Chin Bull Bot ›› 2016, Vol. 51 ›› Issue (1): 68-73.doi: 10.11983/CBB15057

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

Wen Cheng1,2, Zhengjun Xia3, Xianzhong Feng2, Suxin Yang2*   

  1. 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:2016-02-01 Published:2016-01-01
  • Contact: Yang Suxin E-mail:yangsuxin@iga.ac.cn
  • About author:? These authors contributed equally to this paper

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

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

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"

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"

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"

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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