The Diversity Of Common Walnut(Juglans Regia Linn.) Germplasm Resources Planted In Xinjiang And Genome-wide Association Analysis Of Phenotypic Traits

As one of the important walnut producing areas in China,Xinjiang not only has the distribution of wild walnut germplasm resources,but also has a long cultivation history of more than 1000 years.Under the dual effects of nature and man-made,it has bred rich walnut germplasm resources.These germplasm resources provide a rich material basis for carrying out breeding work.However,for a long time,there has been a lack of comprehensive and systematic understanding of the existing walnut germplasm resources in Xinjiang.There are homonymous and homonymous phenomena in walnut germplasm.In addition,many(ancient)real walnut germplasms which are adapted to local environment and contain precious and unique genes are facing deforestation,and their distribution area is shrinking.In view of this,the collection and identification of walnut germplasm resources in Xinjiang are of great practical significance for the utilization of excellent walnut germplasm resources.In this study,230 cultivated walnut germplasm resources collected from the main walnut cultivation areas in Xinjiang were used as research objects.Single nucleotide polymorphism loci(SNPs)were developed using sequence-related amplified polymorphic markers(SRAP),simple repeat sequence markers(SSR)and SLAF-seq sequencing techniques.Combining with morphological investigation,the genetic diversity level of the tested walnut germplasm was explored and its legacy revealed.The DNA fingerprints of the tested germplasms were constructed based on the relationship of transmission and variation,and the molecular markers and important candidate genes affecting phenotypic traits of walnut were searched by genome-wide association analysis.The main results were as follows:A total of 230 walnut germplasm resources were collected in the main walnut cultivation areas in Xinjiang.Among them,85 cultivars,67 individual with good quality and 78 ancient trees were collected.The frequency distribution of quality traits ranged from 0.87%to 9.74%.Among them,the distribution frequency of 3 characters,such as the prominence of apical tip-concave,the shape in longitudinal section through suture-triangle,the shape in longitudinal section perpendicular to suture-ellipse,were less than 1%,and the distribution frequency of 2 characters,such as the shape of leaf base-round,the fruit state on the tree-one to two was more than 90%.The degree of variation of 14 quantitative traits varied from 3.99%to 39.85%.Among them,stem length,kernel shell thickness,single fruit weight and kernel weight had higher degree of variation(CV>20%),while transverse diameter,lateral diameter and mean of three diameters had lower degree of variation(CV<10%).There were some differences among phenotypic traits.Among them,the genetic diversity index of quantitative traits(H'=2.92)was high.The genetic diversity index of quality traits(H'=1.46).The tested walnut germplasms were divided into five categories.The results of classification were related to phenotypic traits such as size,shape and core-shell characteristics of nuts,but not to their geographical distribution.A total of 208 distinct bands were amplified from 230 walnut germplasms by 20 pairs of SRAP primers,including 198 polymorphic bands and 94.92%polymorphic band ratio(PPB).The average observed effective allele number(Ne)was 1.62;the average Nei's gene diversity index(H)was 0.36;and the average Shannon's information index(1)was 0.53.A total of 130 distinct bands were amplified by 15 pairs of SSR primers,including 117 polymorphic bands and 88.72%polymorphic band ratio(PPB).The average observed effective allele number(Ne)was 1.53;the average Nei's gene diversity index(H)was 0.31;and the average Shannon's information index(1)was 0.47.There were differences in the analysis of walnut germplasm resources between the two markers.The results of cluster analysis based on two kinds of molecular markers and two kinds of markers are consistent:the distance between Hetian population(HT)and Kashgar population and Aksu population is relatively long.SLAF-seq simplified genome sequencing technology was based on 686.54 Mb reads data of walnut germplasms tested.The average sequencing depth was 13.66 × and 3,308,806 SNP markers were developed.The tested germplasms were divided into eight groups.Except for the cultivated Germplasms in Hotan area in population 8(Cluster 8),the other seven groups were all distributed with germplasms from different sources.The results of PCA analysis,evolutionary tree analysis and population structure analysis were consistent,but there was no significant difference among the tested walnut germplasms.The detection efficiency of SNP markers was higher than that of SRAP and SSR markers.DNA fingerprints of cultivated varieties were constructed by the shape in longitudinal section perpendicular to suture,prominence of apical tip,the shape of apex perpendicular to suture and the shape of base perpendicular to suture,and two SRAP markers M4/E13,M7/E11 and three SSR markers ZMZ035,WJR291 and WJR100.A total of 63 candidate genes were obtained by genome-wide association analysis(GWAS).No five loci with identical expression of traits were detected.An SNP marker was detected at the level of-lg(P)>6.36,which was related to the number of lobules.A total of 18 functional genes were found on chromosome NW₀17388 856.1.Three SNPs were found to be related to the shape of leaf tips and distributed in leaf tip shape,which was stained with NW₀17443 591.1.1.Twenty-one functional genes were found near three SNPs;one marker was found on NW₀17388926.1 chromosome and four functional genes were found near the SNP;one marker was found on NW₀17388850.1 chromosome and three functional genes were found near the SNP;and one SNP marker was also found to be related to the degree of fruit apex protrusion and distributed in NW 017388850.1 chromosome._Seventeen functional genes were found on chromosome 017389324.1 near the SNP.In summary,Xinjiang cultivated walnut germplasm resources have high phenotypic diversity,which have great potential for exploitation and utilization,and are valuable wealth for walnut breeding and germplasm innovation.At the same time,SRAP and SSR markers can be used as an effective tool for walnut germplasm identification.SNP Markers and candidate genes associated with phenotypic traits in Juglans regia can be found by combining simplified genome sequencing techniques.