高通量提取西红柿基因组DNA方法探究及其在SNP分型中的应用

doi:10.11924/j.issn.1000-6850.casb2022-0005

中国农学通报 ›› 2023, Vol. 39 ›› Issue (9): 45-51.doi: 10.11924/j.issn.1000-6850.casb2022-0005

高通量提取西红柿基因组DNA方法探究及其在SNP分型中的应用

王海英¹(), 韩德俊², 李晓蕊³

¹ 西北农林科技大学园艺学院，陕西杨凌 712100
² 西北农林科技大学农学院，陕西杨凌 712100
³ 西安高新区农业农村和水务局，西安 710077

收稿日期:2022-01-04 修回日期:2022-05-02 出版日期:2023-03-25 发布日期:2023-03-23
作者简介:
王海英，女，1987年出生，河北承德人，助理实验师，硕士，研究方向：园艺植物育种与生物技术。通信地址：712100 陕西省杨凌示范区邰城路3号，Tel：029-87082647，E-mail：wanghaiying@nwafu.edu.cn。
基金资助:
陕西省烟草公司项目“烟草赤星病生物防治与生态调控技术研发与应用”(KJ-2021-02)

A High Throughput Tomato Genomic DNA Extraction Method and Its Application in SNP Genotyping

WANG Haiying¹(), HAN Dejun², LI Xiaorui³

¹ College of Horticulture, Northwest Agriculture and Forestry University, Yangling, Shaanxi 712100
² College of Agronomy, Northwest Agriculture and Forestry University, Yangling, Shaanxi 712100
³ Agriculture, Rural and Water Administration of Xi'an Hi-tech Industries Development Zone, Xi'an 710077

Received:2022-01-04 Revised:2022-05-02 Online:2023-03-25 Published:2023-03-23

摘要/Abstract

摘要：

随着植物分子生物学的发展，植物分子试验对于大规模样品DNA提取的需求越来越大。本研究旨在开发快速、简便、高通量的DNA提取技术。以西红柿叶片为试验材料，用96孔盒对西红柿叶片进行冷冻干燥及粉碎，基于改良的SDS提取DNA方法，采取排枪“两步加样一步抽提”，简化提取步骤，实现DNA的高通量提取。该方法提取DNA样品的平均浓度在138.6 ng/µL左右，满足一般性的PCR扩增要求；琼脂糖凝胶电泳结果显示绝大部分样品泳道可见清晰完整的DNA条带，无明显降解，并伴有少量的RNA污染。利用3个等位基因特异的定量PCR基因分型系统(AQP)分子标记对192个样本进行检测，结果显示3个标记的整体检测结果良好，均能有效区分样品间的不同等位基因位点。样品的有效荧光值占比在97%~99%之间，可以用于分子标记检测或遗传群体的基因分型。本研究在前人基础上探讨了一种简便、快速、高通量的西红柿基因组DNA提取方法，对于提取其他植物叶片组织DNA同样适用，并且该方法能够成功应用于AQP等其他类似的荧光分子标记检测，为大规模分子标记检测等实验DNA样品准备提供了借鉴。

关键词: 西红柿, 改良的SDS方法, 高通量提取DNA, 琼脂糖凝胶电泳, AQP分子标记检测

Abstract:

With the development of plant molecular biology, there is a growing demand for DNA extraction from large-scale samples in plant molecular experiments. In the study, we aim to develop a rapid, simple and high-throughput DNA extraction method. Tomato leaves as test materials were freeze-dried and crushed in a 96-hole box. To simplify the extraction steps and achieve high-throughput DNA extraction, we used the improved SDS-DNA extraction method by “two-step sampling and one-step extraction”. The average concentration of DNA sample extracted by this method was about 138.6 ng/mL, which met the need of PCR amplification. Agarose gel electrophoresis showed that most of the samples had clear and complete DNA bands without obvious degradation, but accompanied by some RNA. A total of 192 samples were detected by using 3 allele-specific quantitative PCR (AQP) markers. The results showed that the overall detection results of the 3 markers were good, and all of them could effectively distinguish different allelic loci between the samples. The effective fluorescence value of samples detected ranged from 97% to 99%, such DNA samples could satisfy molecular marker detection and genetic population genotyping. In this study, a simple, rapid and high-throughput method for DNA extraction from tomato was developed, which was also suitable for DNA extraction from other plant leaf tissues. This method could be successfully applied to AQP and other similar fluorescent molecular marker detection, and provide reference for large-scale molecular marker detection and other experimental DNA sample preparation.

