Development of Cultivar Identification System Using 12 InDel Markers for Widely Distributed Citrus Cultivars in Japan

Citrus cultivar identification systems using cleaved amplified polymorphic site and single-nucleotide polymorphism markers have been developed for nursery trees and flesh fruits, but time and cost efficiency require improvement for the inspection process. Here, we developed a new cultivar identification system using the InDel marker. Twelve InDel markers, which revealed clear bi-allelic PCR fragment patterns, were selected from 185 InDel markers. Their primer sets were redesigned to generate less than 650 bp PCR fragments, and all were confirmed to apply to leaf and fresh fruit samples. It was confirmed that they were inherited in a codominant fashion among cultivars with parent-offspring relationships. At least two differentiating InDel polymorphisms to discriminate any paired combination among 33 citrus cultivars were provided, including 14 ancestral varieties and a reference genome cultivar of the clementine mandarin. Minimal marker subsets to identify the target cultivar are listed for each of the 14 registered cultivars with valid breeder's rights. The developed cultivar identification system features a simple experimental procedure with PCR and electrophoresis, saving time and cost during the inspection process. It could help protect registered cultivars from the illegal distribution of nursery trees and the reimportation of illegal fruits from abroad.