Genome-wide Association Study Of Common Walnut(Juglans Regia L.) Fruit Traits

Persian walnut,also known as common walnut（Juglans regia L.）,is an important perennial deciduous tree with good ecological adaptability and is widely distributed in Eurasia.Due to the high nutritional value of walnut fruit,the good quality of walnut wood,the medicinal properties of the husk and the artistry of some specific varieties of fruit,it is widely cultivated and planted around the world,and the global walnut production is in increasing year by year.So,walnuts have extremely high economic value.The diversification of walnut fruit shape is an important indicator to determine its yield and quality.In the early stage of this study,a lot of researches on walnut morphology,pedigree geography,population genetics and molecular marker opening were carried out.With the rapid development of sequencing technology and genomics,the basic theoretical research and practice of walnut molecular breeding have gradually been executed.However,these studies on the development of new molecular markers for walnut breeding using high-quality reference genomes combined with fruit phenotypes for genome-wide trait associations are rarely reported.As a result,the number of molecular markers related to walnut fruit traits is low,which affects the progress of walnut breeding.In this study,Persian walnut was identified as the research object,and the whole genome of 101 walnut individuals was re-sequenced by high-throughput next-generation resequencing technology.At the same time,10 fruit phenotypes of these samples were measured and analyzed.The high-quality chromosome-level genome was used as the reference sequence,and single-nucleotide polymorphism（SNP）,linkage disequilibrium,population structure,PCA and other Analytical methods were performed.Combined with genome-wide association analysis（GWAS）methods and basic biological experiments,we finished this study The correlation analysis of the phenotype of the Persian walnut fruits was carried out,and then the candidate SNPs loci and genes were associated with the phenotypes.The main findings are as follows:（1）.The results of the phenotypic data analysis showed that the correlation analysis of the phenotypic data showed that the fruit weight was significantly positively correlated with the transverse diameter,longitudinal diameter,and lateral diameter of the fruit,while the shell thickness was significantly negatively correlated with the kernel rate.In addition,shell thickness was significantly negatively correlated with protein content,and protein and fat content were also negatively correlated.（2）Population genetic structure analysis showed that 101 Persian walnut individuals were significantly divided into two subgroups in terms of genetic structure.Phylogenetic tree results confirmed that all samples consisted of two main subgroups.The top 10 principal component analysis（PCA）results showed that all individuals were clearly divided into two main groups,with principal components PC1 and PC2 providing 10.4%explainability.The STRUCTURE results showed that the optimal K value of the population genetic structure of 101 Persian walnut individuals was 2.（3）Population genetic diversity analysis showed that the population nucleotide diversityπvalues differed significantly between the two subpopulations,and secondly,the population genetic differentiation coefficient F_ST results indicated that the degree of population differentiation differed significantly between the two subpopulations.the Tjima’D neutrality test also supported these results.（4）The results of the GWAS analysis showed that after annotating the association analysis results for the 10 phenotypes,we selected 54 SNPs loci and candidate genes that were significantly associated with the phenotypes.Also,for our focus on the shell thickness phenotype,we linked to the homolog of the gene PXC1,which regulates secondary cell wall cellulose thickening genes in Arabidopsis thaliana.Biological results of transcriptome and q RT-PCR experiments showed that the PXC1 gene was significantly expressed in walnut leaves,exocarp,endocarp and flowers,and that its expression had different expression patterns depending on the period of fruit development.In conclusion,based on the analysis of population structure,population differentiation degree,and the correlation between nuclear SNPs and phenotypes,this study proves that GWAS is a more scientific and accurate effective method for screening phenotype-related genes.The results of the correlation analysis between phenotypes provide some theoretical explanation for the interactions found in the diversity of walnut fruit traits.Finally,analysis of the shell thickness phenotype showed that changes in the expression of the PXC1 gene during the developmental stages of walnut fruit demonstrate the possible involvement of this gene in regulating changes in the cellulose content of walnut shells.Meanwhile,the remaining candidate genes related to walnut fruit traits identified in this study provide a theoretical basis and data support for the study of walnut fruit traits and the development of walnut breeding technology.The results of this study will help to reduce the time-consuming development of walnut fruit breeding and provide prospective reference material for the expansion of walnut germplasm resources and the walnut industry.