Determintaion of genomic DNA quantity and purity of different geophiyte genus

Year 2014, Volume: 43 Issue: 1-2, 1 - 8, 05.05.2015

Arif Atak Belinda Aydın Yeşim Doyğacı Adnan Doğan Kutay Coşkun Yıldırım Erdal Kaya

Abstract

Ecology of our country is very convenient in terms of Geophytes. World geophytes (bulbs, tubers, tuberous root, rhizome plants and so on) are an important part in Flora of Turkey. Geophytes grown in Turkey seventy–eight belonging to the genus containing about 1,027 taxa (species, subspecies, varieties, forms) and approximately 40% of them are endemic. A large number of these species within the Yalova Atatürk Central Horticultural Research Institute are under protection. It is required to isolate DNA high purity and enough quantity to successfully carry out the study of molecular biology. Also considering the possibility of extinction of endemic geophyte species increased the importance of this work. In this study, different Geophyte species collected from Turkey ecology DNA isolated from fresh leaves. DNA isolation and cold storage of the total population was 2,517. The results showed that the modified protocol almost successfully produced a sufficient amount of DNA with high quality. It was obtained high purity and sufficient amounts of DNA from Delphinium, Erodium, Cyclamen, Fritillaria, Allium, Crocus, Primula and Anemone. However sufficient purity and quantity of DNA could not be obtained from most of the Asphodelus, Oxalis ve Asphodeline species. 65% of the isolated DNA results were successful but 35 % of the total samples were obtained poor quality DNA. The average amount of samples in a high–quality DNA isolation from 83 ng/μl, and the purity value was 1.89. In the light of these results, Even if studied excessive number of species, different leaf types and their contents although the obtained results can be said to be successful. In this study, future identification, genetic mapping, molecular research all kinds of similarities to the relationship, the source and origin of the genetic material to be protected, and storage of certain realized.

Keywords

DNA isolation, agarose gel, geophytes, spectrophotometer

Farklı geofit cinslerinin genomik DNA miktar ve saflıklarının belirlenmesi

Abstract

Ülkemiz geofitler açısından oldukça elverişli bir ekolojiye sahiptir. Dünya geofitlerinin (soğanlı, yumrulu, yumru köklü, rizomlu ve benzeri bitkiler) önemli bir kısmını barındıran Türkiye florasında geofitlerin 78 cinsine ait yaklaşık 1027 taksonu (tür, alt tür, varyete, form) yetişmekte, olup bunların yaklaşık %40’ı endemiktir. Bu türlerin büyük bir kısmı Yalova Atatürk Bahçe Kültürleri Merkez Araştırma Enstitüsü bünyesinde koruma altına alınmışlardır. Bu ekolojide moleküler biyoloji çalışmalarını başarıyla yürütebilmek için mutlaka yeterli ve yüksek saflıkta DNA izole etmek gerekmektedir. Geofitlerin endemik türlerinin yok olma ihtimalleri de düşünüldüğünde bu çalışmaların önemi daha da artmaktadır. Bu çalışmada Türkiye ekolojisinden toplanan farklı geofit cinslerine ait taze yapraklardan toplam 2517 adet DNA izolasyonu gerçekleştirilmiştir. Bu izole edilen DNA’ların saflık ve miktar tayinleri yapılarak birbirleri ile karşılaştırmaları yapılmıştır. Çalışmada en fazla Allium, Crocus, Ornithagalum, Muscari, Ophrys, Geranium, Cyclamen, Scilla, Anemone, Gagea cinslerine mensup türlerle çalışılmıştır. Bunların dışında çalışılan cinslerle birlikte toplam 26 cinsle çalışma yapılmıştır. Çalışılan cinsler içerisinde elde edilen sonuçlar topluca değerlendirildiğinde DNA izolasyonunun miktar ve saflık açısından en başarılı yapıldığı cinsler Delphinium, Erodium, Cyclamen, Fritillaria, Allium, Crocus, Primula ve Anemone olurken en sorunlu olan cinsler ise Asphodelus, Oxalis ve Asphodeline olmuştur. Çalışılan 2517 örneğin %65’lik kısmında DNA izolasyonu sorunsuz iken %35’lik kısmında ise düşük kalitede DNA elde edilmiştir. DNA izolasyonu sorunsuz olan örneklerin ortalama DNA miktarı 83 ng/μl ve saflık değeri 1.89 olmuştur. Elde edilen sonuçlar ışığında; çalışılan cins sayısının fazlalığı, yaprak tip ve içeriklerinin farklılığına rağmen iyi sonuçlar elde edilmiştir. Bu çalışma ile ileride yapılacak tanımlama, genetik haritalama, benzerlik ilişkisi gibi moleküler araştırmalar için kaynağı ve orijini belli genetik materyalin koruma altına alınması ve depolaması gerçekleştirilmiştir.

