IRAP-analysis for evaluating the genetic stability of endemic and endangered species of the Western Caucasus flora in the collection in vitro

The characterization of genetic diversity is one of the main components of the genetic resources collection and management. Molecular markers are the most effective tool for characterizing and assessing genetic diversity in plant collections. IRAP (inter-retrotransposons amplified polymorphism) markers have proven to be some of the most effective for characterizing and evaluating germplasm, confirming the genetic fidelity of in vitro preserved cultivars and species. In this regard, the aim of this work is to test several IRAP primers to identify genetic polymorphism and study the genetic fidelity of three rare and endemic flora species of the Western Caucasus during in vitro conservation. Approbation of 16 IRAP-primers on the investigated species was carried out for Eryngium maritimum L., Galanthus woronowii Losinsk. and Campanula sclerophylla Kolak. The results made it possible to select the most efficient of them for genetic fidelity analysis of micropropagated plants. Out of 16 IRAP primers 8 amplified PCR products in Eryngium maritimum. In Galanthus woronowii as well 8 of 16 IRAP primers resulted in the amplification with the number of DNA fragments ranging from 2 to 12. In Campanula sclerophylla 9 of 16 IRAP primers amplified 1 to 11 fragments, depending on the marker. The results of the genotyping of regenerants were compared with data on stock in situ plants, which were the source of explants for in vitro conservation. In total, 60 regenerants for each species of the natural flora of the Western Caucasus were involved in the study. The results obtained demonstrated no genetic changes of the regenerants in all the studied species. These results were confirmed using ISSR analysis of an extended sample set of microplants for each species. The results obtained can serve as evidence of a low probability of genetic disorders during in vitro propagation and conservation of the species Eryngium maritimum L., Galanthus woronowii Losinsk. and Campanula sclerophylla Kolak.

Eryngium, Galanthus, Campanula, IRAP, germplasm conservation, biodiversity

1. Boronnikova S.V. Genetic certification of populations of rare plant species of the genus Adonis using ISSR and IRAP markers. Izvestiya Timiryazevskoy Selskohozyaystvennoy Akademii = Bulletin of the Timiryazev Agricultural Academy. 2009;1:82-88. (in Russian)

2. Boronnikova S.V., Calendar R.N. Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants. Russ. J. Genet. 2010;46(10):36-42.

3. Kolomiets T.M., Malyarovskaya V.I., Gvasaliya M.V., Samarina L.S., Sokolov R.N. In vitro propagation of subtropical and ornamental plants and endemic species of Western Caucasus: developed and improved protocols. Selskokhozyaystvennaya Biologiya = Agricultural Biology. 2014;3:49-58. (in Russian)

4. Kolomiets T.M., Sokolov R.N., Malyarovskaya V.I. Micropropagation of Eringium maritimum L. in vitro. Subtropicheskoye i Dekorativnoye Sadovodstvo = Subtropical and Ornamental Horticulture. 2014;50:196-204. (in Russian)

5. Malyarovskaya V.I., Kolomiets T.M., Sokolov R.N., Samarina L.S. Effect of the light spectral distribution on the growth and development of Lilium caucasicum in vitro. Nauchnyy Zhurnal KubGAU = Scientific Journal of KubSAU (Electronic resource). 2013;10(094). Available at http://ej.kubagro.ru/2013/10/pdf/12.pdf. (in Russian)

6. Ryndin A.V., Belous O.G., Pritula Z.V., Malyarovskaya V.I. The Laboratory of Biotechnology, Plant Physiology and Biochemistry, Russian Research Institute of Floriculture and Subtropical Crops: yesterday, today, tomorrow. Subtropicheskoye i Dekorativnoye Sadovodstvo = Subtropical and Ornamental Horticulture. 2015;54: 9-21. (in Russian)

7. Suprun I.I., Kolomiets T.M., Malyarovskaya V.I., Sokolov R.N., Samarina L.S., Slepchenko N.A. Test of ISSR markers for genotyping of rare plant species of Western Caucasus: Lilium caucasicum Miscz. ex Grossh., Galanthus woronowii Kolak., Pancratium maritimum L. Nauchnyy Zhurnal KubGAU = Scientific Journal of KubSAU (Electronic resource). 2014a;103(09). Available at http:// ej.kubagro.ru/2014/09/pdf/37.pdf. (in Russian)

8. Suprun I.I., Kolomiets T.M., Malyarovskaya V.I., Sokolov R.N., Samarina L.S. The search of optimal ISSR markers for sea daffodil genotyping. Plodovodstvo i Vinogradarstvo Yuga Rossii = Fruit Farming and Viticulture in Southern Russia (Electronic resource). 2014b;30(06). Available at http://journal.kubansad.ru/pdf/14/06/02. pdf. (in Russian)

9. Suprun I.I., Malyarovskaya V.I., Stepanov I.V., Kolomiets T.M., Samarina L.S., Slepchenko N.A. Test of ISSR markers for genotyping Galanthus woronowii Losinsk. and analysis of the genetic stability of plants obtained in vitro. Nauchnyy Zhurnal KubGAU = Scientific Journal of KubSAU (Electronic resource). 2017;133(09). Available at http://dx.doi.org/10.21515/1990-4665-133-088. (in Russian)

10. Börner A., Khlestkina E.K., Chebotar S., Nagel M., Arif M.A., Neumann K., Kobiljski B., Lohwasser U., Röder M.S. Molecular markers in management of ex situ PGR – A case study. J. Biosci. 2012;37(5):871-877.

