The common walnut (Juglans regia L.), a species of significant economic and ecological importance, along with its variant, holds developmental value. Simple sequence repeat (SSR) markers are used as an effective method for material identification due to their co-dominant inheritance. In this work, a total of 357629 SSR loci were searched based on previously published walnut genome sequences, with a density of 662.28 (SSRs/Mb). The majority of all these loci were found to be single nucleotide A/T (58.49%), followed by the dinucleotide AT (19.48%). Based on the results of genome-wide SSR site design, 22 effective primer pairs were designed and screened to analyze the genetic diversity of 48 wild walnut samples from three countries. Each locus had an average of 5.17 effective alleles (Ne), with an average polymorphism information content (PIC) of 0.71, average heterozygosity (He) of 0.52, and average genetic differentiation index (Fst) of 0.09. The analysis of 48 wild walnut samples from three countries (Kyrgyzstan, Tajikistan, and China) using the unweighted pairwise clustering method and arithmetic mean (UPGMA) along with principal coordinate analysis revealed the division of all samples into three groups. In summary, these novel SSR markers serve as a reference for the identification of wild walnut germplasm in Central Asia and for the development and utilization of wild walnut seed resources.

Caragana acanthophylla Kom. is an ecologically important drought-tolerant shrub in Xinjiang, China, but species-specific molecular markers for germplasm characterization remain limited. We sampled 93 individuals from 11 localities representing the currently known distribution of C. acanthophylla in Xinjiang. Three individuals per locality (33 in total) were whole-genome resequenced, yielding 2,873,410 high-quality SNPs and 5,679,915 InDels. Genome-wide SNP-based PCA and genetic relationship analysis provided an independent high-resolution assessment of the 33 resequenced individuals. From 34 candidate primer pairs, eight polymorphic InDel markers with stable amplification and clear genotyping profiles were retained and applied to all 93 individuals. The SNP dataset revealed clear regional differentiation and finer locality-associated relationships. Analysis of the same 33 individuals with the eight InDel loci recovered part of this broad pattern, particularly the differentiation of the western YL materials, but showed lower fine-scale resolution. Across all 93 individuals, the InDel panel revealed moderate to low marker-level genetic diversity and detectable regional differentiation. AMOVA attributed 67.00% of the variation to differences among the 11 original sampling localities, while the five exploratory analytical groups showed a similar among-group component (68.37%). The Mantel correlation detected across all 93 individuals (r = 0.801, p < 0.001) disappeared after YL was excluded (r = −0.032, p = 0.724), indicating that the overall spatial signal was largely driven by the geographic separation of YL. These results support the eight-marker panel as a practical, low-cost tool for preliminary germplasm characterization and broader sample screening, while genome-wide SNP data provide substantially greater resolution for population-level inference.