Allozyme polymorphism in chestnut (Castanea) species was investigated using isoelectric focusing in thin-layer polyacrylamide slab gels. Genetic analysis of the progenies of intraspecific crosses and interspecific F2s and backcrosses (BC1s) allowed the verification of 11 polymorphic isozyme loci from 11 enzyme systems. The following loci were defined: Acp, Adh, Est-1, Est-2, Est-5, Me, Prx-1, Prx-2, Prx-3, Skd-3, and Skd-4. All polymorphic loci behaved as single-locus Mendelian genes. Skd showed unique species specificity. Skd-1 and Skd-2 were unique to the American chestnut (C. dentata Borkh.) and the European chestnut (C. sativa Mill.), whereas Skd-3 and Skd-4 were unique to the Chinese chestnut (C. mollissima Bl.) and the Japanese chestnut (C. crenata Sieb.). Linkage analysis revealed linkage for three pairs of loci: Skd-3/Skd-4, Est-1/Est-2, and Est-5/Prx-1. The single-tree progeny method was used successfully for isozyme genetic analysis. Forty-seven chestnut cultivars in six chestnut species were characterized using 12 isozyme loci and can be unambiguously identified by 12 multi-locus genotypes. The interspecific and geographic relationships among species were also discussed.
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