白及SRAP-PCR反应体系正交设计优化及引物筛选

桂腾琴; 李春艳; 韦燕; 武忠亮; 张思平

doi:10.13651/j.cnki.fjnykj.2025.03.003

白及SRAP-PCR反应体系正交设计优化及引物筛选

Orthogonal design of SRAP-PCR reaction system and screening primers for Bletilla striata

摘要

摘要: 建立白及Bletilla striata Rchb.f.最佳SRAP-PCR（sequence-related amplified polymorphism polymerase chain reaction，SRAP-PCR）反应体系，为从分子水平开展白及不同地理群体的分子遗传学评价研究提供相关的技术支持。以采自于贵州省安龙县野生白及嫩叶为材料，应用正交设计L₉（3⁴）对反应体系中4个因素（引物、Taq DNA聚合酶、Mg²⁺和dNTPs）的用量进行优化，正交设计引物组合为（Me1/Em1），探索建立白及最佳SRAP-PCR反应体系并对引物进行筛选，筛选稳定性高、多态性好的引物组合。结果表明：白及的最佳SRAP-PCR反应体系（25 μL）为：2.50 μL10×Buffer、2.50 mmol·L⁻¹Mg2+、0.30 mmol·L⁻¹dNTPs、0.25 μmol·L⁻¹引物、1.50 U Taq DNA聚合酶以及20～80 ng模板DNA。利用21份白及对最佳SRAP-PCR反应体系稳定性检测，该体系稳定性高、重复性好。以野生白及基因组DNA为模板，选用最佳的SRAP-PCR反应体系对80对引物组合进行筛选，最终获得多态性引物13对，共扩增出182个位点，其中多态性位点152个，多态性位点百分比为83.52%。该体系稳定性高、重复性好，为后续遗传多样性研究提供相关的技术支持。

Abstract: The objective of this study was to establish the optimal SRAP-PCR reaction system for Bletilla striata. The results were to provide technical support for the molecular genetic evaluation analysis of Bletilla striata in different geographic populations. It laid the foundation for the study of Bletilla striata Rchb.f. at molecular level. The orthogonal design（L₉（3⁴））was applied to optimize the dosage of four factors（primers, Taq DNA polymerase, Mg²⁺ and dNTPs）in the reaction system. The primer combination for orthogonal design was（Me1/Em1）. The aim was to establish the optimal SRAP-PCR reaction system for Bletilla striata and screen the primer combinations with high stability and good polymorphism. The results showed that the optimal SRAP-PCR reaction system（25 μL）for Bletilla striata was as follows: 2.50 μL 10×Buffer, 2.50 mmol.L⁻¹ Mg2+, 0.30 mmol.L⁻¹ dNTPs, 0.25 μmol·L⁻¹ primers, 1.50 U Taq DNA polymerase and 20-80 ng template DNA. A total of 13 polymorphic SRAP primer combinations were sreened from 80 combinations primer. 13 polymorphic SRAP primer combinations amplified 182 bands, 152polymorphic bands, and 83.52% polymorphicm ratio. The results indicate that PCR products were stably amplified among 21 Bletilla striata. The optimal SRAP-PCR reaction system has good repeatability and high stability.

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