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

doi:10.11983/CBB15057

Abstract

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

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

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB15057

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

Figures/Tables 5

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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