Ribogospod. nauka Ukr., 2017; 4(42): 65-74
Development of a multiplex PCR for the genetic analysis of paddlefish (Polyodon spathula Walbaum,1792) populations
, Ukrainian Laboratory of Quality and Safety of Agricultural Products, Chabany village
Purpose. Paddlefish is commercially important species owing to its biological features and consumer characteristics, namely it produces valuable and delicious fish products, such as high quality meat and black caviar. Consequently, its cultivation under Ukrainian fish farm conditions and further realization in domestic and foreign markets are economically efficient. However, the paddlefish broodstock in Ukraine requires the efficient solution of increasing its productivity, identification and assessment of its genetic variation. Thus, the aim of our study was to develop and implement a multiplex PCR-analysis of paddlefish (Polyodon spathula) for population-genetic monitoring of its artificial broodstocks in Ukraine.
Methodology. A multiplex PCR was used for the study. The multiplex PCR development was performed for four microsatellite DNA markers: Psp12, Psp21, Psp26 and Psp28. Each investigated DNA loci, for which the multiplex PCR was optimized, was selected in such a way that the colored PCR products labeled with fluorescent dye did not overlap the length of the amplified fragments. Evaluation of the multiplex PCR effectiveness and processing of the data were performed by fragment analysis of DNA on the genetic analyzer ABI Prism 3130 (Applied Biosystem, USA). The size of the identified alleles was determined using the "Gene Mapper 3.7" program (Applied Biosystems, USA) and LIZ-500 size standard (Applied Biosystems, USA).
Results. Based on the results of capillary electrophoresis of multiplex PCR products, it was found that the amplified fragments for each of the four studied loci: Psp12, Psp21, Psp26 and Psp28 in one PCR reaction were within the expected size range. Data analysis on the electrophoregram demonstrated that Psp21 had the highest peak intensity at 611 fluorescent units (FU) and the lowest peak intensity at 105 FU was observed for Psp26 locus. In the multiplex PCR after proper interpretation of the data we identified heterozygous allelic variants for Psp12 (218/220 bp), Psp26 (144/146 bp) and Psp28 (256/258 bp). Herewith, the Psp21 locus (150/150 bp) is identified in the homozygous state.
Originality. The optimization was conducted and a multiplex PCR was developed for paddlefish genotyping using microsatellite DNA markers.
Practical value. The obtained results can be used for paddlefish genotyping within selective-breeding programs for artificial reproduction of this fish species.
Keywords: paddlefish, microsatellite DNA markers, multiplex, PCR, genotyping, genetic analysis.
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