SCAR markers for practical application of marker-assisted selection in persimmon (Diospyros kaki Thunb.) breeding

In previous studies, we have developed molecular markers linked to the AST locus that controls fruit astringency type in persimmon; however, these markers are not feasible for practical application to breeding programs since they are not fully effective for discriminating the pollination constant and non-astringent (PCNA) genotype from the non-PCNA genotype in a progeny derived from 'Kurokuma'. Here we developed new sequence characterized amplified region (SCAR) markers that enable easy and reliable selection of the PCNA genotype from breeding populations. Genomic regions adjacent to probe 5R, which showed polymorphic fragments between PCNA and non-PCNA genotypes in restriction fragment length polymorphism (RFLP) analysis, were isolated from the genomic libraries of 'Nishimura-wase', 'Jiro', and 'Kurokuma'-derived offspring. The isolated genomic regions were characterized and 3 insertion/deletion mutations were observed between ast- and AST-linked regions. Several primers were designed in the flanking region of Indel-3 and, in multiplex PCR, it was shown that using 2 forward primers, AST-F and PCNA-F and a reverse primer, 5R3R, is the most useful and reliable primer set. The ASTlinked 220-bp fragment proved to be a common marker of 'Kurokuma'-, 'Nisimura-wase'- and 'Aizumishirazu'- derived progenies. This multiplex PCR is considered the most practical tool for marker-assisted selection (MAS) and can enhance and accelerate progress in persimmon breeding.