Design and validation of an SNP-based assay for detecting Juglans ailantifolia introgression into Juglans cinerea populations or living collections

Abstract Butternut ( Juglans cinerea L.) is a rapidly declining tree species threatened by Ophiognomonia clavigignenti-juglandacearum (Oc-j), the causal agent of butternut canker. The introduction of Japanese walnut ( Juglans ailantifolia Carrière), which hybridizes with butternut and exhibits greater resistance to the disease, presents both a conservation challenge and a potential avenue for genetic improvement. Identifying non-admixed butternuts is critical for conservation efforts and breeding programs aimed at preserving the species’ genetic integrity while exploring resistance strategies. However, morphological identification of first and advanced generation hybrids is unreliable, necessitating the development of efficient detection tools. In this study, we developed and validated a cost-effective, high-throughput SNP genotyping tool using the MassARRAY platform to distinguish between J. cinerea , J. ailantifolia , and their hybrids. The panel consists of 28 nuclear SNPs and two chloroplast SNPs and demonstrates strong concordance with genotyping-by-sequencing (GBS) results using nearly 12,000 SNPs (R² = 0.98), indicating high predictive accuracy. Field testing this genotyping tool to 1,269 trees from natural stands and breeding programs in Canada and the U.S. revealed substantial hybridization, particularly in U.S. plantations, where hybrids comprised up to 18% of tested trees. Chloroplast analysis confirmed asymmetrical hybridization patterns, with J. ailantifolia acting predominantly as the maternal parent. This SNP panel provides a valuable tool for conservation, breeding, and regulatory applications by enabling rapid and accurate hybrid identification. Its implementation will help guide recovery strategies for butternut, ensuring that conservation efforts are informed by precise genetic data.