Development of multiplex panel of microsatellites for genetic studies of Siberian sturgeon (Acipenser baerii) bred in commercial aquaculture

The Siberian sturgeon ( Acipenser baerii Brandt, 1869) is one of the most important aquaculture fish species in Russia. Due to the high demand for valuable commercial products, breeding of Siberian sturgeon is promising in the industry. However, breeding this species is significantly complicated by because of its tetraploid genome, which, together with the need to mix fidh roe and sperm from several producers when obtaining offspring, prevents the introduction into practice of well-proven molecular genetic methods, e.g. microsatellite analysis. In this work, for the first time, the known microsatellite loci in the Siberian sturgeon are characterized from the point of view of the possibility of effectively accounting for the doses of their alleles in the tetraploid genome. Seven loci were found that met this criterion. The goal of our work was to create a panel of microsatellite markers adapted for use in the selection of Siberian sturgeon from the Lena population. The research was carried out in 2023. As biological material, we used sections of fin tissue of the Siberian sturgeon of the Lena population, taken from the fish of an experimental herd kept in a closed water supply installation of the Ernst Federal Research Center for Animal Husbandry. The experimental herd contained fish from the Mozhaisk production and experimental fish hatchery (Goretovo village, Mozhaisk urban District, Moscow Province; group I, n = 42) and fish obtained from RTF Diana LLC (village Kaduy, Kaduysky District, Vologda Province; group II, n = 47). DNA was isolated using the DNA-Extran-2 kit (NPK Synthol, Russia) according to the manufacturer’s protocol. Qualitative assessment of DNA was carried out by electrophoresis in 1.2 % agarose gel. PCR was performed in a Thermal Cycler SimpliAmp amplifier (Thermo Fisher Scientific, Inc, USA). Electrophoretic separation of amplification products was carried out in a Nanofor 05 capillary electrophoresis system (NPK Synthol, Russia). Allele sizes were determined using GeneMarker software (Version 3.0.1). For each locus, the dose of each allele was determined. Twenty seven microsatellite markers known for sturgeon fish species were used (Ls 19, Aox 45, Aox 9, Ls 68, Agu 38, Ag 49a, Agu 37, Agu 41, Agu 15, Agu 51, Agu 59, Agu 34, Agu 36, Agu 46, Agu 56, Agu 54, AoxD 161, AfuG 63, AfuG 51, AfuG 112, An 20, Aru 13, Aru 18, Afu 68 b, Spl 163, AfuG41, and Ls 39). Of these, seven were selected for multiplex panels (Agu 38, An 20, Aru 18, Ls 19, Ag 49a, Agu 37, Agu 41). Based on the polymorphism of seven microsatellite loci for the two studied groups of Siberian sturgeon individuals, classical population genetic indicators were calculated, the average number of alleles per locus (Na = 6.86), the number of effective alleles (Ne = 3.61), observed (Ho = 0.839) and expected (He = 0.6535) heterozygosity. In the studied groups, inbreeding was not revealed (FIS = -0.340 and -0.173) while a significant genetic differentiation occurred (Nei’s GD = 0.1340, Fst = 0.0796). The groups formed two clear, practically non-overlapping PCA clusters despite the fact that the ancestors of Siberian sturgeon individuals in both groups were of related origin. The contribution of the allele dose of the tetraploid locus to the efficiency of microsatellite analysis was assessed. On average, the information content of each locus increased by 32 %. A comparison of the results of genetic analysis with the available research publications allows us to assume that in aquaculture herds of Siberian sturgeon from the Lena population, processes associated with changes in allele frequencies of microsatellite loci occur, which gradually enhance their genetic differentiation. As a result of the work, the high efficiency of the created panels of microsatellite markers and their potential suitability for use in genetic certification were confirmed, each individual Siberian sturgeon had its own genetic profile. The distribution of alleles at seven microsatellite loci indicated a unique genetic structure in Siberian sturgeon stocks in each of the two fish hatcheries that were sources of fish seeding material.