桉树SSR-PCR体系优化及引物筛选应用

Optimization of SSR-PCR Reaction System and Primer Screening of Eucalyptus

以尾叶桉、细叶桉、粗皮桉为材料,采用L16(45)正交设计对SSR-PCR反应体系中的引物浓度、DNA浓度、Mg2+浓度、d NTP浓度和Taq酶含量5种因素的4个水平进行优化实验,确立了适合细叶桉、粗皮桉、尾叶桉SSR-PCR最佳反应体系,最终优化的SSR-PCR反应体系为:0.25μmol/L引物、5 ng模板DNA、3.75 mmol/L Mg2+、0.4 mmol/L d NTP、1.5 U Taq酶、1 m L 10×PCR Buffer,双蒸水补齐10μL;并利用优化的体系从74对SSR引物中筛选出12对多态性较高的引物,12对引物在3种桉树中均能扩增出条带,在尾叶桉、细叶桉、粗皮桉中的多态率分别为100%、92.86%、64.29%,为尾叶桉、细叶桉、粗皮桉的遗传多样性分析、品种鉴定、亲缘关系分析等提供了分子技术基础。

Based on the DNA of E. urophylla, E. tereticornis, E. pellita, an optimizational experiment was taken with orthogonal design L16（45） which concentrated on five factors, primer, template DNA, Mg^（2＋）, d NTPs, and Taq DNA polymerase. We filtered out the optimal 10 μL SSR reaction system consisted of 3.75 mmol/L Mg^（2＋）, 0.4 mmol/L d NTPs, 0.25 μmol/L primer, 1.5 U Taq DNA polymerase and 5 ng template DNA, 1 μL10× PCR Buffer and dd H2 O to 10 μL. In addition, the transferability of 12 polymorphic SSR markers were tested in E. urophylla, E. tereticornis,E. pellita, which were filtered out from 74 EST-SSR markers. 12 SSR markers could effectively amplify in 3species mentioned above. The percentages of polymorphic markers in corresponding species were 100%, 92.86%,64.29%. All these results indicated that the most of given SSR markers should be applied to the genetic diversity analysis and molecular marker-assisted breeding of tested species.