Jul 28, 2025
Version 2
DNA extraction, PCR amplification, and sequencing of multiple loci of Colletotrichum species from an ancient herbarium specimen V.2
- Le Dinh Thao1,2,3,
- Hyeon-Dong Shin4,
- Hyorim Choi1,
- Donghun Kang1,
- Anbazhagan Mageswari1,
- Jae Sung Lee1,
- Daseul Lee1,
- In-Young Choi2,
- Ulrike Damm5,
- Seung-Beom Hong1
- 1Korean Agricultural Culture Collection, Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, South Korea;
- 2Department of Plant Medicine, Jeonbuk National University, Jeonju 54896, South Korea;
- 3Plant Pathology and Phyto-immunology, Plant Protection Research Institute, Ha Noi 100000, Vietnam;
- 4Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, South Korea;
- 5Senckenberg Museum of Natural History Görlitz, PF 300 154, 02806 Görlitz, Germany
Protocol Citation: Le Dinh Thao, Hyeon-Dong Shin, Hyorim Choi, Donghun Kang, Anbazhagan Mageswari, Jae Sung Lee, Daseul Lee, In-Young Choi, Ulrike Damm, Seung-Beom Hong 2025. DNA extraction, PCR amplification, and sequencing of multiple loci of Colletotrichum species from an ancient herbarium specimen. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6znnzgqe/v2Version created by Seung-Beom Hong
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 28, 2025
Last Modified: July 28, 2025
Protocol Integer ID: 223384
Keywords: multiple genes of colletotrichum species, colletotrichum species, ancient herbarium specimen this protocol, ancient herbarium specimen, dna extraction, extraction, multiple gene, pcr amplification, multiple loci of colletotrichum species, dna, multiple loci
Funders Acknowledgements:
Rural Development Administration
Grant ID: PJ01728601
Ministry of Science and ICT in South Korea
Grant ID: RS-2021-NR057643
Abstract
This protocol was developed for DNA extraction, PCR amplification, and sequencing of the ITS2 region and multiple genes of Colletotrichum species from an ancient herbarium specimen.
Ancient herbarium specimen
An isotype specimen of C. humuli (MSC0211749, diseased leaf of H. lupulus) was received from the Michigan State University Herbarium (MSC); it was collected in Manhattan, Kansas, USA, on June 2, 1890.
DNA extraction
Total genomic DNA from the herbarium specimen was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) with a modification.
Approximately 5 mg of symptomatic tissue bearing Colletotrichum conidiomata was collected from the specimen using a sterile scalpel under a SteREO Discovery.V12 microscope (Carl Zeiss, Göttingen, Germany).
The tissue was transferred to a 2-mL Lysing Matrix E tube (MP Biomedicals, Eschwege, Germany) containing 0.1 mm silica spheres, 1.4 mm ceramic spheres, and one 4 mm glass bead.
After adding 400 μL AP1 buffer from the kit and 4 μL RNase, the tissue was disrupted using a FastPrep-24TM instrument (MP Biomedicals, Eschwege, Germany).
The tube was incubated overnight at 65 °C.
The subsequent steps were performed according to the manufacturer’s instructions.
PCR amplification and sequencing
To amplify the ITS2 region, a newly designed forward primer ITSCF1: 5′-GTAATGTGAATTGCAGAATTCAGTG-3′ and the reverse primer ITS4 were used.
A 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and partial sequences of the chitin synthase 1 (CHS-1), histone 3 (HIS3), and actin (ACT) genes were amplified using the primer pairs GDF1/GDR1, CHS-79F/CHS-345R, CYLH3F/CYLH3R, and ACT-512F/ACT-783R, respectively.
PCR reactions were performed in a total volume of 25 μL.
PCR master mix (2X): 12.5 μL
Nuclease-free water: 8.5 μL
Forward primer: 1 μL (4.5 pmol)
Reverse primer: 1 μL (4.5 pmol)
DNA template (100 ng/μL): 2 μL
The PCR conditions for all primer pairs: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min.
PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and checked by gel electrophoresis before being sent to Macrogen (Seoul, South Korea) for sequencing. All primers used for PCR amplification were also used for sequencing.