Authentication of Allium ulleungense, A. microdictyon and A. ochotense based on super-barcoding of plastid genome and 45S nrDNA.

Allium ulleungense (AU) and A. microdictyon (AM) are valuable medicinal and edible vegetables, referred to as mountain garlic in Korea. The identification of AU, AM and a neighboring species A. ochotense (AO) is difficult because of their morphological similarities. We collected samples from three species and 46 cultivated collections to understand the genetic diversity of these valuable Allium species. Among them, we sequenced six collections, including three species and three cultivating collections to obtain data from the plastid genome (plastome) and nuclear 45S ribosomal DNA (nrDNA) for super-barcoding. The AM and AO showed around 60 single nucleotide polymorphisms (SNPs) and 39 Insertion/Deletion (InDels) in the plastome but no variations in the nrDNA sequences. Conversely, the AU and AM showed more than 170 SNPs and 80 InDels in the plastomes, and 20 SNPs and 1 InDel were found in the 45S nrDNA sequences. Among the three cultivating collections, one TB collection was determined to be the AU type in both plastome and nrDNA sequences. However, the other two collections, JB and SA, showed the AM type plastome but were heterozygous in the 45S nrDNA sequences, indicating both AU and AM types (putative AM x AU hybrid). Ten molecular markers were developed based on sequence variations to identify these three species and assess their genetic diversity. A total of 49 collections were genotyped using the ten developed markers and classified into five groups: 14 AU, 22 AM, 1 AO, 3 putative AM x AU hybrids, and 9 putative AU x AM hybrid collections. Super-barcoding with plastomes and nrDNAs revealed the genetic diversity of the three Allium species and putative hybrids between species. The newly developed markers will facilitate species and hybrid identification, thereby benefiting marker-assisted molecular breeding of Allium species.