Optimization of different DNA extraction methods for the molecular detection of resistance genes

Year 2025, Volume: 38 Issue: 2, 69 - 74, 20.08.2025

Hakan Fidan , Ozer Calıs , Esra Nur Sarpyar

https://doi.org/10.29136/mediterranean.1730967

Abstract

Tomato spotted wilt virus (TSWV) and Pepper mild mottle virus (PMMoV) not only cause economic yield losses but also limit production for pepper plants. Resistant genes are the only reliable management strategies to control such viral diseases, but these viruses are able to overcome such resistance mechanisms. Therefore, novel resistance genes should be used to control the TSWV and PMMoV diseases. For molecular detection of resistance genes, DNA quality and purity are extremely vital to obtain proper results. This study aims to identify the best extraction method using resistance gene markers from different leaves of pepper plants. PCR analyzes revealed that fresh first real leaf gave reliable DNA quality with the CTAB DNA extraction method and then the Dellaporta extraction method which yielded lower concentrations. Although the commercial DNA extraction method offered convenient results within PCR analyzes, it is cost effective for molecular breeding such as marker assisted selection programs in developing countries. The study has clearly addressed optimized DNA concentration, using different extraction methods, leaf samples from pepper plants and their storage conditions for ultimate results in breeding programs.

Keywords

DNA quality, Purity, Optimization, Extraction methods, PCR

Project Number

The article did not support with a project

References

• Aka-Kaçar, Y (2003) Bitkilerde DNA İzolasyonu. Alata Bahçe Kültürleri Araştırma Enstitüsü Dergisi 2: 1-3.
• Aleksić JM, Stojanović D, Banović, B et al. (2012) A Simple and Efficient DNA Isolation Method for Salvia officinalis. Biochemical Genetics 50: 881-892.
• Anandakumar P, Kamaraj S, Jagan S, Ramakrishnan G, Vinodhkumar R, Devaki D (2008) Capsaicin modulates pulmonary antioxidant defense system during benzo(a) pyrene-induced lung cancer in Swiss Albino mice. Phytoterapy Research 22: 529-533.
• Christo PJ, Cauley BD (2009) Chapter 36 - Postherpetic Neuralgia. In: Current Therapy in Pain. Elsevier Inc. pp. 261-267. https://doi.org/10.1016/B978-1-4160-4836-7.00036-5.
• Dellaporta SL, Wood J, Hicks JB (1983) A Plant DNA Minipreparation: Version 11. Plant Molecular Biology Reporter 1: 19-21.
• Doyle JJ, Doyle JL (1987) Isolation of Plant DNA Fresh Tissue. Focus 12: 13-15.
• Bozkaya F (2012) DNA İzolasyonunda Fenol-Kloroform Yerine Potasyum Asetat Kullanımının DNA Miktarı ve Kalitesi Üzerine Etkisi. Harran Üniversitesi Veterinerlik Fakültesi Dergisi 1(2): 92-96.
• FAO (2018) Food and Agriculture Organization of The United Nation statistical database. https://www.fao.org/faostat/en/#data/QI. Accessed 28 June, 2025.
• Fidan H, Barut M (2019) Screening of L4 resistance status to pepper mild mottle virus (PMMoV) and characterization by molecular methods. Mediterranean Agricultural Sciences 32 (3): 297-305.
• Furda A, Santos JH, Meyer JN, Van Houten B (2014) Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods in Molecular Biology 1105: 419-437.
• Gallagher SR (2011) Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current Protocols in Molecular Biology Jan; Appendix 3: 3D. Https://doi: 10.1002/0471142727.mba03ds93. PMID: 21225635.
• Glasel JA (1995) Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratios. Biotechniques 18: 62-63.
• İkten H (2019) Molecular screening for TSWV resistance in F2 pepper (Capsicum annuum L.) genotypes from different genetic background. Mediterranean Agricultural Sciences 32(1): 43-48.
• Luciano P, Bertea CM, Temporale G, Maffei M (2007) DNA internal standard for the quantitative determination of hallucinogenic plants in plant mixtures. Forensic Science International Genetics 1: 262-266.
• Martellossi C, Taylor EJ, Lee D, Graziosi G, Donini P (2005) DNA extraction and analysis from processed coffee beans. Agricultural Chemistry Journal 53: 8432-8436.
• Matsunaga HT, Saito M, Hirai T, Yoshida T (2003) DNA markers linked to pepper mild mottle virus (PMMoV) resistant locus (L4) in Capsicum. Journal of the Japanese Society for Horticultural Science 72: 218-220.
• Mitsouras K, Faulhaber EA (2009) Saliva as an alternative source of high yield canine genomic DNA for genotyping studies. BMC Research Notes 2: 219.
• Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H (1986) Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symposia on Quantitative Biology 51: 263-273.
• Nadeem MA, Nawaz MA, Shahid MQ, Doğan Y, Comertpay G, Yıldız M, Baloch FS (2017) DNA molecular markers in plant breeding: current status and recent advancements in genomic selection and genome editing. Biotechnology and Biotechnological Equipment 32(2): 261-285.
• Pierre JS, Giblot-Ducray D, McKay AC (2018) DNA based diagnostic for the quantification of sugarcane root DNA in the field. Scientific Reports 8: 16720.
• Santos TR, José BP, Franco FA, Schuster I (2010) Inheritance and potential use of grain color in the Inheritance and potential use of grain color in the identification of genotypes resistant to pre-harvest identification of genotypes resistant to pre-harvest sprouting in wheat sprouting in wheat. Lilian Crop Breeding and Applied Biotechnology 10: 218-224.
• Sedlackova T, Repiska G, Celec P, Szemes T, Minarik G (2013) Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biological Procedures Online 15: 5.
• Shokere LA, Holden MJ, Jenkins GR (2009) Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA. Food Control 20: 391-401.
• Spittle K, Wang S, Baybayan P (2010) Sample Quality–Effects of Contaminants on SMRTbellTM Library Preparation and Sequencing, Pacific Biosciences at https://www.pacb.com/wp-content/uploads/2015/09/Sample-Quality-and-Contamination.pdf.
• Şimşek Ö, Kanat F, Serçe S, Aka- Kaçar YA (2008) Comparisons of DNA isolation methods for some fruit species. Batı Akdeniz Tarımsal Araştırma Enstitüsü Derim Dergisi 25(1): 59-69.
• TUIK (2021) Turkish statistical Institute Database at https://data.tuik.gov.tr/Kategori/GetKategori?p=tarim-111&dil=1 Verified 27 June, 2025.
• Visvikis S, Schlenck A, Maurice M (1998) DNA extraction and stability for epidemiological studies. Clinical Chemistry and Laboratory Medicine 36: 551-555.
• Yörek N (2005) BİY 4008-Genetik Mühendisliğine Giriş Ders Notları at: https://acikders.ankara.edu.tr/course/info.php?id=1058.