Asmada Verimli DNA Ekstraksiyonu için Geliştirilmiş Bir Yöntem

Yıl 2023, , 21 - 36, 15.04.2023

Tülay Öncü Öner , Melih Temel Seda Pamay , Altin Kardelen Abacı , Hilal Betül Kaya Akkale

https://doi.org/10.38001/ijlsb.1150387

Cited By: 1

Öz

Asma (Vitis vinifera L.), dünya çapında en eski ve en önemli çok yıllık bitkilerden biridir ve gen haritalaması, genetik transformasyon ve DNA parmak izi gibi kapsamlı genetik çalışmalara konu olmuştur. Asmalar, polisakkaritler, polifenolik bileşikler ve birçoğu gıda, zirai kimya ve ilaç endüstrilerinde önemli öneme sahip çeşitli ikincil metabolitler bakımından zengindir. Asma kalitesinin göstergelerinden biri metabolitler olsa da, bunların varlığı asmayı DNA ekstraksiyonu en zor bitkilerden biri haline getirir. Bu metabolitler sadece DNA ekstraksiyon prosedürlerini etkilemekle kalmaz, aynı zamanda restriksiyon enzimleri ile kesimi ve PCR gibi reaksiyonları da etkiler. Genotyping by sequencing (GBS) gibi dizilemeye dayalı yeni genotipleme teknikleri, restriksiyon ve dizileme için yüksek kaliteli DNA gerektirir. Bugüne kadar asmadan DNA ekstraksiyonu için çeşitli protokoller geliştirilmiştir. Bu çalışmada, dört farklı çeşide ait asma yapraklarından DNA ekstraksiyon performansı için üç farklı protokol karşılaştırılmıştır. Bu yöntemlerin etkinlikleri, ekstrakte edilen DNA'nın miktarı ve kalitesi ile belirlenmiştir. GBS'ye uygunluğu doğrulamak için, ekstrakte edilen DNA, restriksiyon enzimleri ile kesilmiştir. Tüm protokoller geleneksel CTAB yöntemine dayanmasına rağmen, farklı DNA verimi ve restriksiyon kesimi verimliliği ile sonuçlanmıştır. PVP-40 ve ß-merkaptoetanol içeren modifiye protokolün, restriksiyona uygun yüksek kalite ve miktarda asma DNA'sı elde etmek için en etkili yöntem olduğu tespit edilmiştir.

Anahtar Kelimeler

asma, DNA, restriksiyon, GBS

Destekleyen Kurum

Manisa Celal Bayar Üniversitesi

Proje Numarası

2017-113

Teşekkür

Yazarlar, Manisa Bağcılık Araştırma Enstitüsü Müdürlüğü'ne materyal sağladıkları için teşekkür ederler.

An Improved Method for Efficient DNA Extraction from Grapevine

Öz

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops worldwide which has been the subject of extensive genetic studies including gene mapping, genetic transformation, and DNA fingerprinting. Grapevines are rich in polysaccharides, polyphenolic compounds, and various secondary metabolites, many of which have significant importance in food, agrochemical, and pharmaceutical industries. While metabolites are one of the indicators of quality of grapevines, the presence of them makes grapevine one of the most difficult plants to extract DNA from. These metabolites not only affect DNA extraction procedures but also downstream reactions such as restriction digestion and PCR. Development of new genotyping techniques based on sequencing such as genotyping by sequencing (GBS) requires high-quality DNA for digestion and sequencing. To date, several protocols have been developed for DNA extraction from grapevine. In this study, three different protocols with modifications were compared for DNA extraction performance from grapevine leaves from four different cultivars. Efficiencies of these methods were determined by extracted DNA’s quantity and quality. To confirm the suitability for GBS, extracted DNA was digested with restriction enzymes. Although all protocols were based on the traditional CTAB method, they resulted in different DNA yield and restriction digestion efficiency. The modified protocol including PVP-40 and ß-mercaptoethanol was found to be the most efficient method to obtain high quality and quantity grapevine DNA that is amenable to restriction digestion.

