DNA Extraction and PCR Optimization of Coffea arabica L. and Coffea canephora Pierre ex A. Froehner
DOI:
10.29303/jppipa.v10iSpecialIssue.7881Published:
2024-08-25Issue:
Vol. 10 No. SpecialIssue (2024): Science Education, Ecotourism, Health ScienceKeywords:
Coffea, CTAB, Extraction DNA, matK, rbcLResearch Articles
Downloads
How to Cite
Downloads
Metrics
Abstract
DNA isolation is an important step for further molecular analysis. Coffee plants contain polysaccharides, polyphenols, and secondary metabolites which can contaminate the results of DNA isolation. This study was conducted to isolate DNA from arabica and robusta coffee leaves using a modified CTAB method and PCR optimization for amplification of matK and rbcL genes. DNA was isolated using buffers (CTAB 10%, PVP 1%, β-mercaptoethanol 1%, Tris-HCl 1M, NaCl 5M, and EDTA 0.5%), and eluted with TE-RNase. Genomic DNA of ten coffee plants was successfully isolated with concentrations ranging from 33-146 nm/µL with purity (A260/A280) 1.7-1.9. Based on the genomic DNA isolated, matK and rbcL genes were amplified with initial denaturation conditions of 94°C (for 1 minute of matK and 4 minutes of rbcL) followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing (adjusted to each primer), and extension at 72°C for 1 minute. The optimal PCR conditions were effective to amplify matK (900 bp) and rbcL (600 bp) genes. Thus, the modified DNA isolation method and PCR optimization can be used as an efficient tool for further molecular analysis of Coffea arabica L. and Coffea canephora Pierre ex A. Froehner.
References
Aga, E. (2005). Molecular genetic diversity study of forest coffee tree (Coffea arabica L.) population in Ethiopia: implications for conservation and breeding. Swedish University of Agricultural Sciences, Alnarp.
Badan Pusat Statistik. (2019). Statistik kopi Indonesia. Jakarta: Badan Pusat Statistik Indonesia.
Badan Pusat Statistik. (2021). Provinsi Aceh dalam angka (Aceh Province in Figures). Banda Aceh: Badan Pusat Statistik.
Benti, T. (2017). Progress in arabica coffee breeding in Ethiopia: achievements, challenges, and prospects. International Journal of Science: Basic and Applied Research, 33, 15–25. Retrieved from https://rb.gy/jubdas
Cao, E. P., Constantino-Santos, D. M., Ramos, L. A. P., Santos, B. S., Quilang, J. P., & Mojica, R. M. (2014). Molecular and morphological differentiation among Coffea (Rubiaceae) varieties grown in the farms of Cavite Province, Philippines. Philippine Science Letters, 7(2), 387–397. Retrieved from https://scienggj.org/2014/PSL 2014-vol07-no02-p387-397 Cao.pdf
Crossley, B. M., Bai, J., Glaser, A., Maes, R., Porter, E., Killian, M. L., Clement, T., & Toohey-Kurth, K. (2020). Guidelines for Sanger sequencing and molecular assay monitoring. Journal of Veterinary Diagnostic Investigation, 32(6), 767–775. https://doi.org/10.1177/1040638720905833
Doyle, J. J., & Doyle, J. L. (1987). a Rapid Dna Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bulletin, 19(1), 11–15. Retrieved from https://webpages.charlotte.edu/~jweller2/pages/BINF8350f2011/BINF8350_Readings/Doyle_plantDN
Dzikrina, H., Sari, D. P., Faridah, N., Saidah, S. S., Nur Alifah, S. A., & Kusumawaty, D. (2022). Penanda DNA: Uji Halal pada Makanan Olahan Daging Menggunakan Primer Multiplex PCR (Polymerase Chain Reaction). Jurnal Bios Logos, 12(1), 1. https://doi.org/10.35799/jbl.v12i1.36437
Fazekas, A. J., Burgess, K. S., Kesanakurti, P. R., Graham, S. W., Newmaster, S. G., Husband, B. C., Percy, D. M., Hajibabaei, M., & Barrett, S. C. H. (2008). Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well. PLoS ONE, 3(7), e2802. https://doi.org/10.1371/journal.pone.0002802
Gupta, N. (2019). DNA extraction and polymerase chain reaction. Journal of Cytology, 36(2), 116. https://doi.org/10.4103/JOC.JOC_110_18
Heckenhauer, J., Barfuss, M. H. J., & Samuel, R. (2016). Universal multiplexable matK primers for DNA barcoding of angiosperms. Applications in Plant Sciences, 4(6), 1500137. https://doi.org/10.3732/apps.1500137
Hulupi, R., Nugroho, D., & Yusianto. (2013). Keragaman beberapa varietas kopi arabika di dataran tinggi Gayo. Pelita Perkebunan, 29, 69–81. Retrieved from https://www.ccrjournal.com/index.php/ccrj/articl
International Coffee Organization. (2020). The value of coffee: sustainability, inclusiveness, and resilience of the coffee global value chain. German Federal Ministry for Economic Cooperation and Development. Retrieved from https://www.ico.org/documents/cy2020-21/ed-2357e-cdr-2020.pdf
Khalid. (2017). ToT Budidaya kopi arabika Gayo Secara berkelanjutan. Badan Penelitian dan Pengembangan Pertanian Kementerian Pertanian.
