Sampling

In total, 147 specimens belonging to the sturgeon family were analyzed in our study (104 Acipenser gueldenstaedtii representatives, 43 Huso huso representatives). All specimens, details of sampling and on the origins, population are indicated in Table 1.

Samples were taken from 40 A. gueldenstaedtii specimens and 26 H. huso specimens. Samples were also taken from the natural population (64 A. gueldenstaedtii specimens and 17 H. huso specimens) caught in the delta of the Ural River and contained in RAS at the Ural-Atyrau Sturgeon Fish Hatchery RSE, which is a complex of engineering structures designed to grow young sturgeon species to replenish the stocks in the basin of the Caspian Sea. The Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University is a modern laboratory aimed at conducting scientific research at aquaculture facilities, including on sturgeon grown in RAS. UNKOPPA is the largest aquaculture complex in Central Asia, where about 35 tons of sturgeon are grown (breeding stock, rearing and breeding stock, commercial fish, and young fish) and which serves as a technical and research basis for the introduction of new developments in fish farming and industrial aquaculture.

DNA extraction

DNA extraction, genotyping, and processing of the results were performed in the Laboratory of Biotechnology and Diagnostics of Infectious Diseases of the Testing Center of the Zhangir Khan West Kazakhstan Agriculture and Technical University.

Fragments of the pectoral fin were selected as material for DNA extraction and short tandem repeat (STR) analysis. Each selected sample was assigned an identification laboratory number. DNA was extracted from fin tissues using a commercial DNA-Extran-2 kit (SYNTOL, Russia). The quality and quantity of the isolated DNA material was determined using a spectrophotometer.

Genetic analysis

For genotyping, we used a GeneXpert-Sturgeon reagent kit (SYNTOL, Russia) designed for the genetic typing of sturgeon species. Genotyping was performed at seven STR loci. Characteristics of all seven loci are indicated in Table 2, including primer sequence. This kit allows the typing of most specimens of sturgeon species. The genotyping results according to these loci can be reproduced in other laboratories; all these loci are amplified in multiplex polymerase chain reaction (PCR).

The amplification reaction was carried out according to the manufacturer’s instructions to the genotyping kit on the ProFlex PCR system thermal cycler (Applied Computer Systems, USA).

The separation of the obtained DNA fragments was carried out by capillary electrophoresis in a polymer gel using the AB 3500 genetic analyzer (Applied Microsystems, USA). The interpretation of the analysis results was carried out using the GeneMapper 6.0 software (Applied Biosystems).

Data analysis

Genetic diversity was analyzed using a specialized add-in for Microsoft Excel, GenAlEx 6.51 [39]. We evaluated the following indicators: allele frequency, number of alleles per locus, effective number of alleles (Na), observed (Ho) and expected (He) heterozygosity, fixation index (Fis), and Nei genetic distance index [40]. Cluster analysis was performed using the Structure 2.3.4 software based on the Markov Chain Monte Carlo (MCMC) method [41]. The number of clusters (C) ranged from 1 to 10. To analyze the results of Structure and identify the most optimal C value, a portable version of the Structure Harvester V. 0.6 software was used, which uses Evanno methods to calculate the Delta K index and determine the optimal number of clusters by sequentially iterating through them [42]. The results of the Structure launches were summarized using the CLUMPP software [43]. The resulting Q-matrices were presented in the form of histograms.  

Results

Phenotypic traits

As a result of the study of DNA polymorphism at seven STR loci, 104 specimens from four studied A. gueldenstaedtii samples showed 16 alleles at the AoxD161 locus, 28 alleles at the Afug41 locus, 13 alleles at the LS19 and Afug135 loci, 23 at the Afug37 locus, 20 at the Spl173 locus, and 17 at the AoxD165 locus. Concerning the 43 specimens in three studied H. huso samples, 5 alleles were found at the AoxD161 locus, 12 at the Afug41 locus, seven alleles each at the LS19, Afug135, Afug37, and Spl173 loci, and eight alleles at the AoxD165 locus . Some of the identified alleles were detected with a low frequency (less than 0.1), which may indicate the risk of loss of these alleles in the future in the studied populations.

