Cevizde Yeni SSR Markörlerin Geliştirilmesi

Abstract

Bu çalışmada ceviz için yeni SSR markörlerin geliştirilmesi amaçlanmıştır. Bunun için Maraş-18 ceviz çeşidi DNA’sı kullanılarak oluşturulan genomik kütüphane dört farklı motif ile (CA, GA, AAC ve AAG) zenginleştirilmiştir. Sekansı yapılan 620 klondan 276 adet primer çifti dizayn edilmiştir. Öncelikle gradient PCR ile her bir primer çiftinin DNA’ya bağlanma sıcaklıkları belirlenmiştir. Bant veren 247 SSR primer çifti 15 ceviz çeşidinde analiz edilerek polimorfizm durumları belirlenmiştir. Toplam 185 primer çifti polimorfizm göstermiş olup bunların 80 tanesi (%44.4) GA kütüphanesinden elde edilmiştir. En az polimorfik primer çifti (14 adet) ise AAC kütüphanesinden elde edilmiştir. CA ve AAG kütüphanelerinden ise benzer oranda polimorfizm elde edilmiştir. Sonuç olarak, cevizde bundan sonra yapılacak genetik kaynakların karakterizasyonu, genetik haritalama, popülasyon genetiği gibi çalışmalarda kullanmak üzere çok sayıda yeni SSR markörleri geliştirilmiştir.

Keywords

• Ceviz
• Juglans regia
• SSR
• mikrosatellit

Development of Simple Sequence Repeat (SSR) Markers from Walnut (Juglans regia L.)

Abstract

In this study, it was aimed to develop SSR markers for walnut. Genomic libraries enriched with CA, GA, AAC and AAG repeats were constructed using genomic DNA of Juglans regia cv. Maraş-18 to develop SSR markers for walnut. A total of 624 clones were sequenced and 276 primer pairs were designed. Amplification of SSR primer pairs was tested by gradient PCR to determine their optimum annealing temperatures, 247 generated PCR products, and 185 polymorphic loci were obtained by characterizing 15 walnut cultivars. A total of 185 primer pairs showed polymorphism and 80 of them obtained from GA-enriched library. The least polymorphism (14 primer) obtained from AAC-enriched library. CA and AAG enriched libraries produced similar polymorphism rates. As a result, the new SSR primer pairs developed in this study can be used in different genetic studies in walnut such as characterization of genetic resources, construction of genetic linkage maps, and population genetics.

Keywords

• Walnut
• Juglans regia
• SSR
• microsatellite

References

1. Bassil, N.V., Botta, R., Mehlenbacher, S.A., 2005. Microsatellite markers in hazelnut: Isolation, characterization, and cross-species amplification. J. Amer. Soc. for Hort. Sci. 130:543–549.
2. Cipriani, G., Lot, G., Huang, H.G., Marrazzo, M.T., Peterlunger, E., Testolin, R., 1999. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L.) Batsch]:isolation, characterization and cross-species amplification in Prunus. Theor. Appl. Genet. 99:65–72.
3. Cuc, L.M., Mace, E.S., Crouch, J.H., Quang, V.D., Long, T.D., Varshney, R.K., 2008. Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea). BMC Plant Biol. 8:55–65.
4. Dangl, G., Woeste, K., Aradhya,M., Koehmstedt, A., Sımon, C., Potter, D., Leslıe, C.A., Mcgranahan, G., 2005. Characterization of 14 microsatellite markers for genetic analysis and cultivar identification of walnut. J. Amer Soc. for Hort. Sci. 130(3):348-354.
5. Doğan, Y., Kafkas, S., Sutyemez, M., Akça, Y., Türemiş, N., 2014. Assessment and characterization of genetic relationships of walnut (Juglans regia L.) genotypes by three types of molecular markers. Scientia Hort. 168:81–87.
6. Doyle, J.J., Doyle, J.L., 1987. A rapid isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19: 11-15.
7. FAOSTAT, 2015. Food and Agriculture Organization of The United Nations. http://faostat.fao.org/default.aspx
8. Fjellstrom, R.G., Parfıtt, D.E., Mcgranahan, G.H., 1994. Genetic relationships and characterization of Persian walnut (J. regia L.) cultivars using restriction fragment length polymorphisms (RFLPs). J. Amer. Soc. for Hort. Sci., 119(4): 833-839.