Kress, W. J., & Erickson, D. L. (2007). A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region. PLoS ONE, 2(6), e508. https://doi.org/10.1371/journal.pone.0000508
Martellossi, C., Taylor, E. J., Lee, D., Graziosi, G., & Donini, P. (2005). DNA Extraction and Analysis from Processed Coffee Beans. Journal of Agricultural and Food Chemistry, 53(22), 8432–8436. https://doi.org/10.1021/jf050776p
Mawardi, A., & Simonapendi, M. L. (2018). Uji Efektivitas Metode Isolasi DNA Genom Kopi Arabika (Coffea arabica L.) Asal Kabupaten Jayawijaya. Jurnal Biologi Papua, 8(1), 7–12. https://doi.org/10.31957/jbp.10
Melayah, D., Bontemps, Z., Bruto, M., Nguyen, A., Oger, P., & Hugoni, M. (2023). Metabarcoding of the Three Domains of Life in Aquatic Saline Ecosystems. In Methods in Molecular Biology (Vol. 2605, pp. 17–35). https://doi.org/10.1007/978-1-0716-2871-3_2
Misra, M., JINGADE, P., & HUDED, A. K. C. (2022). DNA barcoding analysis and phylogenetic relationships of Indian wild coffee species. Turkish Journal of Botany, 46(2), 109–122. https://doi.org/10.55730/1300-008X.2675
Nakagawa, T., Doi, M., Nishi, K., Sugahara, T., Nishimukai, H., & Asano, M. (2019). A simple and versatile authenticity assay of coffee products by single-nucleotide polymorphism genotyping. Bioscience, Biotechnology, and Biochemistry, 83(10), 1829–1836. https://doi.org/10.1080/09168451.2019.1618697
Nandhini, R. M. B., Rahul, R. N., Thilaga, S., Rao, N. S. P., & Ganesh, D. (2013). Molecular distinction of C×R hybrid (Coffea congensis×Coffea canephora) from morphologically resembling male parent using rbcL and matK gene sequences. South African Journal of Botany, 88, 334–340. https://doi.org/10.1016/j.sajb.2013.08.011
O’Neill, M., McPartlin, J., Arthure, K., Riedel, S., & McMillan, N. (2011). Comparison of the TLDA with the Nanodrop and the reference Qubit system. Journal of Physics: Conference Series, 307, 012047. https://doi.org/10.1088/1742-6596/307/1/012047
Panaligan, A. C., Baltazar, M. D., & Alejandro, G. J. D. (2021). Molecular authentication of commercially cultivated coffee (Coffea spp.) in the Philippines using DNA barcodes. Intl J Agric Biol, 25(1), 227–230. Retrieved from http://www.fspublishers.org
Pokharel, S., Khanal, B. C., Basnet, A., Pandey, G. R., & Basnet, S. (2023). DNA Extraction and PCR Optimization for DNA Barcode Analysis of Commercially-Grown Coffee Varieties in Nepal. Kathmandu University Journal of Science, Engineering and Technology, 17(1), 1–7. https://doi.org/10.3126/kuset.v17i1.62399
Rodríguez-Riveiro, R., Velasco, A., & Sotelo, C. G. (2022). The Influence of DNA Extraction Methods on Species Identification Results of Seafood Products. Foods, 11(12), 1739. https://doi.org/10.3390/foods11121739
Setiati, N., Partaya, & Hidayah, N. (2020). The use of two pairs primer for CO1 gene amplification on traded stingray at fish auction Tasik Agung Rembang. Journal of Physics: Conference Series, 1567(3), 032056. https://doi.org/10.1088/1742-6596/1567/3/032056
Sun, J., Wang, Y., Liu, Y., Xu, C., Yuan, Q., Guo, L., & Huang, L. (2020). Evolutionary and phylogenetic aspects of the chloroplast genome of Chaenomeles species. Scientific Reports, 10(1), 11466. https://doi.org/10.1038/s41598-020-67943-1
Sundari, S., & Priadi, B. (2019). Teknik isolasi dan elektroforesis DNA ikan tapah. Buletin Teknik Litkayasa Akuakultur, 17(2), 87–90. Retrieved from http://ejournal-balitbang.kkp.go.id/index.php/btla/article/view/8608/6572
Viljoen, C. D., Booysen, C., & Sreenisvasan Tantuan, S. (2022). The suitability of using spectrophotometry to determine the concentration and purity of DNA extracted from processed food matrices. Journal of Food Composition and Analysis, 112, 104689. https://doi.org/10.1016/j.jfca.2022.104689
Author Biographies
Risa Riani Ramlan, Biology Department, Syiah Kuala University, Banda Aceh, Indonesia
Essy Harnelly, Syiah Kuala University, Darussalam, Banda Aceh
Lenni Fitri, Biology Department, Syiah Kuala University, Banda Aceh, Indonesia
License
Copyright (c) 2024 Risa Riani Ramlan, Harnelly, Lenni Fitri
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with Jurnal Penelitian Pendidikan IPA, agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License (CC-BY License). This license allows authors to use all articles, data sets, graphics, and appendices in data mining applications, search engines, web sites, blogs, and other platforms by providing an appropriate reference. The journal allows the author(s) to hold the copyright without restrictions and will retain publishing rights without restrictions.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Jurnal Penelitian Pendidikan IPA.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
