野牛草ISSR分子标记体系的优化及遗传多样性分析

Optimization of ISSR molecular marker system and genetic diversity analysis of Buchloe dactyloides

摘要:

本研究以 L₉ (3⁴ )正交实验为基础，对野牛草(Buchloe dactyloides)的 ISSR 分子标记体系中各因素的最佳用量进行优化，主要包括 Taq PCR Mix、引物、模板 DNA 及引物的退火温度。确定的最佳 10 μL 体系为：5. 0 μL Mix、40 ng DNA 模板、0. 6 μL 引物(1. 0 μmol/L)及 ddH₂O 补齐。最佳扩增条件为 94 ℃预变性 3 min；94 ℃变性 30 s，48 ℃退火 30 s，72 ℃延伸 10 min，共 34 个循环；72 ℃延伸 10 min；4 ℃保存。对收集到的 39 份野牛草资源的遗传多样性进行分析，UPGMA 聚类将所有材料分为两大类，其中 21 号材料和天津滨海国际机场候选雌性野牛草材料的亲缘关系最近，可以作为后续材料扩繁和生产的种质资源。本研究中 ISSR 体系的探索与优化、遗传多样性分析为野牛草遗传多样性分析工作提供了依据。

关键词:野牛草;ISSR 分子标记;体系优化;聚类分析;遗传多样性

Abstract:

In this study，the optimal parameter of each factor in ISSR molecular marker system of buffalograss (Buchloe dactyloides)was explored based on L₉ (3⁴ )orthogonal test. Factors including Taq PCR Mix，primers，the amount of primer and template DNA，primer annealing temperature were examined. The optimal 10 μL system was 5. 0 μL Mix，40 ng DNA template，0. 6 μL primer(1. 0 μmol/L)and ddH2O complement. The best amplification condition was pre‐denaturation for 3 min at 94 ℃，34 cycles of denaturation at 94 ℃ for 30 s，annealing at 48 ℃ for 30 s，and extension at 72 ℃ for 10min，followed by extension at 72 ℃ for 10 min and stored at 4 ℃. The results of ge‐ netic diversity of 39 collected samples showed that all of them were divided into two groups by UPGMA cluster， among which No. 21 had the closest genetic relationship with the target female B. dactyloides of Tianjin Binhai Inter‐ national Airport. The germplasm No. 21 could be used as the basis for subsequent propagation. In this study，ISSR system exploration and optimization and genetic diversity analysis provided a basis for the genetic diversity analysis of buffalograss.

Key words:Buchloe dactyloides;ISSR molecular markers;system optimization;cluster analysis;genetic diver‐ sity