Combined Genome-wide Association Study And Transcriptome Sequencing Analysis Reveals Snps And Candidate Genes Controlling Salt Tolerance In (Brassica Napus L.)

Soil salinity is a major abiotic stress perceived by plants currently ?800 million hectares are affected by salt worldwide.It significantly restricts plant growth,distribution of crops,and also an important factor that limiting crop yield,and it exerts its undesirable effects via osmotic reticence,disturbing the uptake and translocation of nutritional ions and by ionic toxicity.Brassica napus L.is an economically important crop that provides high oil percent,protein for food,and other industrial purposes.However,rapeseed growth and development are primarily affected by various biotic stresses during its whole life cycle.Salinity among them is a major problem for rapeseed,which seriously affected the yield and quality.Particularly,salt stress is a seriously increasing menace to B.napus at seedling stage alternatively hampers the germination and seedling growth.Therefore,to cope with this issue and improving salinity tolerance in B.napus,it is crucial to understand the complex and dynamic mechanisms in rapeseed saline conditions.Recently evolved genome wide association studies(GWAS)is capable to identify single nucleotide polymorphisms that are associated with the desired traits in a diverse natural population.In this study,a set of 228 diverse association panel of rapeseed natural population was selected on their previously information from the field.These accessions were used for dissecting the genetic mechanism of salt tolerance molecular level on germination and early seedling establishment.Initially,a pilot experiment was conducted to select optimum stress level for GWAS,plant showed maximum variation at 50 m M stress and therefore this stress level along with control(no stress)was chosen for subsequent experiments.Four traits including germination(GR),root length(RL),seed dry weight(SDW)and seed vigor index(SVI)were measured.All these traits showed large variations under both rowing conditions.To unravel the genetic variants in response to salt stress at early seedling growth phase,GWAS was performed using 201,817 high quality SNP markers which previously developed by SLAF-seq by evaluating the fitness and efficiency using both the model GLM)and MLM.The index values retained from SSI and STI on seed germination and seedling traits were used for GWAS analysis.GWAS analysis has identified in a total of 142 SNPs significantly associated with salt tolerance and these markers were positioned at all chromosomes.However,the average phenotype variance explained(PVE %)by each SNPs is 13.11%,with ranging from the 9.664% to 19.474%.Of the total detected SNPs,71 SNPs were detected by both GWAS models(GLM+MLM)combined,45 SNPs were identified by GLM only,and remaining 26 SNPs were detected randomly.In addition,GWAS also showed uneven distribution of these significant SNPs in genome,as proportion of detected SNPs is higher in the C genomes(78/142)than the A genome(64/142 SNPs).Since GWAS was performed on each of indices values separately,a large number of SNPs(89/62.68%)identified by the SSI-based GWAS analysis,and 53 SNPs(37.28%)were detected by the STI-based GWAS analysis.Beside this,no any SNP was common between both these indices.Furthermore,allelic effects of SNPs associated with seedling traits allelic variations revealed that the highest percentage of the favorable alleles than that of the unfavorable effects and identified several salt tolerant lines carrying more desirable alleles.The highest numbers of favorable alleles were for the GR,and the least favorable alleles were for the SVI.Moreover,pleiotropic SNPs also noticed n this study and their allelic effects were mined.These SNPs showed diverse allelic effects;sixteen SNPs in GLM and 9 SNPs in MLM identified whose one allele has a positive effect were identified,and one allele has negative effect for the trait,same alleles also had the same action for the other trait.However,18 SNPs in GLM and 14 SNPs in MLM found as their both alleles are found with favorable effects as these alleles had significantly and positively regulated the associated traits.The SNPs with elite alleles and lines with these alleles are genetically the rich material.The candidate genes significantly associated with salt tolerance were predicted between the tagged significant SNPs.In total 117 candidate genes strongly associated with salt tolerance were found associated with 40 SNPs,and selection of promising genes was disclosed by gene ontology analysis and GO terms related to salt stress(salt,Na Cl,salinity).The proportion of these genes was higher in A genome,as 66 genes are located in the A genome,and 31 are located on the C genome.Further,the BLAST search revealed that 95 genes were found as orthologous with the Arabidopsis genes,encoding various groups such as transcription factors,protein kinase group aquaporins,binding proteins,and growth responsive genes.In addition,transcriptome analysis was performed to identify genes associated with salt tolerance at seedling stage.For transcriptome analysis,most salt tolerant and most salt susceptible lines(from accession panel used in GWAS based on their indices values)were used.A total of 6016 genes were identified in both lines,where 2992 genes were identified in the salt tolerant line and 3024 genes were identified in salt susceptible lines.In the comparison groups with two different cultivars a total of 532 unique up-and down-regulated genes were identified,230 genes were up-regulated,and 302 genes were down-regulated.Further,202 DEGs highly associated with salt based on their GO and 33 DEGs associated with KEGG analysis.The DEGs identified through transcriptomic analysis have strong roles in salt stress.To date,little is known about the genetic mechanisms as well as the natural variation of seedling development in B.napus.Overall,the results obtained in this study have provided new genetic insights into the complex regulation of seed germination and early seedling growth.The identified SNPs and genes through GWAS and transcriptome analysis represent valuable sources,and with further investigations,these markers and genes would be useful for developing markers for marker-assisted selection in a breeding program to improve mustard lines with enhanced salt tolerance.