Генетическая вариабельность в группах лососевых рыб, выявляемая мультилокусным анализом геномной ДНК

Intensive fishing in open waters has led to an exhaustion of fish resources on a global scale. In this regard, fish breeding in closed reservoirs and in aquaculture conditions using recirculating water supply installations is becoming increasingly important

Selection in trout farming is based on the use of both cultivated previously raised breeds and commercial fish from different habitats. The latter forms include Onega, Baltic and Caspian salmon. Their use in breeding work during crossing requires data on the genetic and population characteristics of these fish. Molecular genetic methods have provided breeders with tools to control the genetic structure of fish populations

To isolate genomic DNA, we used venous blood from the tail vein of fish from Onega, Caspian, and Baltic salmon (n = 10, in groups of Baltic and Сaspian salmon, n = 9 with Onega salmon). DNA was extracted using conventional methods using cell lysis in buffer with sodium dodecyl sulfate; removal of the bulk of proteins was achieved by incubation with proteinase K for 3 hours at 55ºC. After shaking the mixture with phenol and centrifugation, the upper phase with DNA was collected into new tubes. DNA was precipitated with ethanol and washed with 70% ethanol to remove remaining salts. The purified DNA was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The quantitative and qualitative parameters of the samples were monitored in a NanoDrop 2000™ spectrophotometer at absorption wavelengths of 260, 280 and 320 nm.

Multilocus genome analysis was performed through successive steps of DNA digestion, electrophoresis, and molecular hybridization. Genomic DNA was digested with restriction enzyme BsuRI for 3 hours at 37ºC. The resulting samples were dissolved in distilled water and added to the wells of 0.8% agarose gel. Electrophoresis was carried out for 24 hours at 60V. The DNA in the gel was denatured with alkali, neutralized, and transferred to a positively charged nylon filter. Single-stranded DNA fragments were fixed by incubation in a thermostat at 80ºC for 2 hours. The filter with fixed DNA was placed in a bath with a reaction buffer containing a digoxigenin-labeled probe (GGAT)4. During incubation, this probe bound to individual complementary DNA fragments. After washing, these binding sites were detected by immunochemical analysis. In this case, the streptavidin-alkaline phosphatase conjugate was bound to digoxigenin and, after introducing special dyes into the solution, was detected in the form of colored bands on the filter

After carrying out the molecular hybridization reaction and visualizing DNA fragments, from 20 to 30 colored bands were detected in each electrophoretic lane (Fig).

Figure 1 - Genetic profiles of individuals of three groups of fish, revealed by multilocus analysis:

lanes 1-10 – Onega salmon; 11-20 – Caspian salmon; 21-30 – Baltic salmon; M – fragment length marker

Note: lambda phage DNA, digested with HindIII BstNII restriction endonucleases, sizes from 3675 to 23130 base pairs

DOI:10.23649/JAE.2024.43.10.1

Pairwise comparisons were made of the location of the bands, both within each group and between them. Parameters such as the proportion of common and different bands (BS value) characterizing a specific group and the genetic distance between groups were calculated (Table 1).

Due to the fact that salmon fish at early stages of development do not clearly demonstrate their breed characteristics, genetic analysis allows for identification of breeds (certification). This can be done either by calculating the frequency of DNA fragments in each group, or by identifying DNA fragments that are characteristic only for one group (marker fragments). The multilocus analysis made it possible to identify a number of promising DNA marker fragments characterizing individual groups of fish (Table 2).

It can be seen that DNA fragment number 21 (when visualizing fragments, they are numbered on the filter depending on the fragment size) is characteristic for Baltic salmon (frequency 0.9), it is rarely found in Onega salmon and absent in Caspian salmon. Some fragments are found in all individuals of all groups, i.e. are monomorphic, thus, they and not informative for subsequent analysis. Obviously, the geographic remoteness of the Caspian salmon habitat from the water resources of North-West Russia, which prevents any fish crossing, predetermined the accumulation of genetic differences of the Caspian salmon from the Baltic and Onega salmon.

Thus, the results of the study of three populations of salmon fish, obtained by multilocus analysis, indicate the promise of using this analysis to identify the specific features of the genetic and population structure of groups of fish of the salmon family. Genetically distant from the Baltic and Onega, salmon is the group of Caspian fish. Marker DNA fragments have been identified that allow certification of fish breeds and populations. Multilocus analysis of salmon genomic DNA proved to be an efficient approach for breed identification (certification), elucidation of population structure of fish groups and determination of intra-population genetic diversity by a criterion of average heterozygosity.