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中国农学通报 ›› 2018, Vol. 34 ›› Issue (4): 43-48.doi: 10.11924/j.issn.1000-6850.casb17010050

所属专题: 生物技术 园艺

• 生物技术科学 • 上一篇    下一篇

辣椒的一种适用于大规模PCR鉴定的DNA快速提取技术

李委,戴祖云,杨中周,夏伟伟   

  1. 合肥学院,安徽江淮园艺种业股份有限公司,安徽江淮园艺种业股份有限公司,安徽江淮园艺种业股份有限公司
  • 收稿日期:2017-01-11 修回日期:2017-03-16 接受日期:2017-03-22 出版日期:2018-02-26 发布日期:2018-02-26
  • 通讯作者: 李委
  • 基金资助:
    2016年安徽省援藏援疆援青计划项目《“嘎玉”辣椒系列新品种及高效栽培技术的示范与推广》(项目编号:1604e0302003)

A DNA Rapid Extraction Method of Pepper: Suitable for High Throughput PCR Identification

杨中周 and   

  • Received:2017-01-11 Revised:2017-03-16 Accepted:2017-03-22 Online:2018-02-26 Published:2018-02-26

摘要: 传统的辣椒DNA提取常采用CTAB法,该技术已难满足大量开展分子生物学工作的需要,为了解决此困境,进行了辣椒上碱煮法提取的尝试。一个可用于新品种的SSR分子标记被用作检验手段,验证碱煮法所提DNA是否可满足实验需要。结果表明针对不同的研究目的需采取不同的组织部位来进行DNA的提取,当面临种子纯度测定的任务时,需要截取种子的芽根。当面临成熟植株的基因型筛选的任务时,需要截取植株的顶端嫩叶。以上这些植物材料置PCR管中,在向管中加入80 μL 0.2 mol/L NaOH溶液后,置PCR仪上以100℃处理30分钟, 再以40 μL Tris-HCl缓冲液将pH值调节至7~8之间后,以之为模板可在后续的SSR-PCR中得到良好的实验结果。综上,本研究表明相较其他耗时耗力的传统DNA提取方法,碱煮法确是更适合解决辣椒大规模鉴定问题的技术手段。

关键词: 白肋烟, 白肋烟, 有机肥, 经济性状, 品质性状

Abstract: The traditional way to extract DNA from pepper is using CTAB method. Now, the CTAB method could not meet the need for massive molecular biology work, in order to solve the problem, we testified alkalin-heating method in pepper DNA extraction. A SSR molecular marker that was recently identified from a new pepper variety was used as examining approach for checking whether the Alkalin-heating method can provide qualified DNA satisfying experimental needs. The results showed that different tissues should be used according to different experimental purposes. Roots of germinated seeds were needed when dealing with seed-purity-identifying. Young leaves of mature plants were needed when dealing with plant genetic screening. These plant materials were then put into PCR tubes. After adding 80 μL 0.2 mol/L NaOH solution, the tubes were treated under 100℃ for 30 mins in PCR amplifier. Then 40 μL Tris-HCl buffer was used to adjust pH value to a range between 7 and 8. Then a good experimental result could be retrieved using the DNA solution as template. In conclusion, it is confirmed that, comparing with other time and labor consuming methods, the Alkalin-heating method would be a more suitable way to solve the problem of high throughput genetic identification in pepper.