Research Articles
Early Access
Optimised DNA isolation protocols for key arid and semi-arid fruit trees to enhance genetic improvement
ICAR-Krishi Vigyan Kendra, Tamil Nadu Agricultural University, Salem 636 203, Tamil Nadu, India
Department of Plant Biotechnology, Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India
Regional Sericultural Research Station, Central Silk Board, Mulugu 502 279, Telangana, India
Horticultural College & Research Institute, Tamil Nadu Agricultural University, Paiyur 635 112, Krishnagiri, Tamil Nadu, India
Horticultural College & Research Institute, Tamil Nadu Agricultural University, Paiyur 635 112, Krishnagiri, Tamil Nadu, India
Dr. M.S. Swaminathan Agricultural College & Research Institute, Tamil Nadu Agricultural University, Thanjavur 614 902, Tamil Nadu, India
Abstract
The study was conducted at Forest College & Research Institute, Mettupalayam, Tamil Nadu to optimize the DNA extraction protocols for important arid and semi-arid fruit trees. Arid and semi-arid fruit trees, including Emblica officinalis, Aegle marmelos, Ziziphus mauritiana, Buchanania lanzan and Carissa grandiflora, play a crucial role in India’s rural economy by providing nutritional security and supporting agroforestry systems. However, the availability of quality planting materials resilient to climate variability remains a big challenge. DNA analysis is a valuable tool for the genetic improvement of long-rotation tree species, aiding in the selection of high-yield and climate-resilient genotypes. Effective DNA extraction is essential for molecular studies but is hindered by the presence of high levels of polysaccharides, polyphenols and tannins. This study aimed to standardize species-specific DNA isolation protocols using three methods CTAB, modified CTAB and SDS. DNA yield and purity were assessed using a Thermo Scientific NanoDrop™ 1000 Spectrophotometer and agarose gel electrophoresis. The highest DNA yield for E. officinalis was obtained using the modified CTAB method (1074 ng/µL), while A. marmelos, B. lanzan and C. grandiflora showed optimal results with the CTAB method, yielding 508 ng/µL, 1106.6 ng/µL and 474 ng/µl, respectively. For Z. mauritiana, the SDS method was superior, yielding 1159.6 ng/µL with a purity ratio (A260/A280) of 1.6. These optimized protocols facilitate molecular breeding, stress-resistance screening and genetic enhancement of these vital species.
References
- 1. Barta CE, Bolander B, Bilby SR, Brown JH, Brown RN, Duryee AM, et al. In situ dark adaptation enhances the efficiency of DNA extraction from mature pin oak (Quercus palustris) leaves, facilitating the identification of partial sequences of the 18S rRNA and isoprene synthase (IspS) genes. Plants (Basel). 2017;6(4):52. https://doi.org/10.3390/plants6040052
- 2. Azmat MA, Khan IA, Cheema HM, Rajwana IA, Khan AS, Khan AA. Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L. J Zhejiang Univ Sci B. 2012;13(4):239–43. https://doi.org/10.1631/jzus.B1100194
- 3. Arif IA, Bakir MA, Khan HA, Ahamed A, Al Farhan AH, Al Homaidan AA, et al. A simple method for DNA extraction from mature date palm leaves: impact of sand grinding and composition of lysis buffer. Int J Mol Sci. 2010;11(9):3149–57. https://doi.org/10.3390/ijms11093149
- 4. Klass MR. In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes. Cell Differ. 1975;3(6):335–45. https://doi.org/10.1016/0045-6039(75)90002-0
- 5. Attikora AJP, Silue S, Yao SDM, De Clerck C, Shumbe L, Diarrassouba N, et al. An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant shea tree (Vitellaria paradoxa) plant and its suitability for downstream applications. Mol Biol Rep. 2024;51(1):171. https://doi.org/10.1007/s11033-023-09098-6
- 6. Kerio S, Terhonen E, LeBoldus JM. Safe DNA-extraction protocol suitable for studying tree-fungus interactions. Bio Protoc. 2020;10(11):e3634. https://doi.org/10.21769/BioProtoc.3634
- 7. Costa J, Melo VS, Santos CG, Oliveira MB, Mafra I. Tracing tree nut allergens in chocolate: a comparison of DNA extraction protocols. Food Chem. 2015;187:469–76. https://doi.org/10.1016/j.foodchem.2015.04.073
- 8. Eon-Duval A, Gumbs K, Ellett C. Precipitation of RNA impurities with high salt in a plasmid DNA purification process: use of experimental design to determine reaction conditions. Biotechnol Bioeng. 2003;83(5):544–53. https://doi.org/10.1002/bit.10704
- 9. Masoomi-Aladizgeh F, Jabbari L, Khayam Nekouei R, Aalami A, Atwell BJ, Haynes PA. A universal protocol for high-quality DNA and RNA isolation from diverse plant species. PLoS One. 2023;18(12):e0295852. https://doi.org/10.1371/journal.pone.0295852
- 10. De Silva S, Ocana-Rios I, Cagliero C, Gostel MR, Johnson G, Anderson JL. Isolation of DNA from plant tissues using a miniaturized matrix solid-phase dispersion approach featuring ionic liquid and magnetic ionic liquid solvents. Anal Chim Acta. 2023;1245:340858. https://doi.org/10.1016/j.aca.2023.340858
- 11. Tamm T, Kristjuhan A. Protocol for rapid and cost-effective extraction of genomic DNA from a wide range of wild yeast species for use in PCR-based applications. STAR Protoc. 2024;5(3):103282. https://doi.org/10.1016/j.xpro.2024.103282
- 12. Van Caenegem W, Haelewaters D. New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales). IMA Fungus. 2024;15(1):14. https://doi.org/10.1186/s43008-024-00146-9
- 13. Pearce DL, Edson JE, Jennelle CS, Walter WD. Evaluation of DNA yield from various tissue and sampling sources for use in single nucleotide polymorphism panels. Sci Rep. 2024;14(1):11340. https://doi.org/10.1038/s41598-024-56128-9
- 14. Scott KD, Playford J. DNA extraction technique for PCR in rain forest plant species. Biotechniques. 1996;20(6):974–9. https://doi.org/10.2144/96206bm07
- 15. Ribeiro RA, Lovato MB. Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia. Genet Mol Res. 2007;6(1):173–87.
