一种快速、无损大豆种子DNA提取方法的建立和应用

doi:10.11983/CBB15057

植物学报 ›› 2016, Vol. 51 ›› Issue (1): 68-73.DOI: 10.11983/CBB15057

一种快速、无损大豆种子DNA提取方法的建立和应用

程文^1,², 夏正俊³, 冯献忠², 杨素欣^2,^*()

¹山东师范大学生命科学学院, 济南 250014
²中国科学院东北地理与农业生态研究所中国科学院大豆分子设计育种重点实验室, 长春 130102
³中国科学院东北地理与农业生态研究所中国科学院大豆分子设计育种重点实验室, 哈尔滨 150081

收稿日期:2015-03-31 接受日期:2015-09-25 出版日期:2016-01-01 发布日期:2016-02-01
通讯作者: 杨素欣
作者简介:? 共同第一作者
基金资助:
基金项目: 国家自然科学基金(No.31171571, No.91131008, No.31271743)

A Rapid and Nondestructive Method for Soybean DNA Extraction and Its Application

Wen Cheng^1,2, Zhengjun Xia³, Xianzhong Feng², Suxin Yang^2*

¹College of Life Sciences, Shandong Normal University, Jinan 250014, China
²Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China
³Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China

Received:2015-03-31 Accepted:2015-09-25 Online:2016-01-01 Published:2016-02-01
Contact: Yang Suxin
About author:? These authors contributed equally to this paper

摘要/Abstract

摘要： 基因分型是进行植物基因功能的遗传分析和分子标记辅助育种的重要环节。该研究以大豆(Glycine max)成熟种子为材料, 建立了通过钻孔采集样品、快速提取DNA进行基因型鉴定的方法。用此方法, 一个熟练的工作人员可以在1个小时内完成120个样品的采集和DNA提取; 同时种子钻孔取样后, 不会对大豆种子的萌发造成影响。利用该方法获得的DNA可满足PCR扩增的要求。实验重复性好, 成功率在98%以上。这种快速且无损的大豆种子基因型鉴定方法可以用于鉴定杂交种子、品种纯度以及遗传分析等研究工作。

关键词: 大豆种子, DNA提取, 无损基因型鉴定

Abstract: Genotyping is an important work in plant genetic analysis and molecular marker-assisted breeding. This study established a simple method for genotyping soybean from mature seeds. By this method, a skilled worker can perform the sample collection and DNA extraction of 120 samples from soybean seeds in 1 h and the treated seeds can germinate normally. The DNA obtained by this method can meet the requirement for PCR amplification, and the success rate of PCR reaction is over 98%. This method can be widely used for identifying soybean hybrid seeds, seed purity and genotyping.

Key words: soybean seed, DNA extraction, nondestructive genotyping

程文, 夏正俊, 冯献忠, 杨素欣. 一种快速、无损大豆种子DNA提取方法的建立和应用. 植物学报, 2016, 51(1): 68-73.

Wen Cheng, Zhengjun Xia, Xianzhong Feng, Suxin Yang. A Rapid and Nondestructive Method for Soybean DNA Extraction and Its Application. Chinese Bulletin of Botany, 2016, 51(1): 68-73.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB15057

https://www.chinbullbotany.com/CN/Y2016/V51/I1/68

图/表 5

表1 分子标记的引物序列

Table 1 Primer sequences of molecular markers

Primer ID	Forward primer (5′-3′)	Reverse primer (5′-3′)
MOL0051	CTCACACCCTTTCATTATCTA	AAATCGCGCATCTAAATTTAC
MOL0489	CTAAAAGGTGGTACTTTTGGTGTAGG	GAGCCTTCTGTCTAGTAGCATGACCTT
MOL0533	ACACTTGAAAACGCCACT	ACACTCGAAGGGTCACAT
MOL0591	GAGATCGTTAATGTCGCATCGTG	GGTGCTAATACCTTCCTCAAACG
MOL0629	CCGTATTACATACTGTTGGGTGA	ACTCGGGTTTGTAGAGTGATACAGT
MOL0679	TACAAGTCACCACTAGCCATCGT	GCTCTAATGACGTGGTTGACAGA
MOL0697	AAGGATCACAGGACGTGAGGACA	TCCCTTCCCTTAAATGTGGTCAA
MOL0765	ATTTGGTGTCCTTTTCGCTTTGTG	AGCATGACATTCATCTGCGTTGT
MOL0897	ACAAACTTGAGTCTATAACTCCCCC	GATGGAAGATGAAGTGATGAGTGA
MOL0911	GAAGAGCATGTTGTGATGCTTTG	TGCGTGGATAATGCCAGGATA
MOL0981	GCAGGGACTGAGCAATAACAG	AGCATAGCCTGAATACACGGA
MOL0997	ACAAGCCCAATAAAGTCCATGTG	GGATTCCACGGTGATGGGTG
MOL1225	TCTTTCAAATGAGATTGCGTTGC	CGCATAACTTGGAGGCTCTTCTT
MOL1227	GAGCCTTTCCCAAGCCAATG	GGTCACTTTGAAAGACACGAAACATA