Among the belugas, allele 100 at the AoxD161 locus (more than 0.6 in each population), allele 198 at the Afug135 locus (frequency from 0.4 to 0.7 depending on the population), and allele 190 (AoxD165) with a frequency from 0.6 to 0.92 in the artificial population of UNKOPPA (HHa1) were distinguished with high frequency. As for the samples of Russian sturgeon, the maximum frequency of alleles did not exceed 0.4, and a decrease in the number of alleles and, accordingly, an increase in frequencies was more typical for the artificial population of A. gueldenstaedtii (AGa).

The I index characterizes genetic diversity; the higher this indicator, the higher the level of diversity in the sample. In our case, the values of I are high in the samples of Russian sturgeon from natural populations (more than 2) and slightly lower in the sample from an artificial population, decreasing up to 1.8 at the Afug135 locus. Samples from beluga populations are characterized by lower I index values from 0.345 to 1.691, while the lowest index values were observed in the HHa1 sample.

The Ho level in almost all loci in the Russian sturgeon samples was below the He level, which indicates a deficiency of heterozygotes, probably as a result of inbred crossing in the population. This is also indicated by the positive value of the F index. The situation differs among the three samples from the beluga populations. In the HHn and HHa1 samples, Ho is higher than He in most loci (the F index is negative), while in the HHa2 sample, only one locus (LS19) out of seven is characterized by a higher level of Ho (0.786) over He (0.673). This fact suggests that the HHn and HHa1 samples have a higher level of heterozygosity compared to the HHa2 sample.

There are not many works in scientific literature on the genetic structure of sturgeons, and even fewer used STR markers for genotyping; there is a wide variety of microsatellite loci suitable for study. Therefore, there is not much work to compare the data we obtained with those available in world databases.

In the samples of the Russian sturgeon, compared with the samples of the beluga, a significantly higher number of alleles is observed for each locus, which is associated with the tetraploidy of these sturgeon species. However, in this image, a decrease in the number of alleles in the AGa sample can be traced for almost every locus except AoxD161, where the number of alleles is comparable to samples from natural populations. The reverse situation is typical for samples from beluga populations: the number of alleles in the sample from the artificial HHa2 group differs favorably in almost all loci from the other two samples, while the situation is similar in the HHn and HHa1 samples. This fact indicates a lack of brood fish in the natural environment and RAS at UNKOPPA and a better selection of parent pairs in Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University compared with the second RAS studied by us.

The He value was established for the study of genetic diversity. The He values are high, especially for the Russian sturgeon, as a polyploid species, while for the beluga, which is a diploid, it was significantly lower and ranged from 0.554 to 0.663. For the total sample of sturgeon, the He was 0.750. For the samples of Russian sturgeon, this indicator is the highest in the sample AgN3 (He=0.871), and the lowest in the sample AGa (He=0.829). Among the beluga, the highest He value was characterized by the HHa2 sample (He=0.663), and the lowest value was found in the HHa1 sample (He=0.554).

In all groups of Russian sturgeon, the possibility of inbreeding can be considered since they had a lack of heterozygotes for all the studied loci, the only exception was the Afug41 locus in the A. gueldenstaedtii AGa group. In the studied beluga groups in HHa2, six of the seven studied loci had a deficiency of heterozygotes, which indicates the possibility of inbreeding in this group.

The Russian sturgeon has the highest Na and Ne values in the AGn1 sample (Na=14,571; Ne=8,691), and the lowest in the AGa sample (Na=10,429; Ne=5,980). In beluga, the highest Na and Ne values were observed in the sample of artificial origin HHa2 (5,571 and 3,238, respectively), and the lowest in the sample of natural origin HHn (3,714 and 2,822, respectively). The low indicators of genetic diversity of the natural beluga sample can be explained by the fact that, first, the number of brood fish of this species is limited in natural conditions and, second, a small number of specimens were studied.

