Development of an Optimal Set of High-Resolution Melting Markers for the Identification of Nucellar and Zygotic Seedlings in Yuzu (Citrus junos Sieb. Ex Tanaka)

Yuzu (Citrus junos Sieb. Ex Tanaka) is primarily reproduced through nucellar embryony, a type of apomixis. To assess the frequency of zygotic seedlings resulting from normal self-fertilization and to select desirable zygotic seedlings, 837 yuzu seeds were germinated. Out of these seeds, 707 produced seedlings, with multiple seedlings emerging from 244 of them. A total of 973 seedlings were analyzed using ten high-resolution melting (HRM) markers showing heterozygous genotypes in three mother plants of the seedlings. As a result, 197 zygotic seedlings with at least one homozygous genotype were identified. The zygotic origins of these 197 seedlings were confirmed through an analysis of an additional eleven HRM markers, including one for genotyping the CitRWP gene responsible for nucellar embryony. Sixty-one zygotic seedlings were devoid of the mutant CitRWP allele. Significant segregation distortions were observed in twelve out of the 21 HRM markers. In particular, one of two alternate homozygous genotypes was completely absent in the YH471 marker, indicating the presence of lethal alleles. The construction of a linkage map using the 21 HRM markers resulted in nine independent linkage groups. Based on the linkage relationship and the presence of segregation distortions, an optimal set of six HRM markers was ultimately selected for the efficient identification of zygotic seedlings in yuzu.