| Rapeseed is the major source of vegetable oil,and Brassica napus L.（B.napus）is the main cultivating species.B.napus is often affected by drought stress because of high water demanding.At seedling,drought stress could cause stomatal closure,transpiration weakening,photosynthesis decreasing and leaf water content decreasing,and impair the accumulation of organic compounds,result in small seeding with few leaves,and affect rearing strong seedling for overwintering and developing in later stages.In addition,B.napus is an allotetraploid with complex genetic background.Drought-resistance is a comprehensive trait containing multiple quantitative traits associated with drought responding.At present,there has been few reports on identification and function of drought-resistance candidate genes and drought adaption mechanism,and the drought-resistance alleles have been lacked for breeding.In our study,genome wide association analysis（GWAS）was conducted based on traits associated with drought responses and polymorphic SNPs obtained by 60 K Illumina Infinium SNP and resequencing in three environments to identify SNPs significantly related to drought-resistance.For screening candidate intervals,the SNP reported in recently were integrated with this study.Moreover,the phenotype investigation and transcriptome analysis were developed in drought-resistance materials（R01 and R02）and drought sensitive materials（S01 and S02）for identifying the difference in phenotype and gene expression among materials with different drought-resistance.The candidate genes were predicted and analyzed based on GWAS and RNA-seq in R01,R02,S01 and S02.In order to explore drought adaption mechanism in B.napus,the seedlings of Zhongshuang 11 were pre-drought stress and rewatered.The drought tolerance and gene expressions were compared in seedlings with pre-drought and seedlings without pre-drought,and terms and pathways of genes with different expression trends were analyzed.Finally,function verification of candidate genes was carried out using qRT-PCR and constructing over-expression transgenic Arabidopsis.The results are of great significance for revealing the mechanism of drought resistance and adaptation and breeding new varieties with drought-resistance.The main results of this thesis are as follows:1.Effects of drought stress on traits associated with drought responsesIn B.napus,the shoot fresh weight,shoot dry weight,leaf relative water content,root fresh weight and soluble protein content were significantly decreased under drought stress.Drought stress also apparently increased soluble sugar content,malondialdehyde content and peroxidase activity,but the root dry weight did not been changed significantly.2.The phenotype difference of drought-resistance and drought-sensitive materialsDrought-resistance materials（R01 and R02）had stronger water holding capacity and weaker membrane oxidation comparing with drought-sensitive materials（S01 and S02）.Stronger drought stress induced more soluble sugar content and proline content accumulation in S01 and S02.Moreover,the PSⅡ reaction center were damaged with different degrees in four materials.The heat dissipation of drought-sensitive materials was stronger than drought-resistance materials.3.Genome wide association analysisThe linkage disequilibrium,genetic relationship and population structure of 229 B.napus germplasms and 75 core B.napus germplasms were analyzed.The results showed that average attenuation distance of A-sub genome was about 200 kb,and that of C-sub genome was about 500 kb.The attenuation rate of A-sub genome was significantly faster than C-sub genome.The genetic relationship was weak among germplasms.The229 accessions were divided two subgroups（P1 and P2）.Most accessions in P1 were winter and semi-winter varieties or lines from China,and the majors in P2 were spring and semi-winter varieties or lines from Qinghai and Gansu,as well as foreign countries such as Denmark and Canada.The main accessions in 75 core germplasms were winter and semi-winter materials,which were distributed in P1 and P2.A total of 7526 SNPs associated with drought-resistance were identified by GWAS for drought resistance index of each trait and the best linear unbiased prediction of each drought resistance index based on 60K Illumina Infinium SNP chip and resequencing,containing 626 SNPs in 60 K SNP chip and 6900 SNPs in resequencing.Inside,295SNPs were detected repeatedly in our study,13 SNPs were co-located with previous researches.A total of 894 candidate intervals covering 141 Mb chromosomes were obtained by combining the attenuation distance and haplotype analysis in A-sub genome and C-sub genome.There were 133 intervals containing SNPs identified in previous studies.4.Different expression genes analysis in drought-resistance materials and drought-sensitive materialsAt 24 h,36 h and 48 h,there were more than 2000 different expression genes（DEGs）among materials or between each drought time and control.During drought stress,the genes associated with photosynthesis were downregulated,especially drought-sensitive material S01 and drought-resistance material R02.In the terms and pathways of response to oxidative stress,stomatal closure,ABA biosynthesis and peroxisome,the genes were upregulated,especially drought-sensitive materials S01 