Anahtar Kelimeler

grapevine, DNA extraction, restriction digestion, GBS

Proje Numarası

2017-113

Kaynakça

• Li-Mallet, A., A. Rabot, and L. Geny, Factors controlling inflorescence primordia formation of grapevine: their role in latent bud fruitfulness? A review. Botany, 2016. 94(3): p. 147-163.
• Pertot, I., et al., A critical review of plant protection tools for reducing pesticide use on grapevine and new perspectives for the implementation of IPM in viticulture. Crop Protection, 2017. 97: p. 70-84.
• Petronilho, S., M.A. Coimbra, and S.M. Rocha, A critical review on extraction techniques and gas chromatography based determination of grapevine derived sesquiterpenes. Analytica Chimica Acta, 2014. 846: p. 8-35.
• Sat, I., M. Sengul, and F. Keles, Use of grape leaves in canned food. Pakistan Journal of Nutrition, 2002. 1(6): p. 257-262.
• Cosme, F., T. Pinto, and A. Vilela, Oenology in the kitchen: The sensory experience offered by culinary dishes cooked with alcoholic drinks, grapes and grape leaves. Beverages, 2017. 3(3): p. 42.
• Anđelković, M., et al., Phenolic compounds and bioactivity of healthy and infected grapevine leaf extracts from red varieties Merlot and Vranac (Vitis vinifera L.). Plant Foods for Human Nutrition, 2015. 70(3): p. 317-323.
• Fernandes, B., et al., Volatile components of vine leaves from two Portuguese grape varieties (Vitis vinifera L.), Touriga Nacional and Tinta Roriz, analysed by solid-phase microextraction. Natural Product Research, 2015. 29(1): p. 37-45.
• Dani, C., et al., Phenolic content of grapevine leaves (Vitis labrusca var. Bordo) and its neuroprotective effect against peroxide damage. Toxicology in Vitro, 2010. 24(1): p. 148-153.
• Lodhi, M.A., et al., A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter, 1994. 12(1): p. 6-13.
• Sefc, K.M., et al., Genetic analysis of grape berries and raisins using microsatellite markers. VITIS-GEILWEILERHOF-, 1998. 37: p. 123-126.
• Marsal, G., et al., Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L. OENO One, 2013. 47(4): p. 227-237.
• Ojeda, H., et al., Berry development of grapevines: relations between the growth of berries and their DNA content indicate cell multiplication and enlargement. Vitis, 1999. 38(4): p. 145-150.
• Alfonzo, A., et al., A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis. African Journal of Biotechnology, 2012. 11(45): p. 10305-10309.
• Friar, E.A., Isolation of DNA from plants with large amounts of secondary metabolites. Methods in Enzymology, 2005. 395: p. 1-12.
• Agrawal, A., A. Sharma, and N.P. Shukla, Genomic DNA extraction protocol for Artemisia annua L. without using liquid nitrogen and phenol. International Journal of Applied Sciences and Biotechnology, 2016. 4(4): p. 448-451.
• Sahu, S.K., M. Thangaraj, and K. Kathiresan, DNA extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. International Scholarly Research Notices, 2012. 2012.
• Adams, R.P. and N. Do, A simple technique for removing plant polysaccharides contaminants from DNA. BioTechniques, 1991. 10(2): p. 162-164.
• Agarwal, M., N. Shrivastava, and H. Padh, Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Reports, 2008. 27(4): p. 617-631.
• Khlestkina, E., Molecular markers in genetic studies and breeding. Russian Journal of Genetics: Applied Research, 2014. 4(3): p. 236-244.
• Theocharis, A., et al., Study of genetic diversity among inter-intraspesific hybrids and original grapevine varieties using AFLP molecular markers. Australian Journal of Crop Science, 2010. 4(1): p. 1-8.
• Li, Z.T., S. Dhekney, and D. Gray, Molecular characterization of a SCAR marker purportedly linked to seedlessness in grapevine (Vitis). Molecular Breeding, 2010. 25(4): p. 637-644.
