大明竹属部分植物ISSR-PCR反应体系优化及有效引物筛选

Optimization of ISSR-PCR reaction system and selection of effective primers for ISSR marker in some species of Pleioblastus

采用单因素试验方法,建立了适合大明竹属25个竹种的ISSR-PCR反应体系,优化了各影响因子的用量,筛选出18条有效引物,并确定它们的退火温度,最后对体系进行了检验.优化后大明竹属植物ISSR-PCR的20μL反应体系含2.0μL10×PCR Buffer、2.5 mmol·L-1Mg2+、0.15 mmol·L-1dNTPs、0.5μmol·L-1引物、1.0 U Tap DNA聚合酶、50 ng模板DNA、10.6μL灭菌ddH2O.扩增程序为:94℃预变性5 min;94℃变性60 s,55℃复性45 s,72℃延伸90 s,循环40次;72℃延伸7 min,4℃保存.该ISSR-PCR反应体系可用于大明竹属部分植物的遗传多样性及遗传结构研究.

By using single factor test method,the ISSR-PCR reaction system suitable for 25 kinds of bamboo in Pleioblastus were established; the availability of every influence factor was optimized; besides,18 effective primers were screened and their annealing temperatures were identified to test the system.In conclusion,the optimizational ISSR-PCR 20 μL reaction system of bamboos in some species of Pleioblastus was 2.0 μL 10 × Buffer,2.5 mmol· L-1 Mg2＋,0.15 mmol· L-1 dNTPs,0.5 μmol · L-1 primers,1.0 U DNA polymerase,50 ng DNA template,10.6 μL sterile ddH2O; in the reaction process of 94 ℃ for5 min,40 circles （94℃ for 60 s,55 ℃ for 45 s ＆ 72 ℃ for 1.5 min）,72 ℃ for 7 min.The ISSR-PCR reaction system can be used in the study of genetic diversity and genetic structure of 25 kinds of bamboo in Pleioblastus.