| Plants encounter a wide range of complex abiotic and biotic stress during the life cycle and drought is a foremost stress which affects the growth and decreases the production of agricultural crops worldwide.Plants are affected by drought at germination and early seedling establishment.However,evaluation of drought stress at the early seedling stage is meaningful because it affects germination and all successive stages which results in low yield.Brassica napus,is one of the economically essential oil crops,have become a significant agricultural species and ranked as third highest source of vegetable oil after soybean and palm oil,and oil mean after soybean and cotton.But it is strictly affected by drought from seed germination to the seed development and seriously curtail its growth,physiology,and productivity of B.napus.Efficient phenotyping and genotyping technique lead the identification of novel marker-trait associations underlying drought.Thus,it is necessary to dissect the genetic variation for drought tolerance,investigate the possible mechanism of the drought tolerance at seedling stage and identify genomic regions responsive to drought stress at early developmental growth stages in Brassica napus.Genome wide association studies at present is a powerful and robust that dissect and provide better understanding of these complex traits mechanism,isolate,characterize and utilization of genes of interest.In the context to aforementioned facts,this study is focused on the dissection of genomic regions regulating drought stress in Brassica napus.The association panel consisting of 228 natural accessions of B.napus was used based on their pedigree,released regions,agronomic performance,importance,and cultivated area.Additionally,the response of genotypes for drought tolerance was evaluated by the calculating two indices: stress tolerance index(STI)and stress susceptibility index(SSI)for all evaluated traits.Further,this study is split into two parts;first part included GWAS analysis with high quality SNP markers and indices values for evaluated traits.In second part,transcript analysis was performed using drought tolerance and drought susceptible lines to identify drought associated genes.In order to get more insights and uncover to represent a wide range of genetic characteristics and morphological diversity of seedling traits of B.napus natural population response to drought tolerance,and to determine whether genetic analysis could facilitate dissection of this complex trait drought.The genome-wide association studies(GWAS)were performed with 201,817 high quality SNP markers discovered through specific locus amplified fragments(SLAFs)separately on each index;STI and SSI values for four seedling traits.The general linear model(GLM)and standard mixed linear model(MLM)were included.Using the larger accessions number and more markers,the combination of highquality SNP markers and indices values,GWAS successfully has identified 314 SNPs with higher resolution on nineteen chromosomes,strongly linked with four seedling traits.All of identified SNPs are new;did not match with previously QTLs.Of the total 314 SNPs,131 SNPs were identified by GLM and MLM combined,and 183 SNPs were detected by GLM model only.These SNP markers were distributed exceptionally in the genome,A genome harbored 108 SNPs and 206 SNPs detected in the C genome.Chromosome C02 harbored largest number of the SNPs(52),A04 harbor only one SNP associated with the RL and SVI.Since the SSI and STI are very distinct to assess the status of drought,our results demonstrate that the proportion of SNPs detected is higher in the SSI(192/313)than the STI(122/313).However,GWAS in this study was performed on each of indices values separately,so according to expectations,no any SNP was detected common between both the indices.Moreover,the allelic effects of these SNPs were attained;the relative percentage of superior alleles was higher in both models of GWAS.The average of superior and inferior alleles was calculated from all possible ways of GWAS(SSI_GLM,SSI_MLM,STI_GLM,STI_MLM)analyses.Though,highest number of marker-trait associations were identified in SSI based GWAS,but surprisingly proportion of favorable alleles were higher in STI based analysis.Overall,highest number of the superior alleles was detected for the RFW followed by SVI and least percentage of superior alleles was in the GR.Thus,future breeding programs on drought may carried out on shoot and root characteristics by involving RFW and SVI.Moreover,several regions of the genome showed pleiotropic associations for drought tolerance.In total,37 SNPs were found to be associated with more than one trait(RL and SVI)mainly distributed on the 13 chromosomes.Present study also identified ten hotspot regions carrying several SNPs in small genomic region.In addition,based on the significant SNPs associated with four traits,candidate genes further were mined.On the basis of GO annotation,102 genes were detected for drought tolerance closely associated with 55 SNPs,distributed on all chromosomes,where 67 genes were on the A genome and 35 genes were distributed on the C genome.In addition,for the confirmation of the function of these genes,sequence of these genes was used for a BLAST search on the reference genome of B.napus and Arabidopsis,85 of these genes are orthologous with Arabidopsis genes likewise NAC genes,PIP2,HB7,CPK1,MYC2,CDPK etc.tightly linked with drought tolerance.The genes identified in this study are fall into various groups such as protein kinase groups,transcription factors(TFs),DNA binding proteins,DNA binding domains,Aquaporins etc.Most of these genes were found very close to the significant SNPs,within 100 kb.It is worth noted that SNPs identified in this study are new,thus alleles and genes found associated with these SNPs could be considered as novel sources for the future drought tolerance breeding programs in Brassica.Furthermore,transcriptome analysis was also performed to identify genes associated with drought stress.Transcriptome study identified 6304 DEGs which have important roles for various biological,cellular and molecular processes.Transcriptome analysis also revealed that more genes were identified in highly susceptible line and more genes were down-regulated than up-regulated ones.Among the total identified DEGs,339 DEGs are closely associated drought stress directly or indirectly.Overall,genes identified through GWAS transcriptome analysis are very important and may serve as novel source while studying the drought tolerance in B.napus. |
