[01] Macsamuel Sesugh Ugbaa, Department of Plant Breeding and Genetics, College of Agronomy, Federal University of Agriculture Makurdi, Makurdi, Nigeria; Molecular Biology Laboratory, Federal University of Agriculture Makurdi, Makurdi, Nigeria; Center for Innovation in Procurement, Environmental and Social Standards (CIPESS), Federal University of Agriculture Makurdi, Makurdi, Nigeria. [02] Godspower Chibuike Ekeuro, Department of Plant Breeding and Genetics, College of Agronomy, Federal University of Agriculture Makurdi, Makurdi, Nigeria; Molecular Biology Laboratory, Federal University of Agriculture Makurdi, Makurdi, Nigeria. [03] Judith Ogechi Ezugwu, Molecular Biology Laboratory, Federal University of Agriculture Makurdi, Makurdi, Nigeria. [04] Tersoo Kaanjo, Department of Botany, College of Science, Federal University of Agriculture Makurdi, Makurdi, Nigeria. [05] Lucky Osabuohien Omoigui, Department of Plant Breeding and Genetics, College of Agronomy, Federal University of Agriculture Makurdi, Makurdi, Nigeria; Molecular Biology Laboratory, Federal University of Agriculture Makurdi, Makurdi, Nigeria.

This study was to identify which protocol yielded much genomic DNA and to optimize genomic DNA isolation methods to determine the annealing temperature of SSR markers suitable for PCR amplification. 22 tomato accessions from Western and North Central Nigeria were planted in 4 liter plastic pots containing top soil in a screen house at University of Agriculture Makurdi. FTA card was used to isolate DNA from 14 days old leaves while CTAB method was used to extract DNA from 23 days old leaves of accessions at the Molecular Biology Laboratory of University of Agriculture Makurdi. Genomic DNA from FTA Plant Saver card and CTAB protocol was verified on 2% agarose gel and the quality of DNA was ascertain using Standard DNA ladder of 50kb. 4 SSR primers were used for PCR amplification and the products were run on 2% agarose gel stained with ethidium bromide, resolved for 2 hours at 100V and visualized under UV light. Gel image revealed sharp DNA bands from CTAB protocol unlike the DNA from FTA card, with faintly visible bands. PCR optimization revealed sharp bands with annealing temperature of 55°C, and an extension temperature of 72°C for 35 cycles. Therefore, CTAB protocol and annealing temperature of 55°C is recommended for tomato genomic DNA extraction and amplification with SSR primers.

