A development of a PCR-RFLP test system for the identification of mitochondrial lines of the Pelophylax ridibundus lake frog in Kazakhstan

Abstract

Background. Molecular typing by PCR-RFLP method allows to identify the spe-cific attribution of an organism that has a weak phenotypic difference. The main advantage of this research method is the capacity to analyze a large number of samples without apply-ing the sequencing method. The purpose of the work is to develop a test-system for the identification the matrilines of marsh frogs of the Pelophylax ridibundus complex from Kazakhstan. Materials and methods. The analysis was based on the variability of the prima-ry structure of the mitochondrial gene the second subunit of dehydrogenase (ND2) which is a species-specific marker. Subsequently a search for marker nucleotide substitutions speci-fic for each lineage and recognition sites for the HaeIII and TasI restriction endonucleases was conducted. Results. As a result of the research, it was confirmed that on the territory of Kazakhstan inhabiting three main forms of lake frogs where two of them are native (Balkhash, Syrdarya), and invasive Anatolian form – P. cf. bedriagae. Conclusions. The authorsʼ PCR-RLFP test technique is a straightforward and reliable tool for detecting mitochondrial lineages in the Pelophylax ridibundus complex marsh frogs and may be used successfully in mass screening investigations.

Keywords

• restriction analysis
• nadh dehydrogenase (nd2)
• mitochondrial dna
• pelo-phylax ridibundus complex