Genome-wide Association Analysis Of Leaf Cuticular Wax Traits In Brassica Napus L.

Brassica napus L.is one of the most important oil crops in the world.It has been developed into a multi-purpose crop for oil,energy,vegetable,forage,green manure,honey source and flower.Plant stratum corneum wax plays an important role in plant antibiotic and abiotic stress,and is the first barrier for plants to resist external environmental stress.The research on wax metabolism,transportation and related regulation network of canola stratum corneum will be of great significance to regulate the stratum corneum through breeding and cultivation methods to improve the resistance of rapeseed.In this study,the leaf wax components of 193 Brassica napus materials from all over the world and planted in Chongqing were quantitatively analyzed.Based on the analysis of 34 phenotypic traits for two years.The 60K SNP chip was used for genotyping.Identification of SNP loci associated with wax traits was performed using genome-wide association analysis for screening candidate genes.The results will be used to further explore the important wax related genes of rapeseed and accelerate the breeding process of rapeseed.The main findings are as follows:1.Analysis of phenotypic variation of leaf wax traitsIn this study,GC-MS was used to identify the leaf wax traits of natural populations consisting of 193 Brassica napus inbred lines.The results showed that the wax layer of the stratum corneum in the associated population consisted mainly of long wax fatty acids,aldehydes,alkanes,secondary alcohols,ketone(C₂₉),primary alcohols and esters.Contains 24 waxy compounds.We statistically analyzed 34 wax traits of these 24 wax compounds,each wax content,29 carbon homologues,Alkane pathway wax content,1-primary alcohol pathway content and total waxes.The results showed that most traits were significantly different between environment,material,environment and material interaction.Most wax traits in natural populations exhibit extensive genetic variation.Among them,the total variation of wax content in 2016 and 2017 was 11.65-73.63μg cm^-2and 2.64-4.01μg cm^-2,respectively,and the coefficient of variation was 29.12%and 28.91%;the variation of the main waxy compound C₂₉9 alkane The amplitudes were5.82-35.28μg cm^-2and 0.72-40.77μg cm^-2,and the coefficient of variation was 31.75%and 33.90%;the variation of C₂₉9 secondary alcohol was 1.40-8.68μg cm^-2and0.21-3.42μg cm^-2,the coefficient of variation was 38.72%and 37.97%;the variation range of C₂₉9 ketone was 2.57-16.63μg cm^-2and 0.14-7.84μg cm^-2,respectively,and the coefficient of variation was 31.28%-34.74%.The coefficient of variation differs from the environmental traits in a single environment.The results of generalized heritability analysis showed that waxiness,wax content,alkane content,and wax traits such as C₂₉ketone showed higher heritability（H²>0.7）.2.Analysis of group structure and kinshipAccording to the distribution of SNPs on 19 chromosomes in Brassica napus genome,4623 SNP markers（MAF>0.2）were selected,and the genetic structure of 193Brassica napus germplasm resources was analyzed by SRUCTURE 2.3.4.The analysis results show that the research materials are divided into four subgroups,in which 68%of the relationship between the materials within the population is less than 0.05,and the55%kinship is 0,indicating that there is no obvious phylogenetic relationship among the 193 materials.The LD attenuation distance analysis of the A and C genomes of Brassica napus L.showed that the LD values of the A and C genomes decreased with the increase of the physical distance,but the attenuation degree was different.The A genome had a faster decay rate than the C genome.When the threshold of r²is set to 0.1,the average attenuation distances of the A and C genomes are approximately 500 Kb and 1000 Kb.3.Genome-wide association analysis of wax traitsUsing the Brassica 60K SNP array,we performed a genome-wide association study of oil and protein contents in a population of 193 accessions.Using the model of naive,PCA,Q based on general linear model（GLM）and K,P+K,Q+K based on mixed linear model（MLM）.Correlation analysis was performed on the BLUP data of wax content.Among them,P+K or Q+K were selected as the best model for each trait.With P<1/31846 as the threshold,we identified 277 SNPs loci were significantly associated with of the 34 wax traits.The marker is also significantly associated with three or more traits.The total wax content screened 3 significant SNPs loci in the Q+K model,explaining 12.7%-13.9%of the phenotypic variation;C₂₉9 alkane screened 7 significant SNPs loci in the Q+K model.Explaining 11.0%-13.6%of phenotypic variation,C₂₉alcohol-2 and C₂₉9 ketone screened 1 significant SNP locus in the P+K model,respectively,explaining 24.4%,12.0%of phenotypic variation;The wax content screened 3 significant SNPs loci in the Q+K model,explaining 17.2%-21.4%of the phenotypic variation;the primary alcohol pathway screened 1 significant SNP loci in the Q+K model.Explaining 12.2%-15.8%of phenotypic variation.4.Candidate gene predictionUsing the genomic annotation information of Brassica napus L.,145 candidate genes related to waxy traits were obtained by analyzing the interval sequences of LD regions of significant SNP loci and Brassica napus.These candidate genes may play a role in regulation,involvement in wax biosynthesis,wax transport,and cuticle development.Among them,there are 90 candidate genes related to cuticular wax biosynthesis;22 candidate genes related to the cuticular wax transportation;31 genes related to the regulation of cuticular wax and cuticle development.Some of these genes are homologous to the reported genes such as VLCFAa biosynthetic gene KCS,KCR,HCD/PAS2 and ECR,alkane related gene CER3 and CB5-B,secondary alcohol and ketone related gene MAH1,wax transporter gene ABCG11 and LTPG1,and wax regulation gene Myb96,Myb30,Myb16,DEWAX and SHIN1/WIN1.