Haplotype Transition at the <i>Freestone-Melting flesh</i> Locus during Japanese Peach Cultivar Improvement

The Freestone-Melting flesh (F-M) locus in peach controls two strongly linked traits that have significant effects on fruit quality: pit adhesion (freestone/clingstone) and flesh type (melting/non-melting). Recently DNA markers have been developed to identify major haplotypes of M0, M1, M2, M2b, and M3 at this locus. Japanese peach cultivars were historically developed using cultivars introduced from overseas; however, how the haplotypes have changed at the F-M locus during major cultivar improvement remains unknown. In this study, we aimed to clarify the transition of haplotypes at the F-M locus in major varieties of peach cultivars from the past to the present. First, we modified the F-M locus haplotype genotyping method by multiplexing the PCR to genotype many cultivars efficiently. Then, we genotyped the F-M locus of 63 cultivars, including major cultivars in 1934, 1980, and 2020 using this method. The results show that the number of cultivars harboring the M1 haplotype, which is a dominant freestone haplotype, decreased significantly, and cultivars homozygous for the M0 haplotype dominated. Haplotypes other than M0 and M1 were infrequent among major cultivars. Cultivars harboring M1 have not always been evaluated as freestone in the literature, while we found a strong relationship between the presence of the M1 haplotype and the freestone trait. All the genetically non-melting cultivars were evaluated as non-melting. There has been only one case in which a genetically melting cultivar was previously evaluated as semi-melting, although all other genetically melting cultivars were evaluated correctly. This result suggests that DNA markers could be effective for freestone breeding and there were no major problems in flesh texture prediction using this marker system. The haplotype transition at the F-M locus during Japanese peach breeding elucidated in this study could lead to new research on freestone/clingstone and flesh texture traits and their application in breeding.