Germplasm identification of Lonicerae Japonicae Flos by DNA melting curve analysis
【Abstract】To establish a new high resolution melting analytical method for identification of Lonicera japonica germplasm, the screening of 7 pairs of simple sequence repeats (SSRs) primers, determining the suitable diagnostic primers by the differences of peak pattern and Tm were conducted. Then into the DNA template concentration, annealing temperature and the suitable range of cycle number were investigated. Combined with SIMCA-P software for data processing analysis, the results showed that three main germplasm honeysuckle could be divided by four sets of primers. It provided methodology for improving L. japonica germplasm identification.
【Keywords】 high resolution melting curve; Lonicerae Japonicae Flos; germplasm identification;
 Gao F Q, Li J, Ran R, et al. Investigate on the germplasm resources for main produce area of flos Lonicerae japonicae in China [J]. Research and Practice on Chinese Medicines, 2010 (3): 21 (in Chinese with English abstract).
 Gao F Q, Zhang Y Q, Zhang F, et al. Investigation and research on germplasm resources of Shandong honeysuckle [J]. Shandong Journal of Traditional Chinese Medicine, 2006, 25 (4): 268 (in Chinese).
 Zhang S S, Haung L Y, Yuan Y, et al. Numerical taxonomy of agronomic trait in cultivated Lonicera japonica [J]. China Journal of Chinese Materia Medica, 2014, 39 (8): 1379 (in Chinese with English abstract).
 Zhang S S. Study on germplasm resources and characteristics of honeysuckle [J]. Wuhan Polytechnic University, 2015 (in Chinese with English abstract).
 Chen K, Jaing C, Yuan Y, et al. High resolution melting and its application in identitystudy of traditional Chinese medicine [J]. Acta Pharmaceutica Sinica, 2015, 50 (12): 1581 (in Chinese with English abstract).
 Li X X, Tao Z M, Wu Z G, et al. Evaluation on genetic diversity among species of Lonicera L. in southern Zhejiang Province by ISSR and SRAP [J]. Chinese Traditional and Herbal Drugs, 2012, 43 (10): 2030 (in Chinese with English abstract).
 Reed G H, Kent J O, Wittwer C T. High-resolution DNA melting analysis for simple and efficient molecular diagnostics [J]. Future Med, 2007, 8: 597.
 Gan S M, Li F G, Weng Q J, et al. High resolution melting and its applications in plant DNA variation detection [J]. Genomics and Applied Biology, 2010 (29): 1 (in Chinese with English abstract).
 Chen K S, Li F, Xu C J, et al. An efficient macro-method of genomic DNA isolation from Actinidia chinensis leaves [J]. Hereditas, 2004, 26 (4): 529 (in Chinese with English abstract).
 Mader E, Lukas B, Novak J. A strategy to setup codominant microsatellite analysis for high-resolution-melting-curve-analysis (HRM) [J]. BMC genetics, 2008, 9 (1): 69.
 Ganopoulos I, Bosmali I, Madesis P, et al. Microsatellite genotyping with HRM (High Resolution Melting) analysis for identification of the PGI common bean variety Plake Megalosperma Prespon [J]. Eur Food Res technol, 2012, 234 (3): 501.
 Hofinger B J, Jing H C, Hammond-Kosack K E, et al. High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley [J]. Theor Appl Genet, 2009, 119 (5): 851.
 Farrar J S, Reed G H, Wittwer C T. High resolution melting curve analysis for molecular diagnostics [M]. 2nd ed. Amsterdam, 2009: 229.
 Gundry C N, Vandersteen J G, Reed G H, et al. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes [J]. Clinical chemistry, 2003, 49 (3): 396.