Key words: tomato, improved SDS method, high throughput DNA extraction, agarose gel electrophoresis, AQP molecular detection

王海英, 韩德俊, 李晓蕊. 高通量提取西红柿基因组DNA方法探究及其在SNP分型中的应用[J]. 中国农学通报, 2023, 39(9): 45-51.

WANG Haiying, HAN Dejun, LI Xiaorui. A High Throughput Tomato Genomic DNA Extraction Method and Its Application in SNP Genotyping[J]. Chinese Agricultural Science Bulletin, 2023, 39(9): 45-51.

图/表 5

参考文献 30

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标记名称	引物名称	引物序列(5'-3')
Sol_KASP_01	AQP-01A	GATATCATTCCACAGGTTCCAT
	AQP-01B	GATATCATTCCACAGGTTCCAA
	AQP-01C	AGCAGACACAACAAACTGAATTG
Sol_KASP_09	AQP-09A	CGAGAACTGTGATGAGCCATATT
	AQP-09B	CGAGAACTGTGATGAGCCATATC
	AQP-09C	GCCTGCTCGATATTGTGATCAA
Sol_KASP_23	AQP-23A	TTCTAACTCGTGCTTGCTCATA
	AQP-23B	TTCTAACTCGTGCTTGCTCATT
	AQP-23C	CTAAAAATGACCCAACTCACAATC

标记名称	引物名称	引物序列(5'-3')
Sol_KASP_01	AQP-01A	GATATCATTCCACAGGTTCCAT
	AQP-01B	GATATCATTCCACAGGTTCCAA
	AQP-01C	AGCAGACACAACAAACTGAATTG
Sol_KASP_09	AQP-09A	CGAGAACTGTGATGAGCCATATT
	AQP-09B	CGAGAACTGTGATGAGCCATATC
	AQP-09C	GCCTGCTCGATATTGTGATCAA
Sol_KASP_23	AQP-23A	TTCTAACTCGTGCTTGCTCATA
	AQP-23B	TTCTAACTCGTGCTTGCTCATT
	AQP-23C	CTAAAAATGACCCAACTCACAATC

样品编号	平均	Ta-001	Ta-002	Ta-003	Ta-004	Ta-005	Ta-006	Ta-007	Ta-008	Ta-009	Ta-010
浓度	138.6±37.4	141.1	138.2	208.7	99.5	186.9	173.4	131	98.5	100.2	109.0
A₂₆₀/A₂₈₀	1.96±0.27	2.23	1.96	2.23	2.19	1.87	1.60	2.11	1.44	2.1	1.86
A₂₃₀/A₂₆₀	1.68±0.10	1.61	1.82	1.72	1.68	1.74	1.60	1.83	1.57	1.73	1.55

样品编号	平均	Ta-001	Ta-002	Ta-003	Ta-004	Ta-005	Ta-006	Ta-007	Ta-008	Ta-009	Ta-010
浓度	138.6±37.4	141.1	138.2	208.7	99.5	186.9	173.4	131	98.5	100.2	109.0
A₂₆₀/A₂₈₀	1.96±0.27	2.23	1.96	2.23	2.19	1.87	1.60	2.11	1.44	2.1	1.86
A₂₃₀/A₂₆₀	1.68±0.10	1.61	1.82	1.72	1.68	1.74	1.60	1.83	1.57	1.73	1.55

高通量提取西红柿基因组DNA方法探究及其在SNP分型中的应用

A High Throughput Tomato Genomic DNA Extraction Method and Its Application in SNP Genotyping

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