Molecular identification of Allium ochotense and Allium microdictyon using multiplex-PCR based on single nucleotide polymorphisms
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Allium ochotense and Allium microdictyon are commonly known as ‘Mountain garlic’ and are popular, economically important species in many countries such as Korea, China, and Mongolia. Their leaves are used as culinary side dishes and in traditional medicines. In Korea, these two species are at risk of extinction due to damage to their natural habitat and thus, conservation and breeding programs are needed. However, their identification relies mostly on morphological data, which is limited and until recently, led to classifying these two species under A. victorialis. In the present study, a simple and reliable method of molecular identification was developed to distinguish A. ochotense from A. microdictyon that targets four barcoding regions: the internal transcribed spacer (ITS), the maturase K gene (matK), the chloroplast psbA-trnH intergenic region, and the ribulose-bisphosphate carboxylase large subunit gene (rbcL). Single nucleotide polymorphisms (SNPs) were found in ITS and matK regions, and species-specific primers were designed based solely on the SNP at position 680 of the ITS region that could differentiate A. ochotense from A. microdictyon. Using these primers in amplification refractory mutation system (ARMS)-PCR, A. ochotense, and A. microdictyon could be simultaneously and efficiently distinguished. This study is the first to report a simple, rapid, and efficient method for discriminating A. ochotense and A. microdictyon, indicating the utility of species-specific markers in the development of conservation and breeding programs.
KeywordsITS matK psbA-trnH rbcL Amplification refractory mutation system (ARMS)-PCR
This study was supported by a 2014 research Grant from Kangwon National University, and by the Rural Development Administration (RDA) and Cooperative Research Program for Agricultural Science and Technology Development (PJ012011032017).
- Fritsch RM, Blattner FR, Gurushidze M (2010) New classification of Allium L. subg. Melanocrommyum (Webb & Berthel.) Rouy (Alliaceae) based on molecular and morphological characters. Phyton 49:145–220Google Scholar
- Ganopoulos I, Aravanopoulos F, Madesis P, Pasentsis K, Bosmali I, Ouzounis C, Tsaftaris A (2013) Taxonomic identification of mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing. PLoS ONE 8:e60945. https://doi.org/10.1371/journal.pone.0060945 CrossRefPubMedPubMedCentralGoogle Scholar
- Han BK, Rhee SJ, Jang YJ, Sim TY, Kim YJ, Park TS, Lee GP (2016) Identification of a causal pathogen of watermelon powdery mildew in Korea and development of a genetic linkage marker for resistance in watermelon (Citrullus lanatus). Korean J Hortic Sci Technol 34:912–925Google Scholar
- Kim J, Kim DS, Lee ES, Ahn YK, Chae WB, Lee SS (2017) The construction of a Chinese cabbage marker-assisted backcrossing system using high-throughput genotyping technology. Hortic Sci Technol 35:232–242Google Scholar
- Li Q-Q, Zhou S-D, He X-J, Yu Y, Zhang Y-C, Wei X-Q (2010) Phylogeny and biogeography of Allium (Amaryllidaceae: Allieae) based on nuclear ribosomal internal transcribed spacer and chloroplast rps16 sequences, focusing on the inclusion of species endemic to China. Ann Bot 106:709–733CrossRefGoogle Scholar
- Sarker D, Johnson MAT, Reynolds A, Brandham PE (1997) Cytology of the highly polyploid disjunct species, Allium dregeanum (Alliaceae), and of some Eurasian relatives. Bot J Linn Soc 124:361–373. https://doi.org/10.1111/j.1095-8339.1997.tb02002.x CrossRefGoogle Scholar
- Semagn K, Bjørnstad Å, Ndjiondjop MN (2006) An overview of molecular marker methods for plants. Afr J Biotechnol 5:2540–2568Google Scholar
- Whatmore AM, Murphy TJ, Shankster S, Young E, Cutler SJ, Macmillan AP (2005) Use of amplified fragment length polymorphism to identify and type Brucella isolates of medical and veterinary interest. J Clin Microbiol 43:761–769. https://doi.org/10.1128/JCM.43.2.761-769.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- Yang JY, Jang SY, Kim H-K, Park SJ (2012) Development of a molecular marker to discriminate Korean Rubus species medicinal plants based on the nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL-F intergenic region sequences. J Korean Soc Appl Biol Chem 55:281–289. https://doi.org/10.1007/s13765-012-1044-6 CrossRefGoogle Scholar
- Yoo KO, Kim WB, Park HJ, Lim SC, Jang HT (1998) Investigation on the ultrastructure of epidermis, anatomical, palynological, and cytological characteristics of Allium victorialis var. platyphyllum collected from three different habitats. J Korean Soc Hortic Sci 39:260–265 [in Korean] Google Scholar