RAD-Seq analysis of typical and minor Citrus accessions, including Bhutanese varieties

We analyzed the reduced-representation genome sequences of Citrus species by double-digest restriction site-associated DNA sequencing (ddRAD-Seq) using 44 accessions, including typical and minor accessions, such as Bhutanese varieties. The results of this analysis using typical accessions were consistent with previous reports that citron, papeda, pummelo, and mandarin are ancestral species, and that most Citrus species are derivatives or hybrids of these four species. Citrus varieties often reproduce asexually and heterozygosity is highly conserved within each variety. Because this approach could readily detect conservation of heterozygosity, it was able to discriminate citrus varieties such as satsuma mandarin from closely related species. Thus, this method provides an inexpensive way to protect citrus varieties from unintended introduction and to prevent the provision of incorrect nursery stocks to customers. One Citrus variety in Bhutan was morphologically similar to Mexican lime and was designated as Himalayan lime. The current analysis confirmed the previous proposition that Mexican lime is a hybrid between papeda and citron, and also suggested that Himalayan lime is a probable hybrid between mandarin and citron. In addition to Himalayan lime, current analysis suggested that several accessions were formed by previously undescribed combinations.