Keywords

DNA izolasyonu, agaroz jel, geofitler, spektrofotometre

References

• Aljanabi, S. M., L. Forget and A. Dookun, 1999. An Improved Rapid Protocol for the Isolation of Polysaccharides and Polyphenols–Free Sugarcane DNA. Plant Mol. Biol. Rep. 17:1–8.
• Al–Saghir, M. G., 2009. Rapid and Efficient Method of Genomic DNA Extraction from Pistachio Trees (Pistacia vera L.). Research Journal of Botany 4:70–73.
• Arbi, G., B. Naceur, C. Messaoud, M. Boussaid and M. Neffati, 2009. A Simple Rapid and Efficient Method for the Extraction of Genomic DNA from Allium roseum L. (Alliaceae). African Journal of Biotechnology 8(17):4020–4024.
• Bashir, A., 2010. Antioxidant Activity and Phenolic Compounds from Colchicum luteum Baker (Liliaceae). African Journal of Biotechnology 9(35):5762–5766.
• Beiki, A. H., F. Keify and J. Mozafari, 2010. Genetic Differentiation of Crocus Species by Random Amplified Polymorphic DNA. Genetic Engineering and Biotechnology Journal GEBJ–18:1–10.
• Beiki, A. H., F. Keify and J. Mozafari, 2011. Rapid Genomic DNA Isolation from Corm of Crocus Species for Genetic Diversity Analysis. Journal of Medicinal Plants Research 5(18):4596–4600.
• Camellia, M. O. and A. I. Malikah, 2011. A DNA Isolation Protocol Suitable for RAPD Analysis from Fresh or Herbarium–Stored Leaves of a Historic Quercus virginiana L. Journal of Plant Sciences. 6:77–87.
• Channarayappa, 2007. Molecular Biotechnology. Principles and Practices. 1st Edn. University press. London. 1228 p.
• Dehestani, A. and S. K. K. Tabar, 2007. A Rapid Efficient Method for DNA Isolation from Plants with High Levels of Secondary Metabolites. Asian Journal of Plant Sciences 6:977–981.
• Ebrahimzadeh, M. A., S. Y. Nabavi, S. F. Nabavi, F. Bahramian and A. R. Bekhradnia, 2010. Antioxidant and free Radical Scavenging Activity of H. officinalis L. var. angustifolius. V. odorata. B. hyrcana and C. speciosum. Pak. J. Pharm. Sci. 23(1):29–34.
• Fleischmann, A. and G. Heubl, 2009. Overcoming DNA Extraction Problems from Carnivorous Plants. Anales Jard. Bot. Madrid 66(2):209–215.
• Göçmen Taşkın, B., N. Vardareli, E. Doğaç, R. Mammadov ve V. Taşkın, 2012. Genetic Diversity of Natural Cyclamen alpinum Populations. Turk. J. Biol. 36:413–422.
• Khan, M. F., 2003. Evaluation of Hexaploid Wheat Genotypes by Using DNA Isolation and Gel–electrophoresis. Asian Journal of Plant Sciences 2:212–215.
• Mirmomeni, M. H., S. Sajjadi Majd, S. Sisakhtnezhad and F. Doranegard, 2010. Comparison of the Three Methods for DNA Extraction from Paraffin–embedded Tissues. Journal of Biological Sciences 10:261–266.
• Murray, M. G. and W. F. Thompson, 1980. Rapid Isolation of High Molecular Weight Plant DNA. Nucleic Acids Research 8(19):4321–4325.
