芦笋SSR-PCR反应体系的优化及验证

Optimization and validation of SSR-PCR reaction system for Asparagus officinalis L.

摘要

以芦笋幼叶为材料，采用单因素与L16(4³)正交试验相结合的方法，对影响芦笋SSR-PCR反应的3个因素(2× Taq Master Mix, 模板DNA，引物)进行优化，并以此为基础通过退火温度和循环次数试验筛选引物最佳退火温度和循环次数。结果表明，芦笋基因组DNA的SSR-PCR最优反应体系为:10 µL反应体系中，50 ng·µL^-1 DNA模板0.125 µL，10 µmol·L^-1上下游引物各0.5 µL，2× Taq PCR Mix 3 µL，灭菌ddH₂O 5.875 µL。PCR扩增程序为:94 ℃预变性4 min；94 ℃变性30 s, 60 ℃退火30 s, 72 ℃延伸35 s, 循环25次；72 ℃延伸10 min, 4 ℃保存。经5对引物组合和5个不同芦笋品种DNA扩增验证，该体系稳定可靠，可用于芦笋遗传多样性分析、种子纯度鉴定、分子标记辅助育种及重要农艺性状的图位克隆等工作。

Abstract

Based on DNA of asparagus young leaves, a single factor screening test combined with a L16(4³) orthogonal design method was employed to optimize the factors of 2× Taq Master Mix, template DNA, and primer combination applied for SSR-PCR and followed by gradient annealing temperature test and gradient cycle times test to screening the optimum annealing temperature and cycle numbers. The results showed that the optimal SSR-PCR system for asparagus included 50 ng·µL^-1 DNA template 0.125 µL, 10 µmol·L^-1 primers 0.5 µL, 2×Taq PCR Mix 3 µL, sterilized ddH₂O 5.875 µL, with a total reaction volume of 10 μL. The PCR amplification procedure for asparagus was: 94 ℃ pre-denaturation for 4 min, 94 ℃ denaturation for 30 s, 60 ℃ annealing 30 s, 72 ℃ renaturation for 35 s, 25 cycles, 72 ℃ extension for 10 min, 4 ℃ for storage. This reaction system was proved to be stable and reliable by PCR amplification with 5 pairs of primer combinations and DNA templates of five different asparagus cultivars. The optimized SSR-PCR reaction system could be satisfactorily used for genetic diversity analysis, seed purity identification, marker-assisted breeding and map-based cloning of important agronomic traits of asparagus.

关键词

芦笋 / SSR-PCR反应体系 / 正交设计

Key words

Asparagus officinalis / SSR-PCR reaction system / Orthogonal design

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基金资助

山西省高校科技创新项目(2022L104)

山西省农业科学院农业科技创新研究课题(YCX2020YQ07)