• Zhao, M., et al., A new strategy for complete identification of 69 grapevine cultivars using random amplified polymorphic DNA (RAPD) markers. African Journal of Plant Science, 2011. 5(4): p. 273-280.
• Guo, D., et al., Genetic variability and relationships between and within grape cultivated varieties and wild species based on SRAP markers. Tree Genetics & Genomes, 2012. 8(4): p. 789-800.
• Lorenzis, G.d., et al., Study of genetic diversity in V. vinifera subsp. sylvestris in Azerbaijan and Georgia and relationship with species of the cultivated compartment. Acta Horticulturae, 2015(1074): p. 49-53.
• Alipour, H., et al., Genotyping-by-sequencing (GBS) revealed molecular genetic diversity of Iranian wheat landraces and cultivars. Frontiers in Plant Science, 2017. 8: p. 1293.
• Elshire, R.J., et al., A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PloS One, 2011. 6(5): p. e19379.
• Poland, J.A., et al., Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PloS One, 2012. 7(2): p. e32253.
• Peterson, G.W., et al., Genotyping-by-sequencing for plant genetic diversity analysis: a lab guide for SNP genotyping. Diversity, 2014. 6(4): p. 665-680.
• Matasyoh, L.G., et al., Leaf storage conditions and genomic DNA isolation efficiency in Ocimum gratissimum L. from Kenya. African Journal of Biotechnology, 2008. 7(5).
• Till, B.J., et al., Low-cost methods for molecular characterization of mutant plants: tissue desiccation, DNA extraction and mutation discovery: protocols. 2015, Springer Nature.
• Khan, S., et al., Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. African Journal of Biotechnology, 2007. 6(3): p. 175-178.
• Doyle, J., DNA protocols for plants, in Molecular techniques in taxonomy. 1991, Springer. p. 283-293.
• Harding, K. and K. Roubelakis-Angelakis, The isolation and purification of DNA from Vitis vinifera L. plants and in vitro cultures. Vitis, 1994. 33(4): p. 247-248.
• Steenkamp, J., et al., Improved method for DNA extraction from Vitis vinifera. American Journal of Enology and Viticulture, 1994. 45(1): p. 102-106.
• Satyanarayana, S.D., M. Krishna, and P.P. Kumar, Optimization of high-yielding protocol for DNA extraction from the forest rhizosphere microbes. 3 Biotech, 2017. 7(2): p. 1-9.
• Salgotra, R.K. and B.S. Chauhan, Comparison of genomic DNA extraction methods to obtain high DNA quality from barnyard grass (Echinochloa colona). 2020.
• Ali, K., et al., Metabolic constituents of grapevine and grape-derived products. Phytochemistry Reviews, 2010. 9(3): p. 357-378.
• Obi, Q.N., et al., Development of Efficient Genotyping Workflow for Accelerating Maize Improvement in Developing Countries. 2020.
• Ali, F., et al., Genetic diversity, population structure and marker-trait association for 100-seed weight in international safflower panel using silicoDArT marker information. Plants, 2020. 9(5): p. 652.
• Gbedevi, K.M., et al., Genetic Diversity and Population Structure of Cowpea [Vigna unguiculata (L.) Walp.] Germplasm Collected from Togo Based on DArT Markers. Genes, 2021. 12(9): p. 1451.
• Rodrigues, P., A. Venâncio, and N. Lima, Toxic reagents and expensive equipment: are they really necessary for the extraction of good quality fungal DNA? Letters in Applied Microbiology, 2018. 66(1): p. 32-37.
• Karakousis, A., et al., An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR. Journal of Microbiological Methods, 2006. 65(1): p. 38-48.
• Rezadoost, M.H., M. Kordrostami, and H.H. Kumleh, An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants. 3 Biotech, 2016. 6(1): p. 61.
• Deepa, K., et al., A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.). Physiology and Molecular Biology of Plants, 2014. 20(2): p. 263-271.
