Zheng Xiuting, Hoegenauer Kevin A, Maeda Andrea B V, Wang Fei, Stelly David M, Nichols Robert L, Jones Don C
Department of Soil and Crop Sciences, Texas A&M University, College Station, TX.
Cotton Incorporated, Cary, NC.
Biotechniques. 2015 May 1;58(5):234-43. doi: 10.2144/000114286. eCollection 2015 May.
Extensive use of targeted PCR-based genotyping is precluded for many plant research laboratories by the cost and time required for DNA extraction. Using cotton (Gossypium hirsutum) as a model for plants with medium-sized seeds, we report here manual procedures for inexpensive non-destructive high-throughput extraction of DNA suitable for PCR-based genotyping of large numbers of individual seeds and seedlings. By sampling only small amounts of cotyledon tissue of ungerminated seed or young seedlings, damage is minimized, and viability is not discernibly affected. The yield of DNA from each seed or seedling is typically sufficient for 1000 or 500 PCR reactions, respectively. For seeds, the tissue sampling procedure relies on a modified 96-well plate that is used subsequently for seed storage. For seeds and seedlings, the DNA is extracted in a strongly basic DNA buffer that is later neutralized and diluted. Extracts can be used directly for high-throughput PCR-based genotyping. Any laboratory can thus extract DNA from thousands of individual seeds/seedlings per person-day at a very modest cost for consumables (~$0.05 per sample). Being non-destructive, our approach enables a wide variety of time- and resource-saving applications, such as marker-assisted selection (MAS), before planting, transplanting, and flowering.
对于许多植物研究实验室来说，基于靶向PCR的基因分型因DNA提取所需的成本和时间而无法广泛应用。以中等大小种子的植物棉花（陆地棉）为模型，我们在此报告了用于廉价、无损、高通量提取DNA的手动方法，该方法适用于对大量单个种子和幼苗进行基于PCR的基因分型。通过仅对未发芽种子或幼苗的少量子叶组织进行采样，损伤可降至最低，且活力不会受到明显影响。每个种子或幼苗的DNA产量通常分别足以进行1000次或500次PCR反应。对于种子，组织采样程序依赖于一种改良的96孔板，该板随后用于种子储存。对于种子和幼苗，DNA在强碱性DNA缓冲液中提取，随后进行中和和稀释。提取物可直接用于基于PCR的高通量基因分型。因此，任何实验室每人每天都可以以非常低的耗材成本（每个样本约0.05美元）从数千个单个种子/幼苗中提取DNA。由于无损，我们的方法能够实现各种节省时间和资源的应用，例如在种植、移栽和开花前进行标记辅助选择（MAS）。