9. Forde, H.I., Griggs, W.H., 1972. Pollination and blooming habits of walnuts. Agriculture Experimental Station, University of California Leaflet 2753.
10. Foroni, I., Rao, R., Woeste, K., Gallitelli, M., 2005. Characterization of Juglans regia L. with SSR markers and evaluation of genetic relationships among cultivars and the ‘Sorrento’ landrace. J. of Hort. Sci. & Biotechnology 80(1):49-53.
11. Foroni, I., Woeste, K., Monti, L.M., Rao, R., 2007. Identification of ‘Sorrento’ walnut using simple sequence repeats (SSR). Springer Science + Business Media B.V. Genet Resour Crop Evol. 54:1081-1094.
12. Gürcan, K., Mehlenbacher S.A., Botta, R., Boccacci, P., 2010. Development, characterization, segregation, and mapping of microsatellitye markers for European hazelnut (Corylus avellana L.) from enriched genomic libraries and usefulness in genetic diversity studies. Tree Genet. Genomes 6:513–531.
13. Hoban, S., Anderson, R.M.C., Cleary, T., Schlarbaum, S., Romero-Severson, I., 2008. Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.). Molecular Ecology Resources 8:643–646.
14. Kafkas, S., Akcan, S., Türemiş, N., Sütyemez, M., Eti, S., Akça, M., 2008. Kaman cevizlerinde apomiksis olasılığının dişi çiçeklerin izolasyonu ve moleküler yöntemlerle araştırılması. Alatarım 7(1):1-10.
15. Kafkas, S., Ozkan, H., Sutyemez, M., 2005. DNA polymorphism and assessment of genetic relationships in walnut genotypes based on AFLP and SAMPL markers. J. Amer. Soc. for Hort. Sci., 130: 585-590.
16. Liu, K., Muse, S.V., 2005. Power Marker: Integrated analysis environment for genetic marker data. Bioinformatics 21: 2128–2129.
17. Manning , W.E., 1978. The classification within the Juglandaceae. Ann. Mo. Bot. Gard. 65:10581087.
18. Missio, R.F., Caixeta, E.T., Zambolim, E.M., Zambolim, L., Sakiyama, N.S., 2009. Development and validation of SSR markers for Coffea arabica L. Crop Breed. Appl. Biotechnol. 9: 361–371.
19. Nicese, F.P., Hormaza, J.I., McGranahan, G.H. 1998. Molecular characterization and genetic relatedness among walnut (Juglans regia L.) genotypes based on RAPD markers. Euphytica 101: 199-206.
20. Novelli, V.M., Cristofani, M., Souza, A.A., Machado, M.A., 2006. Development and characterization of polymorphic simple sequence repeats (SSRs) in sweet orange (Citrus sinensis L. Osbeck). Genet. Mol. Biol. 29: 90–96.
21. Peakall, R., Smouse P.E., 2012. GenAlEx 6.5: genetic analysis in excel. Population genetic software for teaching and research-an update. Bioinformatics 28:2537–2539.
22. Pollegioni, P., Woste, K., Major, A., Mugnozza, S.G., Malvolti, M.E., 2008. Characterization of Juglans nigra (L.), Juglans regia (L.) and Juglans x intermedia (Carr.) by SSR markers. A case study in Italy. Silvae Genetica 1: 6878.
23. Potter, G., Gao, G., Aiello, C., Mcgranahan, G., 2002. Intersimple sequence repeat markers for fingerprinting and determining genetic relationships of walnut (J.regia) cultivars. J. Amer. Soc. for Hort. Sci., 127: 75-81.
24. Robichaud, R.L., Glaubitz, J.C., Rhodes Jr, O.E., Woeste, K., 2006. A robust set of black walnut microsatellites for parentage and clonal identification. New Forest 32:179-196.
25. Rom, R., Carlos, R.F., 1987. Rootstock for Fruit Crops. University of California, Davis, California, pp:415-450.
26. Victory, E.R., Glaubitz, J. C., Rhodes Jr, O.E., Woeste, K.E., 2006. Genetic Homogeneity in Juglans nigra (Juglandeae) at Nuclear Microsatellites. American Journal of Botany 93(1):118–126.
27. Wang , H.D., Pei, D., Gu, R.S., Wang, B.Q. 2008. Genetic diversity and structure of walnut populations in central and southwestern China revealed by microsatellite markers. J. Amer. Soc. for Hort. Sci., 133:197-203.
28. Woeste, K., Burns, R., Rhodes, O., Michler, C. 2002. Thirty polymorphic nuclear microsatellite loci from black walnut. The Journal of Heredity 93: 58-60.
29. You, F.M., Huo, N., Gu, Y.Q., Luo, M., Ma, Y., Hane, D., Lazo, G.R., Dvorak, J., Anderson, O.D., 2008. Batch Primer 3: A high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253.
30. Zaloğlu, S., Kafkas, S., Doğan, Y., Güney, M., 2015. Development and characterization of SSR markers from pistachio (Pistacia vera L.) and their transferability to eight Pistacia species. Sci. Hort. 189: 94–103.