Genotypic Differences Of Waterlogging Tolerance At Germination Stage And Identification Of The Candidate Genes In Brassica Napus

Due to the unique cropping system of “rice-rapeseed” and subtropical climate in the Yangtze-River Valley of China,rapeseed(Brassica napus L.)is frequently subjected to waterlogging stress at germination stage.However,the mechanisms of rapeseed at this stage in response to waterlogging stress are unrevealed.In this study,198 global core accessions of rapeseed and Zhong Shuang11(ZS11),a typical cultivar in China,were treated with 12 h waterlogging stress at germination stage,and four related physiological traits(shoot length,root length,shoot fresh weight,and root fresh weight)were measured after 6 d of recovery culture.The differences of cell ultrastructure and reactive oxygen scavenging system were compared between waterlogging-tolerant and-sensitive genotypes.On the other hand,genome-wide association analysis(GWAS),selective sweep analysis,and transcriptome analysis were used to identify candidate genes responsible for waterlogging tolerance.The main research results are as follows:1.Difference of physiological responses to waterlogging stress among genotypes in Brassica napus at germination stage.By evaluating the waterlogging tolerance of 198 accessions of Brassica napus at germination stage,we found that there were extensive genotype differences in waterlogging tolerance.Among the four tested physiological traits,the relative root length showed the highest correlation with the other three traits,which were seriously affected by waterlogging stress.Therefore,according to the results of the relative root length,three typical waterlogging-sensitive genotypes and three waterlogging-tolerant genotypes were selected for the observation of cell ultrastructure and the comparison of differences in the ROS scavenging system.The results of the observation of cell ultrastructure were showed that the sensitive genotypes had obvious cellular cavity tendency,nuclear condensation or degradation,organelles disappearance,and plasmolysis after waterlogging stress.However,the cell morphology of the waterlogging-tolerant genotype was normal,the nucleus was complete,and the nuclear membrane and nucleolus were visible.For the active oxygen scavenging system,the content of malondialdehyde(MDA)was increased significantly and the activities of superoxide dismutase(SOD)and peroxidase(POD)were decreased in waterlogging sensitive genotypes after waterlogging stress.But,there was no significant change in MDA content,while SOD and POD activities were significantly increased and decreased in the tolerant genotypes,respectively.The above results indicated that there were obvious differences in physiological responses of different genotypes to waterlogging stress at germination stage.2.Identification of candidate genes responsible for waterlogging tolerance in Brassica napus at germination stage by multi-omics methods.In this study,GWAS,selective sweep analysis,and transcriptomic analysis were used to identify candidate genes related to waterlogging tolerance in rapeseed at germination stage.The GWAS analysis identified 6,5,and 2 SNPs linked with relative shoot length,relative shoot fresh weight,and relative root length using 2,235,511 high-quality SNPs within 198 accessions,and then identified 133 candidate genes,including many genes encoding transcription factors.Meanwhile,15 waterlogging-sensitive genotypes and 15 waterlogging-tolerant genotypes were selected as materials for selective sweep analysis,and 1,479 SNPs segments with Fst values greater than 0.5(the first 1% Fst threshold)were identified.Transcriptomic analysis of control and treatment groups after waterlogging stress at germination stage of ZS11 identified 11,341 differentially expressed genes(DEGs).KEGG analysis showed that these DEGs were mainly enriched in “plant hormone signal transduction”,“phenylpropanoid biosynthesis”,and “starch and sucrose metabolism” pathways.Among these genes,we identified 41,19,and 26 DEGs related to root,shoot,and hypocotyl development,including CLAVATA1(CLV1),CORYNE(CRN),CRINKLY4(ACR4),and FASCIATA2(FAS2).Taken together,29 candidate genes were identified by GWAS,selective sweep analysis,and transcriptomic analysis.It is suggested that these candidate genes may play important roles in tolerance to waterlogging stress of rapeseed.This study not only enriches the physiological mechanism of waterlogging stress at germination stage but also provides gene resources for genetic improvement of waterlogging tolerance in Brassica napus in the future.