The European grayling Thymallus thymallus (Linnaeus, 1758) is listed in the Red Book of Ukraine and has the status of a vulnerable species. Therefore, the protection and reproduction of this indigenous salmonid species for Ukraine is an important task. The work in this field was initiated by specialists of the Institute of Fisheries in 2006 at the farm of the Synevyr National Nature Park from individuals caught in the Chorna River (a tributary of the Tereblya River), and since 2014 the work continues at the Lopushno trout farm (Chernivtsi region, Ukraine) (Kucheruk et al., 2018). This fish farm participates in the program of the National Academy of Sciences of Ukraine, aimed at creating a brood stock of grayling with increased productivity and its further use for stocking the river network of the Western region of Ukraine to preserve the species diversity of local ichthyocenoses. Comprehensive work with this species includes both the classical assessment of morphological and productive parameters of the obtained generations of fish on the farm, monitoring of infectious and invasive fish diseases, and also genetic analyses. A set of molecular tools allows studying allelic diversity, assessing stock heterozygosity, and determining phylogenetic origin or relationships with other populations, and thus contributing to the effective management of the genetic resources of graylings on farms and their further use for the preservation of ichthyocenoses in rivers. This work aimed to analyze grayling generations from the Ukrainian fishery using microsatellites and mitochondrial DNA analysis.
Materials and methods
For molecular genetic studies, fish fin clips were collected from individuals of the first generation of grayling from 2017 and the second generation from 2021 of the Lopushno trout farm (Ukraine) with the aim to analyze and monitor changes in the genetic structure of the stock. The study of the genetic diversity was carried out using microsatellite loci (BFRO 005, BFRO 006, BFRO 010, BFRO011) with data processing in Genalex 6.5 software and related programs to obtain basic genetic parameters. Phylogenetic analysis of the existing stock was carried out using the gene of the control region of mitochondrial DNA. The obtained results of the analysis of mtDNA fragments were edited in Sequencher v5.1 and analysed together with reference sequences from the NCBI, BOLD databases in MEGA X, DNAsp and PopART.
The number of alleles for all loci varied from 4 to 6, with values of the effective number of alleles per locus from 2.985 to 5.128. The average value of the allelic richness index (Ar) in the first generation was 4.7 ± 0.5 less than in the second generation 5.7 ± 0.5. Shannon’s diversity index ranged from 1.192 to 1.543 in the 2017 generation and 1.392 to 1.692 in the 2021 generation. In the first generation, the observed heterozygosity (Ho) = 0.600 ± 0.147 and the expected (He) = 0.729 ± 0.025, while in the second generation Ho = 0.767 ± 0.053 and He =0.778 ± 0.021. The average value of the fixation index (F) was lower in the second generation (0.014) than in the first (0.184). Phylogenetic analysis based on sequenced fragments of the control region suggested that the Ukrainian samples belong to the Mixed Central Europe clade defined by Weiss et al., 2002. This clade corresponds to lineage II of European graylings according to the separation proposed by Gum et al., 2009.
In the comparative analysis of two grayling generations from the fish farm, it can be stated that the genetic diversity did not decrease in the second generation when taking into account indicators such as the number of alleles (Na), the effective number of alleles (Ne), allelic richness (Ar) and Shannon’s biodiversity index (I). However, further monitoring is needed to increase the number of monitoring points and to assess changes in effective population size. The predominance of expected heterozygosity (He) over observed heterozygosity (Ho) in both groups indicates the presence of inbreeding. However, the values of the fixation index (F) suggest, that the inbreeding decreased in the second generation.
Based on the assessment and comparison of the morphological and productive parameters of generations at the stage of young-of-the-year (0 +), the most characteristic features observed were that the yield was at the level of 30% in the group of individuals of 2017 generation during the growing period from May to September, which is less than that obtained in the 2021 generation. However, the second generation was characterized by a lower average weight (4.6 g) than the first (9.73 g). Considering the principles of the identity of growing conditions, this fact may be related to technological shortcomings. To fully characterize the differences in the data of the two generations further studies of the 2021 generation are planned after the onset of puberty (for example, estimation of males and females, their fertility), as well as a comprehensive analysis of the variability of morphometric parameters. The study of the genetic features of the obtained generations of graylings is planned to be used in the future to preserve the species diversity of the ichthyocenoses of the river network. Phylogenetic studies of grayling allow establishing the belonging of the Ukrainian samples to the Mixed Central Europe clade and lineage II, which corresponds to previous generally presented schemes and, as indicated, is a mandatory step in the planning of conservation and stocking programs (Gum et al., 2009).
The results of the molecular genetic analysis will allow creating a basis for monitoring and controlling changes in the genetic structure and further selection work with these briding stock to maintain genetic biodiversity and rational economic management of them. Knowledge of the phylogenetic origin of the material will help avoid negative consequences during the stocking of rivers.
This contribution was funded by National Academy of Agrarian Sciences of Ukraine, DR № 0121U108916, and the European Union NextGenerationEU, project No. 09I03-03-V01-00075.
Kucheruk A., Mruk A., Buzevitch I. 2018. Forming a replacement‐brood stock of european grayling Thymallus thymallus (Linnaeus) in artificial conditions. Fisheries Science of Ukraine, 4: 28-38. doi: 10.15407/fsu2018.03.028
Gum B., Gross R., & Geist J. 2009. Conservation genetics and management implications for European grayling, Thymallus thymallus: synthesis of phylogeography and population genetics. Fisheries Management and Ecology, 16(1): 37–51. doi:10.1111/j.1365-2400.2008.00641.x
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