台湾杉ISSR反应体系的建立及检测  

Establishment of ISSR system for the relict plant Taiwania cryptomerioides Hayata

作　　者：杨琴军[1] 袁继林 付强[1] 陈龙清[1] 

机构地区：[1]华中农业大学园艺植物生物学教育部重点实验室,武汉430070 [2]贵州雷公山国家级自然保护区管理局,雷山557100

出　　处：《华中农业大学学报》2011年第4期432-437,共6页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(30901130)

摘　　要：在保持1 UTaq酶和0.2 mmol/L dNTP两个条件不变的情况下,通过对模板DNA的浓度、引物浓度与Mg^2＋浓度的筛选,建立了台湾杉的优化ISSR反应体系,即在25μL的反应体系中,含有40 ng模板DNA、1 UTaq酶、0.2 mmol/L dNTP、0.4μmol/L引物、1.8 mmol/L Mg^2＋、2.5μL 10×buffer。扩增程序为：94℃,4 min;94℃,45 s,51-56℃,45 s,72℃,2 min,40个循环;72℃,7 min。利用这种优化的反应体系,筛选出了10条稳定性强、清晰度高并表现出一定多态性的ISSR引物,并进一步筛选出了这10条引物的最佳退火温度。应用这些引物对分布于湖北利川市星斗山保护区的台湾杉野生居群的20个样品进行了扩增,结果表明,利川星斗山台湾杉居群的多态位点百分率为61.97%,Nei’s基因多样性和Shannon’s信息指数分别为0.136 9和0.216 0。An optimal ISSR reaction system for Taiwania cryptomerioides was established with 25.0 μL reaction mixture made up of 40 ng genomic DNA,0.4 μmol/L primer,1.8 mmol/ L Mg^2＋,1.0 U Taq,0.2 mmol/L dNTP,and 2.5 μL 10×buffer.The PCR were performed as follows：4 min at 94℃,40 cycles of 45 s at 94℃,45 s at 51℃ to 56℃,and 2 min at 72℃,with a final extension of 7 min at 72℃.Ten polymorphic primers were screened using this reaction system.Twenty DNA samples from the wild population of T.cryptomerioides distributed in Lichuan City of Hubei Province were amplified by the 10 selected primers.The percentage of polymorphic bands was 61.97 %,Nei＇s gene diversity and Shannon＇s information measure were 0.136 9 and 0.216 0,respectively.

关 键 词：台湾杉 ISSR PCR条件 反应体系 引物筛选 

分 类 号：S791.280.4[农业科学—林木遗传育种]