Development of a DNA marker for cultivar identification of "Harushizuka", a new satsuma mandarin cultivar created by heavy-ion irradiation.

'Harushizuka' is a novel late-maturing satsuma mandarin (Citrus unshiu Marcow.) cultivar developed through heavy-ion irradiation-induced mutagenesis. The S1152 line, selected from a nucellar seedling of the 'Aoshima unshu' cultivar for superior fruit quality, was irradiated with both carbon and neon ions. Mutations in fruit shape, color, and peel characteristics were observed in the neon-ion-irradiated group. From this group, we isolated the 'Harushizuka' cultivar, which exhibits delayed fruit coloring. The harvest season for 'Harushizuka' is approximately one month later than that of regular C. unshiu, allowing for a more spread-out harvesting period. To develop DNA markers for cultivar protection and identification, we performed whole-genome mutation analysis of 'Harushizuka' and its original cultivar 'Aoshima unshu'. Two mutations were identified: a 30-bp deletion and a 20-bp insertion. The PCR assay targeting the deletion successfully differentiated 'Harushizuka' from 21 other C. unshiu cultivars. Our findings demonstrate that whole-genome mutation analysis is a powerful tool for developing DNA markers, even in citrus cultivars with low genetic diversity caused by bud mutations or nucellar embryogenesis. The established marker enables rapid and accurate identification of 'Harushizuka', contributing to the protection of breeders' rights and the prevention of unauthorized propagation.