Results demonstrated that Na is the average number of alleles per locus, Na freq. ≥5% is the average observed number of alleles with a frequency of ≥5% per locus, Ne is the effective number of alleles, No. of private alleles is the average number of unique alleles in a population, and He is the expected heterozygosity. Thus, the average number of alleles with a frequency of ≥5% was ≈50% for samples from natural populations of A. gueldenstaedtii (AGn1, AGn2, AgN3) and more than 60% for the AGa sample. For samples from H. huso populations, the number Na freq. ≥5% was a significant 70% in the HHa1 sample, 79% in the HHa2 sample, and 88% in the natural HHn population sample. The effective number of alleles was more than 50% of the average number of alleles for each sample, while unique alleles were found in each group of samples (No. of private alleles) except a sample of H. huso from UNKOPPA (HHa1).

Analysis of genetic distances

The average intra-group distances of the Russian sturgeon and beluga for each of the species were approximately the same, and the distances between natural and artificial groups were greater, as expected, as Nei Genetic Distance indicated in Table 3.

There is a clear division of natural and artificial samples in both A. gueldenstaedtii and H. huso. There is a high similarity of two samples caught from natural conditions at different times, namely, AGn1 selected on 23.11.2022 and AgN3 selected on 27.06.2023. Here, repeated sampling from the same specimen is excluded, since newly caught specimens were scanned for the presence of a chip and chipped in case of its absence; all obtained genotypes were checked for duplication.

Cluster analysis

When conducting a cluster analysis of the genotyping results in the Structure program, the C value was set from 1 to 10. However, in all cases, starting from C =4, the clustering process became uninformative. The determination of Delta C values by the Evanno method showed that the most optimal number of clusters in the analysis of all samples (n= 147) of both fish species was three, with Delta C=727.544. Clustering results of the sturgeon fish at C=3 can be seen in Figure 1.

As can be seen from Figure 1, a cluster formed from 3 samples of A. gueldenstaedtii (green) of the natural population of the Ural-Atyrau Sturgeon Fish Hatchery is distinguished. A sample from the artificial population of Russian sturgeon of the Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University is distinguished in a separate cluster (blue). A group of one natural and two artificial populations of beluga is formed in a separate cluster (red). As can be seen in the figure, even though all populations are kept in different conditions, there are cross-values ​​from other species. This indicates the presence of common alleles, since genetic markers are preserved due to common origin. Nevertheless, with a separate analysis of the data for each species of studied sturgeon in the samples of belugas, there is also a division into two clusters (C=2 with Delta C=155.679), a sample of belugas from the natural population forms a separate cluster. In the case of Russian sturgeon samples, no changes are observed in a separate analysis, and two clusters are clearly distinguished separating samples from natural and artificial populations. Results of clustering of Russian sturgeon at C=2 are indicated in Figure 2.

As a result of the analysis of the structure of the studied groups of Russian sturgeon using modeling in STRUCTURE v.2.3.4 [25] based on the results of genotyping for seven STR loci, we showed two distinct clusters (C=2). In Figure 2, 2 clusters are distinguished. Formed from 3 samples of A. gueldenstaedtii (red) of the natural population of the Ural-Atyrau Sturgeon Fish Hatchery and a cluster of the artificial population of Russian sturgeon of the Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University (blue).

Based on the data on the genetic polymorphism of the studied samples of Russian sturgeon and beluga, the following recommendations are given:

1. To preserve the genetic resources of A. gueldenstaedtii, it is recommended to use a sample from the confluence of the Ural River with the Caspian Sea and contained in RAS at the Ural-Atyrau Sturgeon Fish Hatchery (AGn1 and AgN3) with the highest indicators of genetic diversity (He=0.867; Ne=8.691 and He=0.871; Ne=8.268);
2. To compensate for the damage caused to reservoirs by fishing and human economic activity, it is recommended to release sturgeons from the rearing and breeding stock of the Ural-Atyrau Sturgeon Fish Hatchery, the Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University, and UNKOPPA into the Ural River.