图1 大豆种子钻孔DNA提取过程 (A) 大豆种子钻孔所用器材; (B)-(F) 种子钻孔和样品采集的过程; (G) 将样品材料加入0.1 mol∙L-1 NaOH, 于99°C处理10分钟, 碱解样品; (H) 钻孔的种子播种后10天形成的幼苗; (I) 未钻孔的种子播种后10天形成的幼苗

Figure 1 Procedure of sample collection and DNA extraction from soybean seed (A) Equipment used in the sample collecting; (B)-(F) The major steps of sample collection; (G) Incubation of soybean powder in 0.1 mol∙L-1 NaOH at 99°C for 10 minutes; (H) Seedling of drilled seed after sowed 10 days grown in greenhouse; (I) Seedling of soybean seed after sowed 10 days grown in greenhouse

图2 PCR鉴定所提取DNA质量对分子标记MOL0679、MOL0981、MOL0697、MOL0489、MOL0897、MOL0591、MOL0997、MOL0629、MOL0765和MOL0533进行PCR引物扩增。M: 100 bp DNA Ladder

Figure 2 Electrophoretograms of PCR products for examination of DNA quality With MOL0679, MOL0981, MOL0697, MOL0489, MOL0897, MOL0591, MOL0997, MOL0629, MOL0765 and MOL0533, for each molecular marker detected. M: 100 bp DNA Ladder

图3 大豆杂交种和品种纯度的PCR检测 (A) 杂交种子鉴定(分子标记MOL0051), 泳道1: 菏豆12; 泳道2, 3: 杂交种(菏豆12×Williams 82)阳性对照; 泳道4: Williams 82; 泳道5-32: 待检测的28个杂交种, 白色箭头(泳道11, 32)所示为假杂交种DNA的扩增产物, 与母本菏豆12基因型一致; (B) 种子纯度鉴定(分子标记MOL0911), 泳道1-4: Williams 82阳性对照; 泳道5-48: 待检测的44个样品, 白色箭头(泳道9、36和38)示污染种子的带型

Figure 3 Electrophoretograms of PCR products for examination of soybean hybrid seeds and seed purity(A) Identification of hybrid seeds (molecular marker MOL- 0051 was used for PCR amplification), Lane 1: Hedou12; Lane 2, 3: Positive control of hybrids (Hedou 12 × Williams 82); Lane 4: Williams 82; Lane 5-32: 28 examined hybrids, the white arrows in lanes 11 and 32 indicated PCR products amplified from failed crossings; (B) Identification of seed purity (molecular marker MOL0911 was used for PCR amplification), Lane 1-4: Williams 82 positive control; Lane 5-48: Tested seeds, the white arrows in lane 9, 36 and 38 indicated PCR products from contaminated seeds

图4 MOL1227和MOL1225两对引物在同一反应管中PCR扩增产物的电泳检测结果泳道M: DL500 DNA marker; 泳道1-19: 菏豆12与Williams 82 杂交的F2群体的19个个体。箭头A和B分别示MOL1227扩增Williams 82和菏豆12的单倍体型所得到的PCR产物片段(280 bp和266 bp); 箭头C和D分别示MOL1225扩增菏豆12和Williams 82的单倍体型所得到的PCR产物片段(213 bp和193 bp)。

Figure 4 Electrophoretograms of PCR products for checking the results of MOL1227 and MOL1225 double pairs of primers amplification in one tubeLane M: DL500 DNA marker; Lane 1-19: 19 individual plants from F2 population of Hedou 12 and Williams 82. Arrow A and B indicated the PCR products of MOL1227 from Williams 82 haplotype (280 bp) and Hedou 12 haplotype (266 bp), respectively; Arrow C and D indicated the PCR products of MOL1225 from Hedou 12 haplotype (213 bp) and Williams 82 (193 bp) haplotype, respectively.

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一种快速、无损大豆种子DNA提取方法的建立和应用

A Rapid and Nondestructive Method for Soybean DNA Extraction and Its Application

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