Screening of Oryza sativa L. for Hpt Gene and Evaluation of Hpt Positive Samples Using Houba Retransposon-Based IRAP Markers

Öz

Increasing world population needs to enhance agricultural production because of food starvation. Genetically modified organism (GMO) is a way to solve this problem. During gene transfers, DNA is inserted into a plant’s genome in a random way. This produces spontaneous genetic changes with movement of transposable elements, and even increases variations. Houba was described as one of the active retrotransposons in rice. The aim of this study was to screen rice samples collected from Turkey, and analyse Houba retrotransposon movements with IRAP technique in transgenic ones and their controls. For this purpose, 71 different rice seeds obtained from different regions of Turkey were used for GMO analysis. All samples were screened by real time PCR to test cauliflower mosaic virus (CaMV) 35S promoter (P-35S) regions, T-NOS (nopaline synthase terminator) regions, figwort mosaic virus (FMV) regions, bar, pat and Cry1ab/ac, and hpt (hygromycin resistance) genes. Hpt gene was identified in 6 samples as a result of real time PCR analysis. These 6 transgenic samples with their controls were used for IRAP-PCR analysis and 0-56% polymorphism ratios were observed in analysed samples. This study is one of the first detailed experimental data of transgenic Oryza sativa L. samples in terms of retrotransposon-based variation. 

Anahtar Kelimeler

• Genetically modified organism
• Houba
• IRAP
• Real-Time PCR
• rice

Screening of Oryza sativa L. for Hpt Gene and Evaluation of Hpt Positive Samples Using Houba Retransposon-Based IRAP Markers

Öz

Increasing world population needs to enhance agricultural production because of food starvation. Genetically modified organism (GMO) is a way to solve this problem. During gene transfers, DNA is inserted into a plant’s genome in a random way. This produces spontaneous genetic changes with movement of transposable elements, and even increases variations. Houba was described as one of the active retrotransposons in rice. The aim of this study was to screen rice samples collected from Turkey, and analyse Houba retrotransposon movements with IRAP technique in transgenic ones and their controls. For this purpose, 71 different rice seeds obtained from different regions of Turkey were used for GMO analysis. All samples were screened by real time PCR to test cauliflower mosaic virus (CaMV) 35S promoter (P-35S) regions, T-NOS (nopaline synthase terminator) regions, figwort mosaic virus (FMV) regions, bar, pat and Cry1ab/ac, and hpt (hygromycin resistance) genes. Hpt gene was identified in 6 samples as a result of real time PCR analysis. These 6 transgenic samples with their controls were used for IRAP-PCR analysis and 0-56% polymorphism ratios were observed in analysed samples. This study is one of the first detailed experimental data of transgenic Oryza sativa L. samples in terms of retrotransposon-based variation. 

Anahtar Kelimeler

• Genetically modified organism
• Houba
• IRAP
• Real-Time PCR
• rice

Kaynakça

1. Anonymous, 2003. Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability and labeling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive2001/18/EC, Official Journal L, 268: 0024-0028.
2. Anonymous, 2010a. Turkish Republic Biosafety Law: Law number 5977. Official Journal, 27533/Issue date: 26.03.2010.
3. Anonymous, 2010b. Official Gazette of the Republic of Turkey. Number: 27533. Biosafety Law.
4. Anonymous, 2011a. Official Gazette of the Republic of Turkey. Number: 27827, Biosecurity Committee Decisions, decision no.1, 2 and 3.
5. Anonymous, 2011b. Official Gazette of the Republic of Turkey. Number: 28152, Biosecurity Committee Decisions, Decision No. 4-16.
6. Baltes, N.J., Voytas, D.F., 2015. Enabling plant synthetic biology through genome engineering. Trends in Biotechnology, 33(2): 120-131.
7. Barampuram, S., Zhang, Z.J., 2011. Recent advances in plant transformation. Plant Chromosome Engineering, pp. 1-35.
8. Barroso, M.F., Freitas, M., Oliveira, M.B.P.P., De-Los-Santos-Alvarez, N., Lobo-Castanon, M.J., Deleure-Matos, C., 2015. 3D-nanostructured Au electrodes for the event-specific detection of MON810 transgenic maize. Talanta, 134: 158-164.

