One hundred and forty-seven primer pairs originally designed to amplify microsatellites, also known as simple sequence repeats (SSR), in black walnut (Juglans nigra L.) were screened for utility in persian walnut (J. regia L.). Based on scorability and number of informative polymorphisms, the best 14 loci were selected to analyze a diverse group of 47 persian walnut accessions and one J. hindsii (Jepson) Jepson ex R.E. Sm × J. regia hybrid (Paradox) rootstock. Among the 48 accessions, there were 44 unique multi-locus profiles; the accessions with identical profiles appeared to be synonyms. The pairwise genetic distance based on proportion of shared alleles was calculated for all accessions and a UPGMA (unweighted pair group method with arithmetic mean) dendrogram constructed. The results agree well with what is known about the pedigree and/or origins of the genotypes. The SSR markers distinguished pairs of closely related cultivars and should be able to uniquely characterize all walnut cultivars with the exception of budsports. They provide a more powerful and reliable system for the molecular characterization of walnut germplasm than those previously tested. These markers have numerous applications for the walnut industry, including cultivar identification, verification of pedigrees for cultivar and rootstock breeding programs, paternity analysis, and understanding the genetic diversity of germplasm collections.

Geographic distribution of extant taxa of Juglans (Juglandaceae). The distribution of cultivated species J. regia extends beyond its natural home range.

Phylogenetic analyses of extant species of Juglans (Juglandaceae) with the New and Old World distributions were performed using five cpDNA intergenic spacer (IGS) sequences (trnT-trnF, psbA-trnH, atpB-rbcL, trnV-16S rRNA, and trnS-trnfM) to elucidate the origin, diversification, historical biogeography, and evolutionary relationships within the genus. Overall, 3848 characters were sampled, yielding 105 (2.7%) variable sites, of which, 45 (1.16%) were potentially parsimony informative. In all, seventeen in-group (Juglans) and two out-group (Carya and Pterocarya) taxa were analyzed using the parsimony, likelihood, and minimum evolution methods, which produced concordant trees with three well supported clades corresponding to the sections Juglans, Cardiocrayon (butternuts), and Rhysocaryon (black walnuts). Juglans cinerea from the section Trachycaryon was placed within the black walnut clade, probably suggesting historical introgression of black walnut chloroplast during its evolution in North America. Black walnuts were not well resolved perhaps suggestive of their relatively recent origin and diversification or reticulate evolution within the section. Within butternuts, J. hopeiensis is moderately supported as a sister to J. ailantifolia, J. cathayensis, and J. mandshurica, which were well supported as a clade in all three analyses. The Persian walnut, J. regia and its sister taxon, J. sigillata formed a distinct clade sister to both butternuts and black walnuts. The cladogenesis within the genus Juglans appears to deviate from the evolutionary hypothesis based on the fossil history and biogeographic evidence. The results suggest that: (1) the IGS sequence divergence levels observed within and between different sections of Juglans are low; (2) basal placement of the section Juglans in all the three analyses suggests its ancient origin in contrast to fossil evidence, which suggests the earliest divergence of sections Rhysocaryon and Cardiocaryon; (3) the Persian walnuts and butternuts have evolved at different rates as compared to black walnuts; and finally (4) the extant taxa may not represent the entire evolutionary history of the genus.

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