of gradient salt concentration treatment,relevant indicators were measured and analyzed,it was found that 240 mmol/L Na Cl was the most suitable concentration for identifying the salt tolerance of the test materials at the seedling stage.Three evaluation indicators were used:relative number of fully expanded green leaves,salt injury index,and comprehensive evaluation value D to evaluate the salt tolerance at the seedling stage.Based on the comprehensive evaluation of different ranking and score systems,P71,P9,P15,P18and P24 were identified as the most salt-tolerant germplasm during the seedling stage.（3）The above 20 accessions were subjected to alkaline salt stress during the bud stage using a 75 mmol/L Na HCO₃solution.Using membership function and cluster analysis methods,10 germination stage alkali-tolerant salt materials such as P4,P7,and P9 were screened.Among them,P9 and P15 showed strong salt tolerance during the germination and seedling stages under neutral salt treatment and during the germination stage under alkaline salt treatment.2.Development of molecular markers related to salt tolerance at bud stageBy analyzing the main SNP sites obtained from GWAS of germination potential,germination rate and relative salt injury index of 146 accessions under salt stress by previous researchers,78 pairs of primers were designed for the development of molecular markers related to salt tolerance at the rapeseed bud stage.Seven pairs of primers showed polymorphism in the extremely salt-tolerant and extremely salt-sensitive backbone accessions identified in this study.Among them,S2＿3＿879 marker can distinguish between extremely salt-tolerant and extremely salt-sensitive materials.3.Cloning and expression analysis of salt tolerance-related genes.The salt-tolerant related genes Bna A02g05440D and Bna A03g50460D were cloned from salt-tolerant line P2 and salt-sensitive line P76 respectively,and bioinformatics analysis and expression analysis were performed on them.The nucleotide sequences of the cloned genes differed in the two accessions,but the amino acid sequences had 100%similarity.The gene Bna A02g05440D encodes 246 amino acids and belongs to the J domain protein.The gene Bna A03g50460D encodes 75 amino acids and belongs to the UPF0057 family.The expression analysis of the two genes in the materials showed that as the salt stress treatment time increased,the Bna A02g05440D gene was significantly down-regulated in both accessions,and the down-regulation amplitude in salt-tolerant material was higher than that in salt-sensitive material.The Bna A03g50460D gene showed fluctuating changes in both accessions,showing a trend of first decreasing,then increasing and then decreasing.This study provides a material basis for molecular breeding of B.napus L.by screening salt tolerant germplasm,developing salt tolerant molecular markers,and cloning salt tolerant related genes.