SUMMARY
DOI: 10.15414/afz.2015.18.04.83–89Received 7. July2015 | Accepted 30. September 2015 | Available online 7. December 2015Two types of molecular markers based on randomly amplified DNA by RAPD-based assay and amplified microRNA bymiRNA-based assay were applied for the genotyping of five accessions of Silybum marianum (L.) Gaertn. It was also verifiedthe effectiveness of the isolation of genomic DNA by three commercially available isolation kits and two similar complexmethods and their applicability for this medicinal herb. None of the commercial isolation kits provided the genomic DNAof sufficient quality and quantity. Applied originally designed CTAB method and modified CTAB method for purpose ofDNA isolation from medicinal plants allowed to isolate DNA of required quality and of sufficient yield. RAPD-based DNAfingerprints, allowed to distinguish the individual genotypes of Silybum marianum. MicroRNA-based markers showed thecross-genera transferability potential and displayed sufficient level of polymorphism. Both types of molecular markerscould be used as suitable tool for genotyping of milk thistle. However, because of the size of miRNA amplicons is theefficiency of miRNA-based markers PCR amplifications preferred and consequently less DNA quality-dependent which maybe advantageous in view of the content of undesirable secondary metabolites in medicinal plants.Keywords: molecular markers, RAPD, miRNA, Silybum marianumReferencesABENAVOLI, L. et al. (2010). Milk thistle in liver diseases: past, present, future. In Phytother Res, vol. 24, no.10, pp. 1423-1432.BARVKAR, V. T. et al. (2013). Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes. In Planta, vol. 237, pp. 1149-1161.BEJ, S. and BASAK, J. (2014). MicroRNAs: The potential Biomarkers in Plant Stress Response. In Am J Plant Sci, vol. 5, pp. 748-759.CERVENKA, M. et al. (1986). Slovak botanical nomenclature. Bratislava: Príroda.FU, D. et al. (2013). MicroRNA-based molecular markers: a novel PCR-based genotyping technique in Brassica species. In Plant Breeding , vol. 132, pp. 375-381.GALLA, G. et al. (2013). Computational identification of conserved microRNAs and their putative targets in the Hypericum perforatum L. flower transcriptome. In Plant Reprod, vol. 26, pp. 209-229.GURSANSCKY, N.R., SEARLE, I.R. and CARROLL, B.J. (2011). Mobile MicroRNAs Hit the Target. In Traffic, vol. 12, pp. 1475-1482.HABÁN, M. and VAVERKOVÁ, Š. (2014). Milk thistle (Silybum marianum L. Gaertn) – the most cultivated medicinal plant in Slovakia. In International conference on medicinal plants and their use in therapeutic practice. Lubovna Spa, May 14-16, 2014.HABÁN, M. et al. (2015). Production and quality of milk thistle (Silybum marianum L. gaertn.) cultivated during the vegetation periods 2012–2014 in a warm agri-climatic macroregion. In Scientific conferences Banat´s university of agricultural sciences and veterinary medicine. Timisoara: Banat University of Agricultural Sciences and Veterinary Medicine.HLAVACKOVÁ, L. et al. (2015a). Linum usitatissimum L. genotyping by microRNA-based genetic markers. In Agrobiodiversity for improving nutrition, health and life quality. International conference. Nitra: Slovenská polnohospodárska univerzita v Nitre, pp. 242-246. ISBN 978-80-552-1379-8 (in Slovak).HLAVACKOVÁ, L. et al. (2015b.) Application of miRNA-based markers in flax genotypes characterisation. In International Cooperation for the Future Agricultural Researches. Debrecen: University of Debrecen. In pressKULCHESKI, F. R. et al. (2010). The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean. In Anal. Biochem., vol. 406, pp. 185-192.KUMAR, P. et al. (2009). Potential of molecular markers in plant biotechnology. In Plant Omics Journal, vol. 2, no. 4, pp. 141-162.MELNIKOVA, N. V. et al. (2014). Excess fertilizer responsive miRNAs revealed in Linum usitatissimum L. In Biochimie, vol. 109, pp. 36-41. DOI: http://dx.doi.org/10.1016/j.biochi.2014.11.017MOHAMMADI, S. A. et al. (2011). AFLP-based molecular characterization and population structure analysis of Silybum marianum L. In Plant Genetic Resources: Characterization and Utilization, vol. 9, no. 3, pp. 445-453.MONDAL, T. K. and GANIE, S. A. (2014). Identification and characterization of salt responsive miRNA-SSR markers in rice (Oryza sative). In Gene, vol. 535, pp. 204-209.NEUTELINGS, G. et al. (2012). Identification and characterization of miRNAs and their potential targets in flax. In J Plant Physiol., vol. 169, pp. 1754-1766.PADMALATHA, K. and PRASAD, M. (2006). Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. In African Journal of Biotechnology, vol. 5, pp. 230-234.RAFIEIAN-KOPAIE, M. and NASRI, H. (2012). Silymarin and diabetic nephropathy. In J Renal Inj Prev, vol. 1, no. 1, pp. 3-5.RAINODE, F. (2005). Milk thistle. In American Family Physician, vol. 72, no. 7, pp. 1285-1288.ROGERS, S. and BENDICH, A.. (1994). Extraction of DNA from plant, fungal and algal tissues. In Plant Molecular Biology Manual, vol. 5, no. 1, pp. 193-190.SHARAF, A. A. et al. (2010). Biochemical and genetic characterization of 12 Silybum marianum accessionscollected from Borg El-Arab, an Egyptian habitat. In Functional Plant Science and Biotechnology, vol. 5, Special Issue 1, pp. 22-29.SUCHER, N. J. and CARLES, M. C. (2008). Genome-based Approaches to the authentication of medicinal plants. In Planta Med, vol. 74, pp. 603-623.SUNKAR, R. (2010). MicroRNAs with macro-effects on plant stress responses. In Seminars in Cell & Developmental Biology, vol. 21, pp. 805-811.WILLIAMS, J. G. et al. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. In Nucleic Acid Res, vol. 18, pp. 6531-6535.YADAV, C. B. Y. et al. (2014). Development of novel microRNA-based genetic markers in foxtail millet for genotyping applications in related grass species. In Mol Breeding, vol. 34, no. 4 , pp 2219-2224. DOI: http://dx.doi.org/10.1007/s11032-014-0137-9