ANALYSIS OF THE SPECIFICITY OF THE GENETIC STRUCTURE OF PADDLEFISH (POLYODON SPATHULA (WALBAUM, 1792)) USING ISSR MARKERS
Paddlefish (Polyodon spathula (WALBAUM, 1792)) is reared in aquaculture on Ukrainian farms as an alternative source of gourmet meat and caviar to compensate for the lack of this valuable sturgeon product, which was caused by a drastic reduction of sturgeon stocks in Ukraine's natural waters. However these stocks require analysis of the genetic structure of breeding material cultivated in the aquaculture of Ukraine for further control and maintainance of their polymorphism and heterogeneity. Genetic control will allow monitoring the condition of stocks, selection of the most acceptable source of breeding stock and optimizing the breeding of this object.
The aim of the research was to determine the level of efficiency of the ISSR-PCR method for studying polymorphism and identifying specific features of the genetic structure of the paddlefish reared at fish farms in Ukraine. Materials and methods of research. The analysis was performed using two age groups of paddlefish (younger – age-4 and older – age-13–15), which were reared at the state institution "Production and Experimental Dnieper Sturgeon Breeding Plant named after Academician S.T. Artushchyk". Molecular genetic analysis was performed using three ISSR markers (B – (GAG)₆C; C – (AGC)₆G and E – (AGC)₆C) followed by separation of amplification products by agarose gel electrophoresis. The informativeness of ISSR primers was determined by such indicators as the effective multiplex ratio (EMR), marker index (MI), resolution (Rp), calculated according to the methods for dominant markers.
Research results. The specifics of the genetic structure of the paddlefish of the Kherson stock were described using ISSR markers. The study showed that a total of 32 amplicons were obtained for the three primers, of which 87.5% were polymorphic. The largest number of amplicons was obtained using primer E – (AGC)₆C – 14 specimens, while primers B – (GAG)₆C and C – (AGC)₆G were observed 9 bands. The information polymorphism index (PIC) ranged from 0.227 (marker C) to 0.314 (marker E), with an average value of 0.256.
The informativeness of the selected ISSR primers was assessed during the study of the paddlefish (mean values were: PIC = 0.256; PPB = 85.2%; EMR = 8.4; MI = 2.3; Rp = 9.7). These indicators demonstrated the level of informativeness and efficiency of selected intermicrosatellite loci for the analysis of polymorphisms of the genetic structure of the paddlefish. Marker E – (AGC)₆C had the highest values of PPB, PIC, EMR, MI and Rp compared to other selected ISSR-markers, which indicated the greatest effectiveness of this primer in population genetic studies of paddlefish.
Ranges of amplicons at selected intermicrosatellite loci were determined: for marker B – (GAG)₆C from 215 to 1275 bp; for markers C – (AGC)₆G and E – (AGC)₆C in the range of 125–1265 bp and 320–1470 bp, respectively. Following monomorphic amplicons for all studied specimens of both age groups were determined: for marker C – allele 125, 245 and 395 bp; for marker B – (GAG)₆C – 1085 bp. Identified monomorphic bands in the future can be used for interspecific comparisons of different species and to establish the origin of samples.
Differences between the studied different age groups of paddlefish were characterized, which consisted in: 1) the specificity of the frequency distribution of alleles encountered with high frequency (over 40%), 2) the absence of larger amplicons for markers B – (GAG)₆C and C – (AGC)₆G in older paddlefish groups and 3) the presence of different monomorphic alleles for marker E – (AGC)₆C (allele 380 bp in specimens of the younger and allele 320 bp in the older group). In the younger age group, a higher total number of amplified fragments (NTB) and the number of polymorphic fragments (NPB) were observed at the three loci studied compared to the older group.
The biodiversity level according to the Shannon index I (0.394 ± 0.043 in the younger and 0.321 ± 0.043 in the older age group) and the level of heterozygosity (objective expected heterozygosity – 0.286 ± 0.034 and 0.231 ± 0.035, respectively) of the studied groups of paddlefish were determined.
Conclusions. The obtained results demonstrated the effectiveness of the use of selected ISSR-markers to assess the polymorphism of the genetic structure of the otter, the state of biodiversity and the heterozygosity of stocks. The analysis performed by the ISSR-PCR method allowed characterizing the specifics of the genetic structure of the paddlefish and showing age differences. The obtained gene pool profile can be used as a basis for the certification of paddlefish stocks.
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