9. Broeders, S.R.M., De Keersmaecker, S.C.J., Roosens, N.H., 2012. How to deal with the upcoming challenges in GMO detection in food and feed. Journal of Biomedicine and Biotechnology, 2012: 1-11.
10. Cakmak, B., Marakli, S., Gozukirmizi, N., 2015. SIRE1 retrotransposons in barley (Hordeum vulgare L.). Russian Journal of Genetics, 51(7): 661-672.
11. Cheema, H.M.N., Khan, A.A., Khan, M.I., Aslam, U., Rana, I.A., Khan, I.A., 2016. Assessment of Bt cotton genotypes for the Cry1Ac transgene and its expression. The Journal of Agricultural Science, 154(1): 109-117.
12. Christou, P., Ford, T.L., 1995. The impact of selection parameters on the phenotype and genotype of transgenic rice callus and plants. Transgenic Research, 4(1): 44-51.
13. Feng, C., Yuan, J., Wang, R., Liu, Y., Birchler, J.A., Han, F., 2016. Efficient targeted genome modification in maize using CRISPR/Cas9 system. Journal of Genetics and Genomics, 43(1): 37-43.
14. Gryson, N., 2010. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Analytical and Bioanalytical Chemistry, 396(6): 2003-2022.
15. Holst-Jensen, A., Ronning, S.B., Lovseth, A., Berdal, K.G., 2003. PCR technology forscreening and quantification of genetically modified organisms (GMOs). Analytical and Bioanalytical Chemistry, 375(8): 985-993.
16. Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bulletin de la societe vaudoise des sciences naturelles, 44(163): 223-270.
17. James, C., 2015. 20th Anniversary (1996 to 2015) of the Global Commercialization of Biotech Crops and Biotech Crop Highlights in 2015. ISAAA Brief No. 51, ISAAA: Ithaca, NY.
18. Kalendar, R., Grob, T., Regina, M., Suoniemi, A., Schulman, A., 1999. IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theoretical and Applied Genetics, 98(5): 704-711.
19. Kalendar, R., Schulman, A.H., 2006. IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nature Protocols, 1(5): 2478-2484.
20. Kaya, Y., Yilmaz, S., Gozukirmizi, N., Huyop, F., 2013. Evaluation of transgenic Nicotiana tabacum with dehE gene using transposon based IRAP markers. American Journal of Plant Sciences, 4(8A): 41-44.
21. Kuromori, T., Wada, T., Kamiya, A., Yuguchi, M., Yokouchi, T., Imura, Y., Takabe, H., Sakurai, T., Akiyama, K., Hirayama, T., Okada, K., Shinozaki, K., 2006. A trial of phenome analysis using 4000 Ds-insertional mutants in gene-coding regions of Arabidopsis. The Plant Journal, 47(4): 640-651.
22. Matsuoka, T., Kawashima, Y., Akiyama, H., Miura, H., Goda, Y., Kusakabe, Y., 2000. A method of detecting recombinant DNAs from four lines of genetically modified maize. Journal of the Hygienic Society of Japan, 41(2): 137-143.
23. Miraglia, M., Berdal, K.G., Brera, C., Corbisier, P., Holst-Jensen, A., Kok, E.J., Marvin, H.J.P., Schimmel, H., Rentsch, J., Rie, J.P.P.F. van, Zagon, J., 2004. Detection and traceability of genetically modified organisms in the food production chain. Food and Chemical Toxicology, 42(7): 1157-1180.
24. Pervaiz, Z.H., Turi, N.A., Khaliq, I., Rabbani, M.A., Malik, S.A., 2011. A modified method for high-quality DNA extraction for molecular analysis in cereal plants. Genetics and Molecular Research, 10(3): 1669-1673.
25. Poczai, P., Varga, I., Laos, M., Cseh, A., Bell, N., Valkonen, J.P.T., Hyvönen, J., 2013. Advances in plant gene targeted and functional markers: a review. Plant Methods, 9(6): 1-31.
26. Rao, J., Yang, L., Guo, J., Quan, S., Chen, G., Zhao, X., Zhang, D., Shi, J., 2016. Development of event-specific qualitative and quantitative PCR detection methods for the transgenic maize BVLA430101. European Food Research and Technology, 242(8): 1277-1284.
27. Schnell, J., Steele, M., Bean, J., Neuspiel, M., Girard, C., Dormann, N., Pearson, C., Savoie, A., Bourbanniere, L., Macdonald, P., 2015. A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments. Transgenic Research, 24(1): 1-17.
28. Shan, Q., Wang, Y., Li, J., Gao, C., 2014. Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols, 9: 2395-2410.
29. Turkec, A., Lucas, S.J., Karacanli, B., Baykut, A., Yuksel, H., 2016. Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs). Food Chemistry, 194: 399-409.
30. Van den Elzen, P.J.M., Townsend, J., Lee, K.Y., Redbrook, J.R., 1985. A chimaeric hygromycin resistance gene as a selectable marker in plant cells. Plant Molecular Biology, 5(5): 299-302.
31. Vijayakumar, K.R., Martin, A., Gowda, L.R., Prakash, V., 2009. Detection of genetically modified soya and maize: Impact of heat processing. Food Chemistry, 117(3): 514-521.
32. Weeks, D.P., Spalding, M.H., Yang, B., 2016. Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnology Journal, 14(2): 483-495.
33. Xu, R.F., Li, H., Qin, R.Y., Li, J., Qiu, C.H., Yang, Y-C., Ma, H., Li, L., Wei, P-C., Yang, J-B., 2015. Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Scientific Reports, 5: 1-10.
34. Yuzbasioglu, G., Yilmaz, S., Marakli, S., Gozukirmizi, N., 2016. Analysis of Hopi/Osr27 and Houba/Tos5/Osr13 retrotransposons in rice. Biotechnology & Biotechnological Equipment, 30(2): 213-218.
35. Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou, F., Feng, Z., Mo, Y., Yang, L., Zhang, H., Xu, N., 2014. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 12(6): 797-807.
36. Zhang, M., Yu, Y., Gao, X., Zhang, K., Luan, F., Zhu, Y., Qu, B., 2015. Event-specific quantitative detection of genetically modified wheat B72-8-11 based on the 3′ flanking sequence. European Food Research and Technology, 240(4): 775-782.
37. Zuraida, A.R., Rahiniza, K., Zulkifli, A.S., Alizah, Z., Zamri, Z., Aziz, A., 2013. Hygromycin as selective marker in Agrobacterium-mediated genetic transformation of indica rice MR219. Journal of Tropical Agriculture and Food Science, 41(1): 71-79.