11. Ferreira M.E. Molecular analysis of gene banks for sustainable conservation and increased use of crop genetic resources. Eds. J. Ruane, A. Sonnino. The Role of Biotechnology in Exploring and Protecting Agricultural Genetic Resources. Rome: FAO, 2006;121-127. http://www.fao.org/biotech/docs/ferreira.pdf.

12. Jawdat D., Al-Faoury H., Ayyoubi Z., Al-Safadi B. Molecular and ecological study of Eryngium species in Syria. Biologia. 2010;65(5):796804. DOI 10.2478/s11756-010-0086-7.

13. Kalendar R., Flavell A.J., Ellis T.H.N., Sjakste T., Moisy C., Schulman A.H. Analysis of plant diversity with retrotransposon-based molecular markers. Heredity. 2011;106(4):520-530. DOI 10.1038/hdy.2010.93.

14. Kolomiets T.M., Malyarovskaya V.I., Samarina L.S. In vitro conservation of Campanula sclerophylla Kolak – endemic endangered species of Western Caucasus. Plant Tissue Cult. & Biotech. 2016; 26(2):143-149.

15. Kour B., Kour G., Kaul S., Dhar M.K. In vitro mass multiplication and assessment of genetic stability of in vitro raised Artemisia absinthium L. plants using ISSR and SSAP molecular markers. Adv. Bot. 2014;13(8):1-7. DOI 10.1155/2014/727020.

16. Lanteri S., Barcaccia G. Molecular markers based analysis for crop germplasm preservation. Eds. J. Ruane, A. Sonnino. The Role of Biotechnology in Exploring and Protecting Agricultural Genetic Resources. Rome: FAO, 2006;55-66.

17. Liu X., Yang G. Adventitious shoot regeneration of oriental lily (Lilium orientalis) and genetic stability evaluation based on ISSR marker variation. In Vitro Cell. Dev. Biol. – Plant. 2012;48:72-179.

18. Lucchin N., Barcaccia G., Parrini P. Characterization of a flint maize (Zea mays var. indurata) Italian landrace: I. Morpho-phenological and agronomic traits. Gen. Res. Crop Evol. 2003;50(3):315-327.

19. Mahjoob B., Zarini H.N., Hashemi S.H., Shamasbi V. Comparison of ISSR, IRAP and REMAP markers for assessing genetic diversity in different species of Brassica sp. Russ. J. Genet. 2016;52:1272. DOI 10.1134/S1022795416120073.

20. Murray M.G., Thompson W.F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8(19):4321-4325.

21. Negri V., Tosti N., Falcinelli M., Veronesi F. Characterization of thirteen cowpea landraces from Umbria (Italy). Strategy for their conservation and promotion. Genet. Resour. Crop Evol. 2000;47:141-146.

22. Rahmani M.-S., Rahmani P.M., Pijut P.M., Pijut N., Shabanian N., Shabanian M.N. Genetic fidelity assessment of in vitro-regenerated plants of Albizia julibrissin using SCoT and IRAP fingerprinting. In Vitro Cell. Dev. Biol. – Plant. 2015;51(4):407-419. DOI 10.1007/s11627-015-9692-y.

23. Santana M.F., de Araújo E.F., de Souza J.T., Mizubuti E.S.G., de Queiroz M.V. Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa. Eur. J. Plant. Pathol. 2012;134:497-507. DOI 10.1007/s10658-012-0031-4.

24. Senková S., Žiarovská J., Bežo M., Štefúnová V., Ražná K. Utilization of IRAP technique for plums genotypes differentiation. Biosci. Res. 2013;10(1):1-7.

25. Singh S., Nandhaa P.S., Singh J.S. Transposon-based genetic diversity assessment in wild and cultivated barley. Crop J. 2017;5(4):296-304. DOI 10.1016/j.cj.2017.01.003.

26. Tomás D., Dias A.L., Silva M., Oliveira H.R., Suso M.J., Viegas W., Veloso M.M. Genetic diversity assessment of Portuguese cultivated Vicia faba L. through IRAP markers. Diversity. 2016;8(2):8. DOI 10.3390/d8020008.

27. Yuying S., Xiajunb D., Feia W., Binhuaa C., Zhihonga G., Zhena Z. Analysis of genetic diversity in Japanese apricot (Prunus mume Sieb. et Zucc.) based on REMAP and IRAP molecular markers. Sci. Hortic. 2011;132(5):50-58. DOI 10.1016/j.scienta. 2011.10.005.