Genetic Diversity, Structure and Differentiation in Cultivated Walnut (Juglans regia L.)

An analysis of genetic structure and differentiation in cultivated walnut (Juglans regia) using 15 microsatellite loci revealed a considerable amount of genetic variation with a mild genetic structure indicating five genetic groups corresponding to the centers of diversity within the home range of walnut in Eurasia. Despite the narrow genetic differentiation among groups accounting for only 10 to 15% of the total variation, the groups differed significantly with respect to frequency and composition of alleles for different loci. Moderate to high genetic variability with mild genetic structure is found to be ideal for association genetic analysis. INTRODUCTION The thin-shelled cultivated walnut (Juglans regia) belongs to the section Juglans within the genus Juglans of the family Juglandaceae. Its native range extends from the Carpathian Mountains of Eastern Europe to the Southern Caucasus, northern Turkey, Iran, to the Tien Shan province of western China to the Himalayan states of India, Sikkim, and Bhutan (Zohary and Hopf, 1993). The taxonomic placement of the cultivated walnut within the genus Juglans was problematic and the earlier studies placed it as sister either to the Asian butternuts section, Cardiocaryon (Stanford et al., 2000) or to the black walnut section, Rhysocaryon (Manos and Stone, 2001). But a recent study based on sequences from the chloroplast non-coding regions strongly supports the section Juglans as an independent clade sister to the remaining three sections within the genus Juglans (Aradhya et al., 2004). The evolutionary history of the section Juglans is riddled with widespread extinctions, geographic isolations, and bottlenecks during the Quaternary glaciations. Subsequent expansion and human selection in the Transcaucasia, Central, West and East Asia greatly influenced the genetic structure within the section Juglans (Popov, 1929; Beug, 1975). The modern distribution of Walnut (J. regia) extends beyond its native range occurring under cultivation in both the Old and New World. Its sister taxon, J. sigillata with a hard shell bearing a black kernel may represent a semidomesticated or primitive form within the section Juglans restricted to southern China. Knowledge of genetic diversity, structure and differentiation of cultivated walnut is important for effective conservation, management and utilization of germplasm. Several studies have examined the genetic diversity and relationships among walnut cultivars using allozymes (Arulsekar et al., 1985), restriction fragment length polymorphisms (RFLP; Fjellstrom et al., 1994), randomly amplified polymorphic DNA (RAPD; Nicese et al., 1998), and microsatellite markers (Dangl et al., 2005). Genetic analysis of germplasm collections often provides insights into the complex interactions of evolutionary forces such as mutation, gene flow, selection, and drift shaping the ecogeographic structure and domestication history of a crop species. In outcrossing species such as walnut information on genetic structure and co-ancestral relationships among germplasm accessions have proven to be useful in association genetic analysis. The present study represents a first step towards association or disequilibrium mapping of genes. Here we report results of a preliminary analysis of genetic structure and differentiation in a walnut germplasm collection maintained at the USDA Germplasm Proc. VIth Intl. Walnut Symposium Ed.: D.L. McNeil Acta Hort. 861, ISHS 2010 128 Repository at the University of California, Davis, California, USA based on genetic polymorphism at microsatellite loci. MATERIALS AND METHODS 459 trees representing 203 diverse accessions of walnut (J. regia) germplasm conserved at the USDA repository were genotyped using 15 microsatellite (also known as SSR) loci WGA001, WGA004, WGA009, WGA069, WGA089, WGA106, WGA118, WGA178, WGA202, WGA237, WGA318, WGA321, WGA331, WGA338 and WGA384 using standard PCR protocols with florescent labeled primers (Dangl et al., 2005). The microsatellite loci were originally developed for J. nigra at the Hardwood Tree Improvement Center, U.S. Forest Service, Purdue University, Indiana, USA (Woeste et al., 2002) and adapted here to J. regia. Amplified products were resolved using capillary electrophoresis in an ABI Prism 3100 genetic analyzer with data collection software, version 1.2 (PE/Applied Biosystems). The fragment data were further analyzed using Genescan, version 3.1 and Genotyper, version 2.5 to assess the size of alleles and data assembled as microsatellite genotypes as well as in binary format. The binary data were used to compute a distance matrix using Nei and Li distance (Nei and Li, 1979) based on the proportion of alleles shared between two accessions for all possible pair-wise combinations. The resultant distance matrix was subjected to a cluster analyses (CA) using the neighbor-joining method to produce a phenogram. The multilocus SSR genotype data were pooled into groups based on the results of CA and analyzed for various within-group genetic variability measures such as mean number of alleles per locus and observed and expected levels of heterozygosities. Contingency χ analysis was performed to determine the genetic heterogeneity among groups. Genetic differentiation within and among groups was computed using the Nei’s gene diversity analysis (Nei, 1973). Total gene diversity (HT) was partitioned into gene diversity within groups (HG) and gene diversity between groups (DGT), where HT = HG + DGT. Genetic differentiation between groups is calculated as GGT = DGT/HT, where GGT varies from zero (when HG = HT) and unity (when HG = 0), i.e., groups fixed for different alleles. Analysis of molecular variance (AMOVA; Excoffier et al., 1992) was performed on group-wise genotypic data to partition the total variance into variance within and among groups. RESULTS AND DISCUSSION Although the origin of walnut is obscure, it is considered to be native to the region extending from the Carpathian Mountains to Transcaucasia and parts of West Asia, East Asia into the Himalayan regions comprising Jammu and Kashmir, Himachal Pradesh, and North Eastern regions of India, Sikkim and Bhutan (Dode, 1909; McGranahan and Leslie, 1991). The species went through a series of bottlenecks during the Quaternary glaciations in isolated cryptic refugia in Carpathian, Ponto-Caspian and other central Asian regions rapidly eroding the diversity. Climatic deterioration and human activity during the postglacial range expansion and colonization of new areas by small founder populations have rapidly modified the genetic structure of the species. The germplasm collection assayed in this study of cultivated walnut is somewhat reminiscent of the evolutionary and domestication history of walnut. Genetic diversity and the patterns of distribution within a species germplasm collection determine the potential for improving the species through breeding programs. The walnut collection assayed showed considerable variability with the observed number of alleles per locus ranging from 5 for WGA384 to 19 for WGA202 with an average of 11 alleles per locus (Table 1). There was a significant deficiency of heterozygotes compared to Hardy-Weinberg proportions for 14 out of the 15 loci assayed suggesting substructuring within the collection and obviously indicating some level of inbreeding with restricted gene flow among subpopulations. The CA identified 5 broad groups corresponding to the major areas of distribution of walnut in its native central, west and East Asia (Fig. 1). Further analysis of the affinities among the groups indicated that the West Asia walnuts are the most diverse within which there is a subgroup closely allied