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

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

Abstract

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

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.

Figures/Tables 5

References 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