西洋杜鹃SRAP体系优化及遗传多样性分析

以西洋杜鹃Rhododendron hybridum功能叶片为试验材料，采用改良的十六烷基三甲基溴化铵（CTAB）法提取西洋杜鹃基因组DNA，并且运用L16（4^4）正交试验设计，在4个水平上对影响西洋杜鹃相关序列扩增多态性（SRAP）反应的锾离子（Mg^2＋）浓度、Taq酶用量、三磷酸碱基脱氧核苷酸（dNTPs）浓度、引物浓度等4个因素进行了优化，确立了一套适用于西洋杜鹃的稳定可靠、重复性强的SRAP-PCR反应体系。实验发现，其最佳反应体系（20．0μL）为：Mgn浓度1．750mmol·L^-1，dNTPs浓度0．175mmol．L^-1，砌酶1．500×16．67nkat，引物0．200μmol·L^-1。利用该优化体系对24个西洋杜鹃花品种进行遗传多样性分析。从88对SRAP引物中筛选出10对扩增清晰且多态性高的引物，共扩增出217条带，其中多态性条带212条，多态性比率达97.35％。24个杜鹃品种的遗传相似系数变化范围为0．591～0．708，算术平均数的非加权配对算术平均法（UPGMA）聚类分析结果显示：在相似系数为0．590处将24个供试品种分为2个类群，在相似系数为0．623处又可分为2个亚群，说明该优化体系可应用于西洋杜鹃花品种鉴定及遗传多样性的研究。

An optimal SRAP-PCR system of Rhododendron hybridum and its genetic diversity analysis with SRAP marker

In order to find the optimal sequence-related amplified polymorphism （SRAP）-PCR system of Rhododendron hybridum and analyse its genetic diversity, the functional leaves of Rhododendron hybridum were used to extract its genome DNA, using an improved cetyl trimethyl ammonium bromide （CTAB） method. Concentrations of Mg^2＋, Taq polymerase, dNTPs and primer, which maybe affect the SRAP-PCR reactions, were optimized by L16（＆）orthogonal design experiments to establish the SRAP molecular marker system in Rhododendron hybridum. Then, an optimal, stable and repeatable SRAP-polymerase chain reaction （PCR） of 20.0 μL containing 1.750 mmol. L^-1 Mg^2＋, 0.175 mmol.L^-1 dNTPs, 1.500 ± 16.67 nkat Taq polymerase, and 0.200 μmol.L^-1 primer was established. Finally, genetic diversity and relationships of 24 Rhododendron hybridum cuhivars were analyzed using an Unweighted Pair Group Method with Arithmetic Mean （UPGMA） cluster analysis with this optimized system. Results revealed 10 highly polymorphic and sta- ble primer pairs selected from 88 pairs of SRAP primers with a total of 217 bands being detected from these 10 primer pairs. Of the detected bands, 212 were polymorphic （a 97.35% average） having a genetic similarity coefficient ranging from 0.591 to 0.708. The cluster analysis divided the 24 cultivars into two groups at a 0.590 similarity level, and these were further delineated into two sub-groups at a 0.623 similarity level. These results demonstrated that this optimized SRAP-PCR system can be applied to cultivars identification and genetic diversity research on Rhododendron hybridum.