• Sedlackova, T., et al., Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biological Procedures Online, 2013. 15(1): p. 1-8.
• Simbolo, M., et al., DNA qualification workflow for next generation sequencing of histopathological samples. PloS One, 2013. 8(6): p. e62692.
• Garcia-Elias, A., et al., Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs. Scientific Reports, 2017. 7(1): p. 1-14.
• Labra, M., et al., Extraction and purification of DNA from grapevine leaves. VITIS-GEILWEILERHOF-, 2001. 40(2): p. 101-102.
• Campa, A. and J.J. Ferreira, Genetic diversity assessed by genotyping by sequencing (GBS) and for phenological traits in blueberry cultivars. PloS One, 2018. 13(10): p. e0206361.
• Kumar, S., et al., Genotyping-by-sequencing of pear (Pyrus spp.) accessions unravels novel patterns of genetic diversity and selection footprints. Horticulture Research, 2017. 4(1): p. 1-10.
• Gürcan, K., et al., Genotyping by sequencing (GBS) in apricots and genetic diversity assessment with GBS-derived single-nucleotide polymorphisms (SNPs). Biochemical Genetics, 2016. 54(6): p. 854-885.
• Micheletti, D., et al., Whole-genome analysis of diversity and SNP-major gene association in peach germplasm. PloS One, 2015. 10(9): p. e0136803.
• Larsen, B., et al., Population structure, relatedness and ploidy levels in an apple gene bank revealed through genotyping-by-sequencing. PLoS One, 2018. 13(8): p. e0201889.
• Islam, A., et al., Genetic Diversity and Population Structure Analysis of the USDA Olive Germplasm Using Genotyping-By-Sequencing (GBS). Genes, 2021. 12(12): p. 2007.
• Gardner, K.M., et al., Fast and cost-effective genetic mapping in apple using next-generation sequencing. G3: Genes, Genomes, Genetics, 2014. 4(9): p. 1681-1687.
• Guajardo, V., et al., Construction of high density sweet cherry (Prunus avium L.) linkage maps using microsatellite markers and SNPs detected by genotyping-by-sequencing (GBS). PloS One, 2015. 10(5): p. e0127750.
• Migicovsky, Z., et al., Genomic consequences of apple improvement. Horticulture Research, 2021. 8(1): p. 1-13.
• McClure, K.A., et al., A genome‐wide association study of apple quality and scab resistance. The Plant Genome, 2018. 11(1): p. 170075.
• Kaya, H.B., et al., Genome wide association study of 5 agronomic traits in olive (Olea europaea L.). Scientific reports, 2019. 9(1): p. 1-14.
• Li, Y., et al., Genomic selection for non-key traits in radiata pine when the documented pedigree is corrected using DNA marker information. BMC Genomics, 2019. 20(1): p. 1-10.
• Nsibi, M., et al., Adoption and Optimization of Genomic Selection To Sustain Breeding for Apricot Fruit Quality. G3: Genes, Genomes, Genetics, 2020. 10(12): p. 4513-4529.
• Yang, S., et al., Next generation mapping of enological traits in an F2 interspecific grapevine hybrid family. PloS One, 2016. 11(3): p. e0149560.
• Barba, P., et al., Grapevine powdery mildew resistance and susceptibility loci identified on a high-resolution SNP map. Theoretical and Applied Genetics, 2014. 127(1): p. 73-84.
• Hyma, K.E., et al., Heterozygous mapping strategy (HetMappS) for high resolution genotyping-by-sequencing markers: a case study in grapevine. PloS One, 2015. 10(8): p. e0134880.
• Tello, J., et al., A novel high-density grapevine (Vitis vinifera L.) integrated linkage map using GBS in a half-diallel population. Theoretical and Applied Genetics, 2019. 132(8): p. 2237-2252.
• Jang, H.A. and S.-K. Oh, Development of an efficient genotyping-by-sequencing (GBS) library construction method for genomic analysis of grapevine. Korean Journal of Agricultural Science, 2017. 44(4): p. 495-503.
• Akkurt, M., Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.). Genetics and Molecular Research, 2012. 11(3): p. 2343-2351.