Optimization, CTAB Protocol, Tomato, SSR Primers, FTA Plant Saver Card

[01] Abdullah, F. S., Rongzhi, W. and Shihua, W. (2015). Microsatellites in Pursuit of Microbial Genome Evolution. Frontiers in Microbiology, 6 (6): 1462-1472. [02] Afsana Sharmin, Ekramul Hoque, Masudul Haque and Fahima Khatun (2018). Molecular Diversity Analysis of Some Chilli (Capsicum spp.) Genotypes Using SSR Markers. American Journal of Plant Sciences, 9: 368-379. [03] Aguirre, N. C., López, W., Orozco-Cárdenas, M., Coronado, Y. M. and Vallejo Cabrera, F. (2017). Use of microsatellites for evaluation of genetic diversity in cherry tomato. Bragantia, 76 (2): 220-228. https://dx.doi.org/10.1590/1678 4499.116. [04] Aguoru, C. U. and Olasan, J. O. (2014). Introduction to Techniques in Molecular Genetics. A text for Undergraduate and Postgraduate Students. Kingdom Press, Makurdi, 105 p. [05] Aguoru, C. U., Omoigui L. O. and Olasan, J. O. (2015). Comparative optimized protocols of DNA Extraction and purification using FTA Plant Saver Card and DNA Zol Methods for Eggplant (Solanum species) studies in North Central Nigeria. Open access Library Journal, 02: 1-5, doi: 10.4236/oalib.1101406. [06] Al-Qadumii, L. W., Sadder, M. T. and Migdadi, H. (2012). Assessment of in silico BAC- based simple sequence repeat (SSR) marker development for tomato (Solanum lycopersicum L.). African Journal of Biotechnology 11 (75): 13938-13946. [07] Chandrashekara, K. N., Prasannakumar, M. K., Deepa, M. and Vani, A. (2012). A rapid, sensitive and reliable method for detecting Ralstonia solanacearum using FTA (Whatman) Card. Journal of Plant Pathology 94: 219–221. [08] Doyle, J. J. and Doyle J. L. (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15. [09] Kadry, K. and Gamal, O. (2017). Genetic analysis of Plectranthus (Lamiaceae) in Saudi Arabia based on RAPD and ISSR markers. Pakistani Journal of Botany, 49 (3): 1073-1084. [10] Khorsheduzzaman, K. M., Alam, M. Z., Rahman, M. M., Mian, M. A. K., Mian, M. I. H. and Hossain, M. M. (2008) Molecular Characterization of Five Selected Brinjal (Solanum melongena L.) Genotypes Using SSR Markers. Bangladesh Journal of Plant Breeding and Genetics, 21: 27-34. [11] Marques, A. P., Zé-Zé, L., San-Romão, M. V., Tenreiro, R. (2010). A novel molecular method for identification of Oenococcus oeni and its specific detection in wine. Int J Food Microbiol. 142 (1-2): 251-255. [12] Mondini, M., Arshiya, N. and Mario, A. (2009). Assessing Plant Genetic Diversity by Molecular Tools, 2009; Department of Agrobiology and Agrochemistry, Tuscia University, Italy. [13] Mucksood A. G. and Khan T. A. (2011). Studies on the interactive effects of Meloidogyne incognita and Fusarium solani on Lycopersicon esculentum, Mill. International Journal of Botany, 7: 205-208. [14] Nkiru, H. O. and Ifenkwe, O. P. (2005). Pattern of flowering, fruits distribution and reproduction efficiency in some varieties of tomato (Lycorpericum esculentum Mill.). Nigerian Journal of Botany, 18: 243-252. [15] Padmalatha K, and Prasad M. N. V (2005). Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India African Journal of Biotechnology, 5 (3): 230-234. [16] Pikkart M. J. and Villeponteau, B. (1993). Suppression of PCR amplification by high levels of RNA. Biotechnique 14: 24-25. [17] Sharmin, A., Hoque, M. E., Haque, M. M. and Khatun, F. (2018). Molecular Diversity Analysis of Some Chilli (Capsicum spp.) Genotypes Using SSR Markers. American Journal of Plant Sciences, 9, 368-379. https://doi.org/10.4236/ajps.2018.9302. [18] Sharmin, D., Khalil, M. I., Begum, S. N. and Meah, M. B. (2011). Molecular Characterization of Eggplant Crosses by Using RAPD Analysis. International Journal of Sustainability and Crop Production, 6: 22-28. [19] Siegel, C. S., Stevenson. F. O. and Zimmer, E. A. (2017). Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa. Applications in Plant Sciences, 5 (2): 1-7. DOI: 10.3732/apps.1600109. [20] Singh, B., Aakansha Goswami and Vaishali (2014). Morphological and Molecular Characterization of Tomato (Lycopersicumesculentum Mill) Genotypes. Vegetos. 28 (4): 67-75. [21] Solomon Benor, Mengyu Zhang, Zhoufei Wang and Hongsheng Zhang (2008). Assessment of genetic variation in tomato (Solanum lycopersicum L.) inbred lines using SSR molecular markers. J. Genet. Genomics, 35: 373−379. [22] Suzuki, S, Taketani, H, Kusumoto, K and Kashiwagi, Y. (2006). High-throughput genotyping of filamentous fungus Aspergillus oryzae based on colony direct polymerase chain reaction. J Biosci Bioeng. 102 (6): 572-574. [23] Van der Schoot, J., Pospiskova, M., and Vosman, B. (2000). Development and characterization of microsatellite markers in black poplar (Populus nigra L.). Theor. Appl. Genet. 101: 317−322.