• Qiagen Sample and Assay Technologies, 2006. DNeasy Plant Handbook. (http://www.qiagen.com/literature).
• Ronsted, N., S. S. Law, H. Thornton, M. F. Fay, M. W. Chase, 2005. Molecular Phylogenetic Evidence for the Monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the Infrageneric Classification of Fritillaria. Molecular Phylogenetic and Evolution 35:509–527.
• Sahasrabudhe, A. and M. Deodhar, 2010. Standardization of DNA Extraction and Optimization of RAPD–PCR Conditions in Garcinia indica. International Journal of Botany 6:293–298.
• Salem, H. H., T. H. Huang, B. A. Ali and Q. D. Xie, 2006. Differentiation of Bacillus thuringiensis and Escherichia coli by the Randomly Amplified Polymorphic DNA Analysis. Journal of Applied Sciences 6:1540–1546.
• Şener, B., M. Koyuncu, F. Bingöl and F. Muhtar, 1997. Production of Bioactive Alkaloids from Turkish Geophytes. International Conference on Biodiversity and Bioresources: Conservation and Utilization, 23–27 November 1997, Phuket, Thailand, pp:1–6.
• Shankar, K., L. Chavan, S. Shinde and B. Patil, 2011. An Improved DNA Extraction Protocol from Four in vitro Banana Cultivars. Asian Journal of Biotechnology 3:84–90.
• Shashi, R., K. Garima, S. J. Vikash, J.P. Bhatt and G. Sanjay, 2010. Standardization of Extraction of Genomic DNA and PCR–RFLP Conditions of Allium stracheyi: A High Altitude Plant. Academia Arena 2(7):11–14.
• Silva, J. A. T. D., 2005. Effectiveness of DNA Extraction Protocols for Horticultural and Physiological Model Plant Analyses. International Journal of Botany 1:93–99.
• Srivastava, N., S. Vikas, K. Barkha, A. K. Dobriyal and S. J. Vikash, 2010. Polyphenolics free DNA Isolation from Different Types of Tissues of Aconitum heterophyllum wall–Endangered Medicinal Species. Journal of Plant Sciences 5:414–419.
• Tiwari, K. L., S. K. Jadhav and S. Gupta, 2012. Modified CTAB Technique for Isolation of DNA from Some Medicinal Plants. Research Journal of Medicinal Plant 6(1):65–73.
• Van Tuyl, J. M. and E. Boon, 1996. Variation in DNA–Content in the Genus Lilium. Proc. Int’l Symp. on Flower Bulbs. Acta Hort. 430:829–835.
• Varma, A., H. Padh and N. Shrivastava, 2007. Plant Genomic DNA Isolation: An Art or a science. Biotechnol. J. 2:386–392.
• Vural, H. C., 2009. Genomic DNA Isolation from Aromatic and Medicinal Plants Growing in Turkey. Sci. Res. Essays 4:59–64
• Zhang, J. and J. M. Stewart, 2000. Economical and Rapid Method for Extracting Cotton Genomic DNA. J. Cotton Sci. 4:193–201.
• Ziv, M., 1997. The Contribution of Biotechnology to Breeding Propagation and Disease Resistance in Geophytes. Biotechnology and Propagation. Int. Proc. Int’l Symp. On Flower Bulbs. (Eds.): H. Lilien–Kipnis. A. H. Halevy. A. Borochov. Acta Hort. 430:247–258.