苔草属植物ISSR-PCR体系的建立及优化  

Establishment and Optimization of ISSR-PCR System in Carex

作　　者：宁花 郑成淑[1] 王文莉[1] 朱翠英[1] 李朝晖[2] 张艳敏[3] 

机构地区：[1]山东农业大学园艺科学与工程学院、作物生物学国家重点实验室、国家苹果工程技术中心,泰安271018 [2]山东农业大学林学院,泰安271018 [3]山东农业大学生命科学学院,泰安271018

出　　处：《分子植物育种》2014年第2期349-355,共7页Molecular Plant Breeding

基　　金：山东省自然科学基金(ZR2011CM048)资助

摘　　要：本研究以苔草属植物10个野生种为试材,采用L16(45)正交试验设计,对影响ISSR-PCR反应的Taq DNA聚合酶、Mg2+、dNTPs、引物及模板DNA以及退火温度、循环次数等因素进行优化试验。研究结果表明,苔草属植物的最优ISSR-PCR反应体系,即25μL反应体积中,含Taq DNA聚合酶1.5 U,Mg2+1.5 mmol/L,DNA模板150 ng,引物0.24μmol/L,dNTPs 0.25 mmol/L。苔草属植物的最佳扩增程序,即:94℃预变性3 min;94℃变性30 s,退火45 s,72℃延伸1.5 min,38个循环;72℃延伸7 min;4℃保存。采用优化后的体系对10份苔草属植物进行扩增,能扩增出清晰明亮的条带,表明该反应体系和扩增程序适于苔草属植物的ISSR分析,为进一步开展苔草属植物的遗传多样性分析、分子系统学研究及种质资源评价奠定了基础。In this experiment, a L16（45） orthogonal design was used for the optimal ISSR-PCR reaction system of Carex with 10 species as materials in these factors including Taq DNA polymerase, Mg2＋, dNTPs, primers, template DNA annealing temperature gradient, cycle gradient annealing time gradient and extending time gradient. Results showed that the optimum reaction system in 25μL reaction mixture was as follow： 1.5 U Taq DNA polymerase, 1.5 mmol/L Mg2＋, 150 ng template DNA, 0.24 μmol/L primer and 0.25 mmol/L dNTPs. The best amplification procedure included pre-denaturation at 94℃ for 3min; denaturation at 94℃ for 30 s, annealing for 45 s, extending at 72℃ for 90 s, after 38 cycles and a final extention at 72℃ for 7 min, then kept the temperature at 4℃. With the ISSR-PCR best reaction system and the optimum program for 10 Carex accessions, UBC825 produced clear and bright bands. It indicated that the reaction system and reaction process could be used to analyze genetic diversity on Co：rex with high stability and repeability. The establishment of the PCR reaction conditions could settle favorable basis for the further study on the genetic diversity and molecular systematics of Carex by using ISSR molecular marker techniques.

关 键 词：苔草属 ISSR 分子标记 正交设计 体系优化 

分 类 号：Q948.1[生物学—植物学]