Simple and efficient protocol for DNA extraction from the coffee tree (Coffea canephora Pierre) seeds
Keywords:
DNA, coffee tree, extraction, genetics, breeding, seedsAbstract
The first step in any genetic study is to have a protocol for DNA isolation. Although leaf tissue is the most commonly used plant material for this purpose, seeds are another option when leaf tissue is not available. In addition, during seed transfer from the field to the laboratory, DNA degradation does not occur, and liquid nitrogen is not needed for preservation. However, DNA extraction from seeds of some species, such as coffee trees, is complicated due to their hardness and content of phytochemical compounds. In this work, we propose optimizing a DNA extraction protocol from coffee tree (C. canephora) seeds for molecular analysis. The CTAB method was used as a control and three variants of this method. DNA quality was checked in agarose gels, and its concentration was determined using a nano-spectrophotometer. DNA amplification was performed by PCR with a RAPD primer and checked in electrophoresis. It was possible to obtain DNA free of fats and polysaccharides, with sufficient quality for PCR. The quality was demonstrated by gel observation, concentration values, and absorbance ratios, as well as by obtaining the bands produced by the primer. This protocol allows access to the genetic characterization of the coffee plant, essential for conserving its diversity and for genetic improvement.
References
1. Bramel P, Krishnan S, Horna D, Lainoff B, Montagnon C. Global conservation strategy for coffee genetic resources [Internet]. Crop Trust- World Coffee Research; 2017 [Consultado 24 Sept 2023]. 71 p. Disponible en: https://worldcoffeeresearch.org/media/documents/Coffee_Strategy_Low_Res.pdf.
2. Davis AP, Rakotonasolo F. Six new species of coffee (Coffea) from northern Madagascar. Kew Bull. 2021; 76: 497-511.
3. Krishnan S, Matsumoto T, Nagai C, Falconer J, Shriner S, Long J, Medrano JF, Vega F E. Vulnerability of coffee (Coffea spp.) genetic resources in the United States. Genet Resour Crop Evol. 2021; 68: 2691-710.
4. Aguilar ME, Ortiz JL, Mesén F, Jiménez D, Altmann F. Cafe Arabica Coffea arabica L. En Jain SM, Gupta PPK, editores. Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants [Internet]. Springer Nature; 2018 [Consultado 18 Sept 2023]. p. 39-62. Disponible en: https://link.springer.com/chapter/10.1007/978-3-319-79087-9_3.
5. International Coffee Organization. Coffee Report and Outlook [Internet]. 2023 [Consultado 11 Oct 2023]. Disponible en: https://icocoffee.org/documents/cy2022-23/Coffee_Report_and_Outlook_April_2023_-_ICO.pdf.
6. González ME, Rosales PR, Castilla Y, Lacerra JA, Ferrer M. Efecto del Bioenraiz® como estimulante de la germinación y el desarrollo de plántulas de cafeto (Coffea arabica L.). Cultivos Tropicales. 2015; 36(1): 73-9.
7. Davis AP, Chadburn H, Moat J, O’Sullivan R, Hargreaves S, Lughadha EN. High extinction risk of wild coffee species and implications for coffee sector sustainability. Sci Adv [Internet]. 2019 [Consultado 31 Oct 2023]; 5(1): eaav3473. Disponible en: https://doi.org/10.1126/sciadv.aav3473.
8. Ramírez IM, Rodríguez NN, Valdés-Infante J, Capote M, Becker D, Rohde W. Isolation of genomic DNAs from the tropical fruit trees avocado, coconut, guava and mango for PCR-based DNA marker application. Cultivos Tropicales. 2004; 25(1): 33-8.
9. Patay ÉB, Sali N, Kőszegi T, Csepregi R, Balázs VL, Németh TS, Németh T, Papp N. Antioxidant potential, tannin and polyphenol contents of seed and pericarp of three Coffea species. Asian Pac J Trop Med. 2016; 9(4): 366-71.
10. Speer K, Kölling-Speer I. The lipid fraction of the coffee bean. Braz J Plant Physiol. 2006; 18(1): 201-16.
11. Antoine G, Vaissayre V, Meile JC, Payet J, Conéjéro G, Costet L, Fock-Bastide I, Joët T, Dussert S. Diterpenes of Coffea seeds confer defense against fungi and insects and are transferred from the endosperm to the seedling after germination. Plant Physiol Biochem. 2023; 194: 627-37.
