Development of a simple and easily interpretable cultivar identification system using 18 insertion/deletion (InDel) markers in Japanese pummelo (Citrus maxima)

Pummelo is one of the ancestral species involved in the origin and diversification of citrus and is also an important fruit crop cultivated mainly in Asia. In Japan, cultivar identification systems based on cleaved amplified polymorphic site (CAPS) and insertion/deletion (InDel) markers have been developed for mandarins and acid citrus and are used as practical tools for the protection of breeders’ rights. However, a simple cultivar identification system for pummelo has not yet been sufficiently established. In this study, we designed 2172 InDel markers potentially suitable for pummelo cultivar identification based on comparative analysis of whole-genome sequences and long-read data. Among these, 18 InDel markers showing clear biallelic polymerase chain reaction (PCR) fragment patterns were selected, enabling cultivar identification using only standard PCR and agarose gel electrophoresis. This simple method may also be useful for cultivar identification in customs office and field laboratories where access to specialized genotyping equipment is limited. These markers enabled major pummelo cultivars and their hybrids grown in Japan to be clearly distinguished using a minimum of five markers. In addition, a unique allele shared by ‘Hondabuntan’ and ‘Hassaku’ suggests that ‘Hondabuntan’ is a candidate parent of ‘Hassaku’. The InDel markers developed in this study are expected to contribute not only to the protection of breeders’ rights through cultivar identification but also to the elucidation of the genetic background of pummelo and provide useful knowledge for citrus breeding.