- 16. Huanca-Mamani W, Rivera-Cabello D, Maita-Maita J. A simple, fast, and inexpensive CTAB-PVP-silica based method for genomic DNA isolation from single, small insect larvae and pupae. Genet Mol Res. 2015;14(3):8001–7. https://doi.org/10.4238/2015.July.17.8
- 17. Attitalla IH. Modified CTAB method for high quality genomic DNA extraction from medicinal plants. Pak J Biol Sci. 2011;14(21):998–9. https://doi.org/10.3923/pjbs.2011.998.999
- 18. Del Sal G, Manfioletti G, Schneider C. The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques. 1989;7(5):514–20.
- 19. Xia Y, Chen F, Du Y, Liu C, Bu G, Xin Y, et al. A modified SDS-based DNA extraction method from raw soybean. Biosci Rep. 2019;39(2):BSR20182271. https://doi.org/10.1042/BSR20182271
- 20. Natarajan VP, Zhang X, Morono Y, Inagaki F, Wang F. A modified SDS-based DNA extraction method for high quality environmental DNA from seafloor environments. Front Microbiol. 2016;7:986. https://doi.org/10.3389/fmicb.2016.00986
- 21. Chiong KT, Damaj MB, Padilla CS, Avila CA, Pant SR, Mandadi KK, et al. Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications. Plant Methods. 2017;13:106. https://doi.org/10.1186/s13007-017-0255-6
- 22. Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol. 1996;62(2):316–22. https://doi.org/10.1128/aem.62.2.316-322.1996
- 23. Khanuja SPS, Shasany AK, Darokar MP, Kumar S. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol Biol Rep. 1999;17(1):74. https://doi.org/10.1023/A:1007528101452
- 24. Dellaporta SL, Wood J, Hicks JB. A plant DNA minipreparation: version II. Plant Mol Biol Rep. 1983;1(4):19–21. https://doi.org/10.1007/BF02712670
- 25. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA. 1984;81(24):8014–8. https://doi.org/10.1073/pnas.81.24.8014
- 26. Kim CS, Lee CH, Shin JS, Chung YS, Hyung NI. A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nucleic Acids Res. 1997;25(5):1085–6. https://doi.org/10.1093/nar/25.5.1085
- 27. Weishing K, Wolff K, Meyer W. DNA isolation and purification. In: Weishing K, Wolff K, Meyer W, editors. DNA fingerprinting in plants and fungi. Boca Raton: CRC Press; 1995. p. 45–59.
- 28. Lefort F, Douglas GC. An efficient micro-method of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus and Quercus. Ann For Sci. 1999;56:259–63. https://doi.org/10.1051/forest:19990308
- 29. Shepherd LD, McLay TG. Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue. J Plant Res. 2011;124(2):311–4. https://doi.org/10.1007/s10265-010-0379-5
- 30. Pandey RN, Adams RP, Flournoy LE. Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides. Plant Mol Biol Rep. 1996;14(1):17–22. https://doi.org/10.1007/BF02671898
- 31. Nath O, Fletcher SJ, Hayward A, Shaw LM, Agarwal R, Furtado A, et al. A comprehensive high-quality DNA and RNA extraction protocol for a range of cultivars and tissue types of the woody crop avocado. Plants (Basel). 2022;11(3):242. https://doi.org/10.3390/plants11030242
- 32. Warburg O, Christian W. Isolierung und Kristallisation des Gärungsferments Enolase. Naturwissenschaften. 1941;29(39):589–90. https://doi.org/10.1007/BF01482279
Downloads
Download data is not yet available.