Genotypic Differences In Response To Drought Stress At Seedling Stage And Identification Of The Genes Responsible For The Tolerance In Brassica Napus

Drought is a major abiotic stress in the world,which severely restricts the yield of crops and the sustainable development of agriculture.Brassica napus is one of the most important worldwide oil crops,and also the most important oil crop in China.Therefore,revealing the physiological and molecular mechanisms of drought tolerance in Brassica napus,and digging out candidate genes,are important to generate drought-tolerant cultivars and guarantee the vegetable oil supply.At present,several genes related to drought tolerance have been reported in rapeseed,and more genes can be further identified through omics analysis.In this research,300 core accessions of rapeseed were used to explore the physiological and genetic mechanism of drought tolerance through omics analysis,and to identify candidate genes related to drought tolerance as well as leaf stomatal density.The main results obtained are as follows:1.Genotypic differences of drought tolerance and leaf stomatal density in core accessions of rapeseed at seedling stage.In this study,300 core collections of Brassica napus were used.The drought tolerance of all accessions of rapeseed under drought stress was analyzed using the aboveground water content as the index,and the phenotypic data of leaf stomatal density were collected at seedling stage under normal culture condition.The results indicated that there were a wide range of genotypic divergences of two traits in 300 core collections.Furthermore,there was no significant correlation between the phenotype of rapeseed leaf stomatal density and drought tolerance.In summary,it suggests that drought stress can affect leaf stomatal density by narrowing the size of a plant leaf.However,leaf stomatal density may have no significant impact on drought tolerance of plants,and the two traits are regulated by diverse genetic mechanisms.2.Genetic loci of drought tolerance and stomatal density in rapeseed at seedling stage.In this study,we performed genome-wide association analysis to identify candidate genes of drought tolerance and stomatal density in rapeseed core germplasm.The results showed that 18 SNP loci were identified and 189 candidate genes were obtained from drought tolerance association analysis,including SnRK gene family members.On the other hand,4 significant chromosomal regions were detected for the leaf stomatal density by association analysis,and 71 genes were selected as candidate genes.The significant loci of two traits were divergent,which suggested that the two triats have different genetic regulation pathways.3.Selective sweep analysis between drought tolerant and sensitive genotypes of rapeseed.According to their drought tolerance,we selected 8 drought-tolerant genotypes and 8 drought-sensitive genotypes of rapeseed for selective sweep analysis.The verificationof the drought phenotype showed that the drought tolerance of two groups was stable and markedly different.The selective sweep analysis revealed significant genetic differences between the two groups.There were 33 selected regions including 7111 annotated genes.We performed a crossover analysis between the 7111 selective sweep genes and 189 associated candidate genes,and found 24 overlapped genes including NAC transcription factor,F-box protein-coding gene and CCCH zinc-finger transcription factor.4.Divergence in transcriptome responses to drought stress in rapeseed at seedling stage.A total of 16 genotypes(as described above)were used to conduct transcriptome sequencing analysis under drought stress and normal conditions.Through the comparison of transcriptome data under treatment and normal conditions,1529(855up-regulated,674 down-regulated)and 14117 differentially expressed genes were identified in drought-tolerant genotypes and drought-sensitive genotypes,respectively.Combining the results of selective sweep and transcriptome analysis,142 genes were detected including BnSnRK genes BnaC01g11140 D and BnaC09g48250 D.5.Identification of BnSnRK gene family and functional validation of BnSnRK3.39.According to the above results,we found the SnRK gene family is involved in drought response of rapeseed.Therefore,we conducted genome-wide identification of BnSnRK gene family and performed BnSnRK3.39 functional validation in Arabidopsis.A total of 114 BnSnRK genes were identified and classified into three subfamilies based on gene structural analysis and the domain types.Moreover,the expression patterns of BnSnRKs in various tissues and under diverse abiotic stresses were distinctly different.Furthermore,we selected the BnSnRK3.39 gene for heterologous functional verification in Arabidopsis.This result showed that the expression of BnSnRK3.39 can enhance the drought tolerance of Arabidopsis.In this research,the physiological and molecular characterization of drought tolerance were systematically analyzed using core accessions of rapeseed by physiological,omics,and molecular biology methods.The results are useful for identification of the candidate genes for drought tolerance and the foundation for the cultivation of new drought-tolerant rape varieties.