ABSTRACT
This study was aimed to establish and optimize the reaction system of MSAP for analysis on DNA methylation inCyathula officinalis Kuan of Sichuanmedicinal. The tender leaves ofC. officinalisKuan were used as materials. Orthogonal method was used in the study of main factors which affected the system quality of MSAP. The results showed that the optimal reaction systems of MSAP included: enzyme digestion (20μL), 300 ng DNA, 1.5μL EcoRI, 1.5μL HpaII or 1.5μL MspI (HpaII); ligation (25μL): 15μL digestion products, 1μL EcoR I adaptor, 1μL Hpa II/Msp I adaptor, 0.2μL T4 ligase; pre-amplication mixture (25μL): ligation products 1μL, rTaq polymerase 1 U, each primer 1.5μL, dNTP 2μL; selective amplification mixture (25μL): pre-amplification product was diluted 50 times, rTaq polymerase 1 U, each primer 1.5μL, dNTP 3μL. The optimal MSAP reaction system was used to screen for 6 pairs of effective primers from 256 pairs of primers of MSAP. It was concluded that the optimized system ensured the stable and clear bands and the screened 6 pairs of primers were with good specificity. It provided useful references for further studies of epigenetic onC. officinalisKuan DNA methylation and MSAP analysis for other medicinal plants.