Genetic Analysis Of Salt Tolerance And Functional Verification Of Candidate Genes In Brassica Napus

As the global population continues to increase,the contradiction between food shortage and increasing population has become increasingly prominent.However,soil salinization has severely restricted the normal growth and development of crops,thereby posing a serious threat to the world’s food supply.Brassica napus has important economic value of oil use,animal feeding to use,vegetable use and tourism.Therefore,it is of great significance for the genetic improvement and molecular mechanism analysis of salt tolerance in Brassica napus.From the perspective of forward genetics and artificially simulated salt stress conditions,this study carried out germination,seedling hydroponic and full-growing phenotypic platform pot experiments on 505 Brassica napus germplasm resources.In addition,the phenotypic platform pot experiment of the whole growth period was performed on 91 intervarietal substitution lines(ISLs)by artificially simulating 9 treatment conditions such as salt and alkali stress conditions.Then,through genome-wide association studies(GWAS)and linkage analysis,the genetic analysis of the complex traits of the two Brassica napus mapping populations under salt and alkali stress conditions were carried out.Through the functional verification of the candidate genes,there are many advantages and effectiveness of the combination of GWAS,linkage analysis and phenotyping platform in the evaluation of resistance to salt and alkali stress,screening of extreme germplasm resources and mining of candidate genes in Brassica napus.We totally collected 4 germination indicators and 10 growth and physiological indicators of 505 germplasm resources during the germination and hydroponic seedling stages,such as germination,germination rate,stem length and root length at germination stage,and plant height,root length,leaf area,ground dry weight,root weight,relative conductivity,malondialdehyde,chlorophyll content and proline content at seedling stage under artificially simulated salt stress conditions.It was found that the same traits were generally correlated under normal and salt stress treatment conditions.The correlation coefficient between the germination indexes and the seedling stage indexes was not significant.A total of 5,282 significantly associated SNPs were identified through genome-wide association studies(GWAS),of which 422 SNPs were co-localized by two or more indicators.Combining the differential expression analysis,sequence variation and comprehensive scoring of candidate genes under salt stress conditions,177 and 228 candidate genes related to salt stress were identified in the germination and the hydroponic seedling stages,respectively.Among them,many genes,which have been reported to be associated with salt stress response were also identified,including genes such as Bna A03g43130D(Bn RAB),Bna A04g15550D(Bn ABI1),Bna A05g01580D(Bn ABA1),Bna A07g12170D(Bn ABA1),Bna A10g29660D(Bn SOS3)and Bna C07g25850D(Bn ABI5).In addition,some genes,which are co-localized with the candidate genes in previous studies have been identified,including Bna A01g02240D(Bn MYB32),Bna A01g12890D(Bn OSM34),Bna A01g02100D(Bn TMT2),Bna A05g03980D(Bn BGLU27),Bna A05g05230D(Bn HH2),Bna A05g04990D(Bn SAP18),Bna A07g22240D(Bn MYB95),Bna A07g22790D(Bn TIP3;1)and other genes.In addition,through the high-throughput phenotyping platform,a total of 2,111 traits were collected from 505 Brassica napus accessions at 5 different time points and artificially simulated lowsalt and high-salt stress conditions,including 54 side-view indicators and 17 top-view indicators and2,040 hyperspectral indicators.Through a series of strict screening criteria such as high reproducibility,heritability and treatment effect,a total of 928 high-quality traits closely related to salt stress were identified under different periods and treatment conditions.Then,GWAS was performed on 2,245 salt stress coefficients,and 234 candidate genes related to salt stress were finally identified through strict candidate gene scoring standards.In addition,in order to make up for the false-positive defect of GWAS,a total of 71 visible indicators,including 54 side-view indicators and 17 top-view indicators,were collected from 91 ISLs at 5 time points by artificially simulating salt and alkali potting conditions and using the high-throughput phenotyping platform.Then,2,132 tolerance indexes were performed by linkage analysis,and a total of 204 high-frequency(≥ 10)co-localized QTLs were identified.Some of the identified candidate genes have been reported to be related to salt stress response,such as Sn RK2,RD20,SOS3,ABI5 and WRKY33.Furthermore,two genes,Bn CKX5 and Bn ERF3,which have not been reported to be involved in salt stress response,were verified by transgene function.Bn CKX5 contains a FAD/cytokinin conserved domain,which encodes a cytokinin oxidase/dehydrogenase to regulate the homeostasis of cytokinin.Bn ERF3 encodes a transcription factor of AP2/ERF3 family to participate in multiple abiotic stresses.Through artificial simulation of salt stress and mannitol stress conditions during the germination period,it was finally found that the germination,germination rate,stem length and root length of Bn CKX5 and Bn ERF3 overexpression materials were inhibited compared with the wild type.Interestingly,it was found that the germination potential of Bn ERF3 strains overexpressing Bn ERF3 was inhibited under ABA stress conditions.However,when the cotyledons turn green,the rate of green cotyledons increases significantly under ABA stress.At the same time,from the three verification methods of hydroponics,soil culture and phenotyping platform,it was found that compared with the wild type,the overexpression of Bn CKX5 increased the sensitivity to salt stress at the seedling stage,while the overexpression of Bn ERF3 increased the resistance to salt stress at the seedling stage.In summary,we made full use of GWAS and linkage analysis to analyze 505 accessions in an associated populations and 91 ISLs of Brassica napus at the germination stage,seedling stage,and full growth period of phenotypic platform,respectively,under artificially simulated salt and alkali stress conditions.The following complex agronomic traits are analyzed.We identified a number of complex traits and extreme varieties related to salt stress response,and at the same time,we also unearthed candidate genes related to salt stress and performed functional verification.This research provides an important germplasm resource and theory for the genetic improvement of salt tolerance in Brassica napus.