Analysis Of Lignin Variation Sites And CAD Gene Family In Walnut Endocarp Based On Genome Resequencing

Xinjiang is one of the important walnut producing areas in China and has rich germplasm resources with paper-skinned,thin-shelled and thick-shelled varieties of walnuts.The walnut shell is too thick to affect the kernel yield of walnuts,while the shell is too thin to show incomplete shell development and exposed walnut kernels,affecting the appearance and transportation and storage of walnuts.Genome sequencing technology can uncover information on genetic variation associated with important plant traits and resolve the genetic mechanism of phenotypic differences.In order to investigate the developmental mechanism of walnut endocarp,this study used Xinjiang’s unique thin-shelled and dewy walnut variety ’Xinlu’ as the main target and the thick-shelled walnut variety ’Xincui feng’ as the control for whole-genome resequencing analysis,with the goal of identifying variant loci and candidate genes related to endocarp development.The results show that:(1)In this study,four samples of each of the two varieties of ’Xinlu’ walnut and ’Xincui feng’ walnut were analyzed by genome resequencing,and the sequencing depths were above 20 x.A total of 139.4 G of raw data were generated after sequencing,and after quality control A total of 7.6 million SNP loci were obtained for the two walnuts,with exonic regions accounting for 2.97%,127,043 non-synonymous mutations and 94,740 synonymous mutations;a total of 930,000 In Del loci were obtained,the majority of which were located in intergenic regions,with 585,000(62.72%),8,523(0.91%)In Dels located in coding regions,and 3,810 shifted insertion and deletion sites.(2)Identification of SNP loci between ’Xinlu’ walnut and ’Xincui feng’ walnut based on the frequency of distribution of genotypic differences between populations,26,191,556,510 specific SNP loci were obtained in ’Xinlu’ and ’Xincui feng’ respectively,covering 9,579,12,047 genes.The genes identified were compared with the sequence database and showed high homology with the genes in the NR database.The annotation of the egg NOG database showed that the proportion of secondary metabolite biosynthesis was in the top,and the analysis of the GO and KEGG databases revealed that 39 genes were involved in the formation of plant cell wall,among which 20 genes were involved in the lignin monomer synthesis.The transcriptome expression trends of target candidate genes during endocarp hardening in ’Xinlu’ walnuts were obtained,and different expression patterns of COMT,CAD and GUX were found,which were speculated to have other functions in the endocarp hardening process of walnuts.(3)Eighteen JrCADs were identified in the whole walnut genome using bioinformatics,and the walnut CAD gene family was categorized into three subfamilies.Jr CAD gene family are very conserved in gene structure,motif analysis and conserved structural domain analysis.The cis-acting element prediction results indicate the presence of regulatory elements associated with phytohormone collaboration or resistance in all Jr CAD members.Protein interaction analysis and covariance analysis indicated homology between walnut and Arabidopsis thaliana,and small black walnut,and the existence of an interaction network between the proteins.Transcriptomic data showed that Jr CADs have different expression patterns in the developmental stages of the endocarp of ’Xinlu’ walnuts,with the presence of some highly expressed genes.Combining the q RT-PCR data and the results of phylogenetic tree and evolutionary analysis,the two candidate genes,Jr CAD17 and Jr CAD18 are closely related to lignin accumulation during walnut endocarp hardening.