东兴金花茶SSR-PCR反应体系的优化及引物筛选  

Optimization of SSR-PCR Reaction System and Primer Screening of Camellia tunghinensis Chang

作　　者：唐健民 陈宗游[2,3] 韦霄[2] 史艳财[2] 柴胜丰[2] 孔德鑫[2] 

机构地区：[1]广西师范大学生命科学院,桂林541004 [2]广西壮族自治区中国科学院广西植物研究所,桂林541006 [3]广西大学农学院,南宁530004

出　　处：《基因组学与应用生物学》2014年第2期398-404,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31160137);广西自然科学基金项目(2012GXNSFAA053067);广西科学研究与技术开发计划项目(桂科重1355001-5-4);广西植物研究所基本科研业务费项目(桂植业12011);国际植物园保护联盟(BGCI)(R4331)项目共同资助

摘　　要：采用L16（45）正交设计试验对东兴金花茶SSR-PCR反应的五因素（Mg^2＋,dNTPs,引物,Taq酶和模板DNA浓度）四水平进行了优化试验,然后利用优化后的反应体系,将31对同属植物SSR引物（4对茶树,7对山茶和20对金花茶引物）运用于东兴金花茶材料上,筛选适宜东兴金花茶遗传分析的SSR引物。结果表明,东兴金花茶最佳反应体系（15μL）及扩增条件为：Mg^2＋1.50 mmol/L、dNTP 0.30 mmol/L、引物0.40μmol/L、Taq酶0.75 U、模板DNA 40.00 ng;最佳扩增程序为：94℃预变性5 min;94℃变性30 s,58℃退火30 s（因引物而异,该温度为引物CN10的退火温度）,72℃延伸40 s,共36个循环;72℃最后延伸10 min。山茶属的不同植物之间可以共享部分SSR引物信息,东兴金花茶获得了9对能扩增出条带清晰且具有多态性的引物,引物最适退货温度为54-60℃,观测杂合度（HO）和期望杂合度（HE）分别为0.433-1.000和0.391-0.932。本试验为下一步进行的东兴金花茶遗传多样性分析、遗传图谱的构建等研究奠定了基础。In this paper, the orthogonal design was used to optimize the SSR-PCR amplification system of Camellia tunghinensis Chang in five factors（Mg^2＋, dNTPs, Primers, Taq DNA polymerase and DNA template） at four levels. And then, the optimized reaction system was used to screen the primers by cross-amplification of 31 primers designed in congeneric species. The experimental results showed that, the optimal, 10 μL Camellia tunghinensis Chang SSR-PCR reaction system consisted of 1.50 mmol/L Mg^2＋, 0.30 mmol/L dNTP, 0.40 μmol/L primer, 0.75 U Taq polymerase and 40.00 ng DNA template. And the augmentation procedure was pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 58℃（different primers have different annealing temperatures, this temperatures was for the CN10 primer） for 30 s, extension at 72℃ for 40 s, reaction with 36 cycles, and extension at 72℃ for 10 min. Part of SSR primers information can be shared in congeneric species and 9 primer pairs with clear banding and plentiful polymorphism were selected for Camellia tunghinensis Chang. The annealing temperature of primers were 54-60℃. Observed and expected heterozygosities ranged from 0.433 to 1.000, and 0.391 to 0.932, respectively. The experiment results of this paper could build the basis on further study（e.g. genetic diversity assessment and construction of molecular genetic map etc）of Camellia tunghinensis Chang.

关 键 词：东兴金花茶 SSR-PCR 体系优化 引物筛选 

分 类 号：S685.14[农业科学—观赏园艺]