Genetic Characteristics Of Tibetan Walnut Seedling Population And Genome-wide Association Analysis Of Four Traits

China is one of the origins of walnut（Juglans regia L.）,and Tibet,as an important production area of walnut in China,has rich germplasm resources and is an important genetic resource pool for breeding work.However,with the continuous development of intensive cultivation mode and the increasing trend of local walnut cultivate variety,some old germplasm resources have been variety grafted,and the area of forest land has been gradually reduced,leading to a certain impact on the genetic diversity of local walnut germplasm resources.In this context,it is particularly important to investigate the germplasm resources of walnuts in Tibet and to formulate appropriate conservation strategies.Walnut is an anemophily monoecious plant with different blooming stage and dichogamy.This genetic characteristic can effectively avoid the problems of inbreeding depression,pollen discounting and population diversity reduction,but in production,it also leads to low fruit-setting rate of walnuts,which affects the improvement of economic efficiency.Therefore,using the rich genetic diversity of walnut germplasm in Tibet to locate and explore the main effect genes regulating the dichogamy traits of walnut can provide a reference basis for targeted breeding work.Walnut germplasm in Tibet are mainly distributed in the Brahmaputra River basin,with the most concentrated distribution in Shannan and Nyingchi.In this study,we used the representative seeding landrace walnuts introduced from Gyaca County in Shannan Region,Lang County and Mainling County in Nyingchi Region to provide a basis for the conservation and utilization of walnut germplasm resources in Tibet,and to locate the key genes regulating dichogamy in walnut using genome-wide association analysis.The main results from this study are as follows.1.The germination stage,leaf expansion stage,blooming stage and the dichogamy traits were investigated in Tibetan seeding landrace walnuts populations.Phenotypic data were analyzed in three different geographical groups and clustering trees were constructed.The dichogamy traits were found to be relatively stable in 2019 and 2021,with no difference between the two years of data,but the germination stage,leaf expansion stage and blooming stage were slightly delayed in 2021 compared to 2019,with the largest coefficient of variation in germination stage in 2019.The differences in the proportions of protogynous and protogynous among the three geographical groups were small,and the clustering results based on phenotypes revealed no significant correlation between the proportions of protandrous and protogynous phenotypes and the geographical distribution of single plants.2.A total of 96 Tibetan walnut materials from Gyaca,Mainling and Lang counties in Tibet were subjected to simplified genome sequencing,and 229.93 Gb of raw sequencing data were obtained.The sequencing results after quality control were compared with the reference genome to detect variant sites and it was found that the average sequencing depth of the filtered samples reached 16.9.In the reads of paired-end sequencing results,the proportion of successful matched on the reference genome distribution range from 73.0%to 92.5%,with an average of 84.9%,which indicating the high quality of the sequencing data.A total of 181,487 population SNPs and 91,327 In Dels（61,629 INS variants and29,698 DEL variants）were detected,with an average of 529 variants per 1 Mb of genome,and these sites had high support,with an average of 16.75 reads per sites covered,and the mean square value（MQ）of the covered sequences was 58.6.This indicates that the quality of the detected variant sites is high.Among all the variant sites,variants located upstream and downstream of the genes accounted for the largest proportion,together accounting for53.170%of the overall variation.There were also more（23.786%）variant sites in the intergenic region.There were 3.992%of the variant sites in the exon region and 7.367%of the variant sites in the intron region.3.The population genetic structure of 96 Tibetan walnut materials was analyzed,and the optimal population subpopulation number K=3.Subpopulation 1 contained 56 plants,subpopulation 2 contained 33 plants,and subpopulation 3 contained 7 plants.Subpopulation 1 had an extensive exchange of genetic material with the other two populations,and 98.2%of the plants in this population shared some degree of genetic background with the other two subpopulations.Subpopulation 2 also had 78.8%of plants with genetic backgrounds from the other two subpopulations.These three subgroups also showed obvious subgroup characteristics in the principal component analysis,where PC1well separated subgroup 3 and PC2 could well reveal the distribution characteristics of subgroup 1 and subgroup 2.The results of kinship analysis and phylogenetic tree construction were consistent with the principal component analysis and group structure analysis.The mean value of the neutral test（Tajima’s D）of the population was 2.66,and the overall range of variation was all greater than 0,indicating that this Tibetan walnut population had obvious group selection.The interpopulation fixed index（Fst）of subpopulation 3 was larger than those of subpopulation 1 and subpopulation 2,0.09 and0.106,respectively,indicating that subpopulation 3 was moderately genetically differentiated from the other two subpopulations and more differentiated from subpopulation 2.The nucleotide polymorphisms（π）of the three subpopulations were1.3×10^-4,1.23×10^-4,and 1.21×10^-4.It indicates that all of which were low,and this was generally accompanied by selection.Combined with the results of the neutral test,it indicates that there is a tendency for the genetic background of Tibetan walnut germplasm resources to be gradually homogeneous.4.The LD decay analysis of walnut population variant sites revealed that when r²decayed to half of the maximum value,the LD decay distance of the population was 1.0 kb.The data of germination stage,leaf expansion stage,blooming stage and dichogamy traits in 2019 and 2021 were correlated with the variant sites detection data.It was found that dichogamy traits had 25 significantly associated sites,concentrated at the end of chromosome 7.Based on the LD decay distance and the location of the first and last variant sites,115 candidate genes were found to be affected by significantly associated variant sites in the candidate region.The GO annotation showed that the genes in this region are related to synthesis and metabolism of sugar,plant signal transduction,cellular major component formation,and cell cycle alteration.Based on the GO annotations of these genes,12candidate genes were filtered for possible relevance to the mechanism of dichogamy in walnut.