A Reliable Molecular Diagnostic Tool for CA90 (Castanea sativa × Castanea crenata) Hybrid Identification Through SSR

Chestnut trees are an essential source of both food and timber. However, the severe threats from invasive pests and diseases compromise their existence and productivity. In Europe, chestnut hybridization programs have been initiated to produce resilient rootstocks in response to ink disease. However, the gap in the identification of these hybrid plants is typically based on field observations and morphological features and remains a challenge. Our study presents a marker set for distinguishing between chestnut hybrid CA90 (Castanea sativa × Castanea crenata), a hybrid with demonstrated resistance to Phytophthora cinnamomi, and other varieties using microsatellite (SSR) markers and bioinformatics tools. We used 35 chestnut samples, including three CA90 controls, hybrids sampled within Portugal, with an aim to define the profiles of the chestnut hybrids and varieties in this study based on band patterns and SSR motifs. We selected and modified nine distinct SSR primers with null allelic features from 43 already developed simple sequence repeat (SSR) markers. PCR amplification and agarose gel electrophoresis were used to amplify and visualize the DNA bands. To confirm genetic variations, 27 amplified bands were sequenced by Sanger sequencing. This analysis identified 31 SSRs across 22 SSR-containing sequences, with trinucleotide (67.74%) repeats being the most common, followed by repeats of dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%). A total of 18 alleles were observed for the nine loci. The alleles ranged from one to three per locus for the 35 samples. The novel locus CP4 could only be found in CA90 hybrids. This tool can aid in identifying and selecting disease-resistant hybrids, thereby contributing to chestnut production and management strategies.