Abstract

To evaluate the effectiveness of microsatellite (SSR) markers for verifying the authenticity of walnut controlled-cross offspring, seven controlled-cross combinations were established using improved and widely cultivated walnut varieties from Guizhou Province, China, generating a total of 108 candidate offspring. Among 60 previously reported SSR primer pairs, 15 loci exhibiting stable amplification and clear genotyping profiles were selected for analysis. Parentage identification was conducted using the genotype exclusion and maximum likelihood methods, and the applicability of each method was assessed through genetic diversity and cluster analyses. The 15 SSR loci detected a total of 68 alleles, with an average of 4.53 alleles per locus. The polymorphism information content (PIC) ranged from 0.017 to 0.608, with a mean value of 0.396. The genotype exclusion method confirmed 76 offspring as genetically consistent with their recorded parental combination, corresponding to an overall identification rate of 70.37%. In the maximum likelihood analysis, 27 offspring were assigned to their most likely parent pairs when both parents were unknown, with an identification rate of 25.00%. When the maternal parent was known and only the paternal parent was unknown, 47 offspring were successfully assigned, increasing the identification rate to 43.52%. Cluster analysis showed that most offspring clustered near their corresponding parents, although some individuals deviated from their original cross combinations, suggesting allele sharing or complex genetic origins among the parental materials. The shared maternal parent structure, closely related paternal backgrounds, high-frequency alleles, and null alleles may jointly influence the efficiency of SSR-based parentage assignment. Compared with the maximum likelihood method, the genotype exclusion method showed greater practical stability for verifying recorded cross combinations in a structured walnut breeding population characterized by a shared maternal parent, multiple closely related paternal parents, and moderate SSR polymorphism. These findings indicate that likelihood-based parentage assignment should be interpreted cautiously when candidate parents are genetically similar and marker informativeness is limited. This study provides a technical reference for authenticity verification of walnut controlled-cross offspring, pedigree management, and marker-assisted breeding.

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