ADAPTING CHESTNUT SINGLE NUCLEOTIDE POLYMORPHISMS FOR USE IN BREEDING

The goal of the American Chestnut Foundation (TACF) is the restoration of the American chestnut as a viable species to its native range. Toward that end, TACF is using conventional backcross breeding to introgress blight resistance from Chinese into American chestnut. The identification of species-specific molecular genetic markers could potentially accelerate recovery of the American type by permitting selection against Chinese chestnut genes not associated with disease resistance, and identifying pairings of hybrid trees that can produce progeny with a higher proportion of American genetic background than expected by chance. Therefore, we examined genotypes of forty-six Chinese and forty-nine American chestnut trees using a GoldenGateBeadArray (Illumina) of American chestnut single-nucleotide polymorphisms (SNPs), and identified eighteen SNPs with strongly divergent allele frequencies in the two species. The web-based Allele-Specific Primer (WASP) designing tool successfully designed primers for fifteen of these eighteen SNPs. The corresponding sequences from American and Chinese chestnut were aligned to each other to identify potential problems in primer sites and aligned to peach or apple genomic sequence to identify likely intron positions that might interfere with amplification. The fifteen WASP primer sets were tested on a panel of two American, one interspecific F1, and three Chinese chestnut trees. Twelve primer sets amplified an allele from American and hybrid but not from Chinese chestnut trees (allele potentially specific to American chestnut). Of these twelve, three also amplified an allele from Chinese and hybrid but not American chestnut (allele potentially specific to Chinese chestnut). For general use, species specificity of these alleles will need to be validated in a larger panel of both American and Chinese chestnut trees. However, for immediate use in the TACF breeding program, these alleles only need to differentiate the Chinese (resistance sources) and American chestnut parents (recurrent parents) used in the current backcross generations.