Development of Polymorphic Microsatellite Markers and Identification of Applications for Wild Walnut (Juglans regia L.) in Middle Asia

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.