and S02.The genes contributing to wax biosynthesis were upregulated firstly and downregulated later in drought-resistance R01,and downregulated in other materials,and the downregulated level was largest in S01.In R01,there were not obvious changes in expressions of genes associated with the terms containing flavonoid synthesis,membrane protein localization,amino acid transport,vitamin E biosynthesis and metabolism,inositol phosphate metabolism,chlorophyll catabolism and response to containing oxygen-containing compound,and pathways containing ribosome,phosphate metabolism,glycerol-phosphatidylcholine metabolism,pyruvate metabolism and inositol phosphate metabolism,but they were significantly downregulated or upregulated in other materials.The genes related to stress response,systemic immunity,plant pathogen interaction and MAPK signaling transduction were significantly upregulated at drought stress 36 h in S02 and drought stress 48 h in S01 and R02.5.Identification and analysis of candidate genes associated with drought-resistanceAmong the DEGs between drought stress and control in four materials,6237 genes located in candidate intervals,and enriched in terms of response to oxygen-containing compound and abiotic stimuli and glycometabolism.The KEGG enrichments were conducted for the genes encoding transcription factors and 1793 genes in the above terms.The pathways were enriched including hormone signal,MAPK signal,plant-pathogen interaction,glutathione metabolism and glucose metabolism.The genes participated in auxin,brassinolide,gibberellin,abscisic acid,ethylene,cytokinin and jasmonic acid signal transduction,MAPK signal transduction plant-pathogen interaction,glutathione metabolism and glucose metabolism,expressed different levels among four materials.Among them,the genes related to jasmonic acid signaling transduction and glutathione synthesis and encoding glycoisomerase proteins had the obvious difference between drought-resistance materials and drought-sensitive materials.Among the 6237 candidate genes,1745 were located in the candidate intervals containing SNPs identified repeatedly or reported,including AUX1 and ARF5 in auxin signal pathway,GBF4 in abscisic acid signal pathway,RR1,ARR4 and ARR15 in CTK signal pathway,JAZ6,JAZ2 and JAZ2 in JA signal pathway,GSH1,GPX2,GSH2,ERD9 and GSTU13 in glutathione metabolism pathway,CPK28,CDPK6,CNGC13 and KCS1 in plant-pathogen interaction response and TPS8,BAM1 and VI1 associated with glycometabolism.6.The study results of drought adaptionThe seedlings of B.napus cv.Zhongshuang 11 were pre-drought stress.After rewatering,the seedlings with pre-drought and non-pre-drought were drought stress at the same time.The results of measuring relative water content showed that pre-drought seedlings had stronger resistance than non-pre-drought seedlings.Transcriptome analysis showed that there were more DEGs between pre-drought and non-pre-drought seedlings at drought stress 24 h and 36 h after drought stress,and less DEGs at 48 h.The results of weighted gene co-expression network and expression trend analysis showed that the first genes to respond to drought stress were associated with heat stress protein,jasmonic acid signaling factors Ca2+signaling factors and protein kinase or responding to abiotic stimuli.The genes related to RNA modification,ribosome,amino acid biosynthesis,circadian rhythms,light capture,photosynthesis antennae,oxygen-containing compound and abiotic stimuli were regulated at the middle stage of drought stress.The expressions of genes responding to stress were lower at drought stress in pre-drought seedlings than that in non-pre-drought seedlings,and majors of these genes involved in plant hormone signal transduction,phenylalanine metabolism,glutathione metabolism,arginine and proline metabolism,fatty acid metabolism,MAPK signal transduction,plant pathogen interaction,ribosome,endocytosis and SNARE interaction pathway in vesicle transport.The expressions of genes in chloroplast,photosynthesis,cell wall,cell periphery,Golgi and plasma membrane terms were higher under drought stress in pre-drought seedlings compared with that in non-predrought seedlings,and main participated the pathways containing secondary metabolism,photosynthesis and ribosome.Compared with control,the downregulated genes were enriched in immune response associated terms and pathways,while the upregulated genes involved in flavonoid metabolism,drug transports and light stimulation response associated terms and pathways.7.Preliminary function verification of candidate genesThe expressions genes were verified by RT-PCR,containing drought-resistance candidate genes including BnaC07MYB77 and BnaC02WRKY40 identified by GWAS and transcriptome analysis of drought-resistance and drought-sensitive materials,and the genes with downregulated expressions under rewatering and drought stress in pre-drought and non-predrought seedlings including BnaC03ATAF2,BnaA05WRKY33,BnaC03NAC062-1 and BnaA06b HLH92.The results were consistent with RNA-sequencing.The over-expression vectors of these genes were constructed and transformed wild type Arabidopsis.The over-expression lines of BnaC07MYB77 and BnaA06b HLH92 had weaker drought tolerance compared with wild type.Over-expression BnaC03ATAF2 enhanced drought tolerance in Arabidopsis. |
