A Comparison of the Efficiency of Simple DNA Extraction Methods by Direct Amplification of rbcL gene in  Capsicum annuum and Capsicum chinense

Kanya Anukulthanakorn; Sayrung  Kaewtanmaung

doi:10.53848/ssstj.v13i1.1175

Authors

Kanya Anukulthanakorn Nakhon Sawan Rajabhat University
Sayrung Kaewtanmaung Nakhon Sawan Rajabhat University

DOI:

https://doi.org/10.53848/ssstj.v13i1.1175

Keywords:

DNA extraction, rbcL gene, NaOH, Capsicum, Chilli

Abstract

Chilli landraces of Thailand are an essential spice in Thai cuisine. Taxonomically, they belong to the family Solanaceae, genus Capsicum. This study aimed to (i) survey the biodiversity of chilli varieties in Tambon Saylampong, Amphoe Thatako, Nakhon Sawan Province, and (ii) compare the efficiency of two DNA extraction methods, a commercial kit and NaOH extraction, through the direct amplification of the rcbL gene. A line transect method was used for the biodiversity survey and sample collection, covering a distance of one kilometer. Young leaves from each chilli variety were collected and used for DNA extraction using the commercial kit and NaOH extraction methods. The study found two chilli species comprising seven distinct varieties, Capsicum annuum consisted of five varieties: Chinda, Chee Fah, Khee Noo Si Moung, Khee Noo Khao, and Sai Pla Lai, and Capsicum chinense consisted of two varieties: Khee Noo Hom and Khee Noo Suan. DNA extracted using a commercial kit and NaOH extraction methods were available to measure the yield and purity by spectrophotometer. Regarding amplifying a partial sequence of the rcbL gene, DNA extracted using the commercial kit showed strong amplification in all samples. Some DNA samples of diluted DNA extracted using NaOH method were also successfully amplified, whereas all samples of non-diluted DNA extracted using NaOH method failed to amplify. These findings indicate that the commercial kit yields high-quality DNA suitable for polymerase chain reaction (PCR) applications. However, the NaOH extraction method presents a faster and more cost-effective alternative, though further optimization is needed to enhance its efficiency.

References

Amani, J., Kazemi, R., Abbasi, A. R., & Salmanian A. H. (2011). A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis. Iranian Journal of Biotechnology, 9(1), 69–71.

Basith, A., Arumingtyas, E. L., & Widodo, W. (2021). Genetic variation analysis of four local varieties of Indonesian black rice (Oryza sativa L.) based on partially rbcL cpDNA gene sequence. The Journal of Experimental Life Science, 11(1), 1–5. https://doi.org/10.21776/ub.jels.2021.011.01.01

CBOL Plant Working Group. (2009). A DNA barcode for land plants. PNAS, 106(31), 12794–12797.

El-Ashram, S., Al Nasr, I., & Suo, X. (2016). Nucleic acid protocols: Extraction and optimization. Biotechnology Reports, 12, 33–39. https://doi.org/10.1016/j.btre.2016.10.001

Farooq, Q., Shakir, M., Ejaz, F., Zafar, T., Durrani, K., & Ullah, A. (2020). Role of DNA barcoding in plant biodiversity conservation. Scholars International Journal of Biochemistry, 3(3), 48–52.https://doi.org/10.36348/sijb.2020.v03i03.002

Fay, M. F., Swensen, S. M., & Chase, M. W. (1997). Taxonomic affinities of Medusagyne oppositifolia (Medusagynaceae). Kew Bulletin, 52(1), 111–120. https://doi.org/10.2307/4117844

Ferniah, R. S., & Pujiyanto, S. (2017). Biodiversity of Indonesian red chilli (Capsicum annuum var. longum) based on morphological characters. Advanced Science Letters, 23(7), 6462–6464.https://doi.org/10.1166/asl.2017.9654

He, Y. H., Ning, G. G., Sun, Y. L., Qi, Y. C., & Bao, M. Z. (2009). Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta). Plant Breeding, 128, 92–96. https://doi.org/10.1111/j.1439-0523.2008.01536.x

Hollingsworth, P. M. (2011). Refining the DNA barcode for land plants. PNAS, 108(49), 19451–19452. https://doi.org/10.1073/pnas.1116812108

Kethom, W., Tongyoo, P., & Mongkolporn, O. (2019). Genetic diversity and capsaicinoids content association of Thai chili landraces analyzed by whole genome sequencing-based SNPs. Scientia Horticulturae, 249, 401–406. https://doi.org/10.1016/j.scienta.2019.02.022

Krishnamurthy, P. K., & Francis, R. A. (2012). A critical review on the utility of DNA barcoding in biodiversity conservation. Biodiversity and Conservation, 21, 1901–1919. https://doi.org/10.1007/s10531-012-0306-2

Mazourek, M., Pujar, A., Borovsky, Y., Paran, I., Mueller, L., & Jahn, M. M. (2009). A dynamic interface for capsaicinoid systems biology. Plant Physiology, 150(4), 1806–1821. https://doi.org/10.1104/pp.109.136549

Nelson, E. K. (1919). The constitution of capsaicin, the pungent principle of Capcicum. Journal of the American Chemical Society, 41(7), 1115–1121. https://doi.org/10.1021/ja02228a011

Osmundson, T. W., Eyre, C. A., Hayden, K. M., Dhillon, J., & Garbelotto, M. M. (2013). Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples. Molecular Ecology Resources, 13, 66–74.https://doi.org/10.1111/1755-0998.12031

Popelka, P., Jevinova, P., Smejkal, K., & Roba, P. (2017). Determination of capsaicin content and pungency level of different fresh and dried chilli pepper. Folia Veterinaria, 61(2), 11–16. https://doi.org/10.1515/fv-2017-0012

Porebski, S., Bailey, L. G., & Baum, B. R. (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter, 15(1), 8–15.

Satya, P., Mitra, S., Ray, D. P., Mahapatra, B. S., Karan, M., Jana, S., & Sharma, A. K. (2013). Rapid and inexpensive NaOH based direct PCR for amplification of nuclear and organelle DNA from ramie (Boehmeria nivea), a bast fibre crop containing complex polysaccharides. Industrial Crops and Products, 50, 532–536. https://doi.org/10.1016/j.indcrop.2013.07.049

Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and protein extraction: The past and the present. Journal of Biomedicine and Biotechnology, 2009, 1–10. https://doi.org/10.1155/2009/574398

Wang, H., Qi, M., & Cutler, A. J. (1993). A simple method of preparing plant samples for PCR. Nucleic Acids Research, 21(17). 4153–4154. https://doi.org/10.1093/nar/21.17.4153

Werner, O., Ros, R. M., & Guerra, J. (2002). Direct amplification and NaOH extraction: two rapid and simple methods for preparing bryophyte DNA for polymerase chain reaction (PCR). Journal of Bryology, 24,127–131. https://doi.org/10.1179/037366802125000980

Winter, J., Bevan, S., & Campbell, E. A. (1995). Capsaicin and pain mechanisms. British Journal of Anaesthesia, 75(2), 157–168. https://doi.org/10.1093/bja/75.2.157