12. Kumar M, Sandhya N, Ram AS, Sreenath HL, Jayarama. A simple method of DNA extraction from coffee seeds suitable for PCR analysis. Afr J Biotechnol. 2008; 7(4): 409-13.
13. Diniz LEC, Ruas CF, Carvalho VP, Torres FM, Ruas EA, Santos MO, Sera T, Ruas PM. Genetic diversity among forty coffee varieties assessed by RAPD markers associated with restriction digestion. Braz Arch Biol Technol. 2005; 48(4): 511-21.
14. Bressan EA, Rossi ML, Gerald LT, Figueira A. Extraction of high-quality DNA from ethanol-preserved tropical plant tissues. BMC Res Notes [Internet]. 2014 [Consultado 31 Oct 2023]; 7: 268. Disponible en: http://www.biomedcentral.com/1756-0500/7/268.
15. Suazo T, Miranda S, Rivers E, Lacayo M, Tenorio D. Evaluación de metodologías de extracción de ADN de plantas recalcitrantes. Revista Torreón Universitario. 2020; 9(24): 45-57.
16. Parveen A, Kumar D. Comparative study of different approaches to isolate DNA from Coffea arabica leaves. Glob J Res Anal. 2023; 12(5): 52-3.
17. Pokharel S, Khanal BC, Basnetb A, Pandey GR, Basnet S. DNA extraction and PCR optimization for DNA barcode analysis of commercially-grown coffee varieties in Nepal. J Sci Eng Technol [Internet]. 2023 [Consultado 30 Nov 2023]; 17(1). Disponible en: https://journals.ku.edu.np/kuset/article/view/124/48.
18. Crochemore M L, Nunes ML, Andrade GA, Molinare HBC, Vasconcellos ME. Varietal identification of coffee seeds by RAPD technique. Braz Arch Biol Technol. 2004; 47(1): 7-11.
19. Martellossi C, Taylor EJ, Lee, D, Graziosi G, Donini P. DNA Extraction and analysis from processed coffee beans. J Agric Food Chem. 2005; 53(22): 8432-436.
20. Križman M, Jakše J, Baričevič D, Javornik B. Robust CTAB-activated charcoal protocol for plant DNA extraction. Acta Agric Slov. 2006; 87(2): 427-33.
21. González ME, Rodríguez J. Respuesta de plantas de Coffea canephora a la inoculación con hongos micorrizógenos arbusculares durante la fase de aclimatización. Cultivos Tropicales. 2004, 25(1): 13-6.
22. Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. 1987; 19(1): 11-5.
23. Vargas-Segura C, López-Gamboa E, Araya-Valverde E, Valdez-Melara M, Gatica-Arias A. Sensitivity of seeds to chemical mutagens, detection of DNA polymorphisms and agro-metrical traits in M1 generation of coffee (Coffea arabica L.). J Crop Sci Biotechnol. 2019; 22: 451-64.
24. Sant’Ana GC, Pereira LFP, Pot D, Ivamoto ST, Domingues DS, Ferreira RV, Pagiatto N F, da Silva BSR, Nogueira LM, Kitzberger CSG, Scholz MBS, de Oliveira FF, Sera GH, Padilha L, Labouisse J-P, Guyot R, Charmetant P, Leroy T. Genome-wide association study reveals candidate genes influencing lipids and diterpenes contents in Coffea arabica L. Sci Rep. [Internet]. 2018 [Consultado 30 Nov 2023]; 8, 465. Disponible en: https://doi.org/10.1038/s41598-017-18800-1
25. Ramos SNM, Salazar MM, Pereira GAG, Efraim P. Plant and metagenomic DNA extraction of mucilaginous seeds. MethodsX. 2014; 1: 225-28.
26. Velasco R. Marcadores moleculares y la extracción de ADN. Biotecnología en el Sector Agropecuario y Agroindustrial. 2005; 3(1): 14-8.
27. Heikrujam J, Kishor R, Mazumder PB. The chemistry behind plant DNA isolation protocols. En: Boldura OM, Baltă C, Awwad NS, editores. Biochemical. Analysis Tools - Methods for Bio-Molecules Studies [Internet]. IntechOpen; 2020 [Consultado 16 Sep 2023]. Disponible en: http://dx.doi.org/10.5772/intechopen.92206.
Published
How to Cite
Issue
Section
Categories
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with BioNatura Journal agree to the following terms: Authors retain copyright and grant the BioNatura Institutional Publishing Consortium (BIPC) right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). This allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