One of the main goals of genotyping and studying the genetic diversity of rare species of fish and sturgeons is to preserve the gene pool and prevent its depletion. The selection of parental pairs and the development of sturgeon crossing schemes becomes an important task. The data allows us to determine the degree of kinship of specimens based on microsatellite analysis data. The general sample of sturgeons is characterized by high He (He=0.750) and Ne values (Ne=5.676).

The indicators of genetic diversity in Russian sturgeon are higher in the groups AGn1 (He=0.867; Ne=8.691) and Agn3 (He=0.871; Ne=8.268), A. gueldenstaedtii from the Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University caught at the confluence of the Ural River with the Caspian Sea, and lower values are observed in the sample from the Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University AGa (He=0.829; Ne=5.980).

In beluga, the indicators of genetic diversity are higher in the group from the Laboratory of Ichthyology and Aquaculture of Zhangir Khan West Kazakhstan Agriculture and Technical University (He=0.663; Ne=3.238), and lower in the group from UNKOPPA (He=0.554; Ne=2.704).

In our case, with a maximum degree of kinship equal to 1, 100% of alleles in both specimens will be repeated and, conversely, if both specimens do not have the same alleles, the degree of kinship will be 0. Thus, when selecting parental pairs in RAS, it is recommended to identify (chip), as well as genotyping each specimen, and select pairs with the lowest total number of alleles. The maximum permissible value of the degree of kinship can be set at 0.25. However, it is necessary to separately consider the situation with the state of the gene pool and the number and type of genetic markers studied in each population and strive for lower values of the degree of kinship when developing crossing schemes.

Figures & Tables

Discussion

The study of genetic polymorphism was carried out using microsatellite markers, the effectiveness of which for sturgeon has been shown in many works [23,44-47].

Our findings unveiled notable differences in the genetic diversity between natural and artificial populations of sturgeon species (Acipenser gueldenstaedtii and Huso huso). The data we have obtained are consistent with the opinion of the authors [52], the presence of hexaploid individuals in A. gueldenstaedtii, low I index value for natural beluga populations, lower Ho values in Russian sturgeon compared to Beluga specimens which showed higher Ho values. Our data displayed that AoxD161 locus, Afug135, and AoxD165 from the artificial population of Hh displayed a higher frequency, which is in direct contrast to the data from these authors [52]. As in our previous work, a hexasomal distribution of alleles was found at the LS19 locus in four A. gueldenstaedtii specimens caught from the natural environment [48].

The data we obtained were slightly consistent with the opinion of the authors [53], who studied the six microsatellite loci of which AoxD16, AoxD165, and AfuG41 aligned with ours and revealed that belugas showed higher He values at AoxD161 and AfuG41 loci than those observed. In our research, He values in AfuG41 loci were consistently higher in every Hh population but AoxD161 was significantly low.

The highest genetic distance was recorded in Hhn, which is consistent with the results of the authors [52]; the low genetic distance among AG specimens aligns with these authors [54], and our findings on the clustering of the conclusion that values of C or K > 3 were uninformative was confirmed by [52]. Inbreeding within artificial populations may occur due to the absence of heterozygotes in several loci, which agrees with the work reported by Roques et al. (4) on Acipenser sturio. Thus, annual monitoring and broadening the sturgeon species range are necessary for preventing the formation of such related pairs of parents. These actions are consistent with proposals made by previous studies [55].

A. Dudu et al. [49] conducted a study of 33 samples of belugas caught in the Danube River in different years (2001-2007). The analysis was carried out on seven microsatellite loci, of which only one coincides with the loci used by us (LS19). The number of alleles in this locus was nine, whereas in ours, in each of the three samples, it did not exceed five for each sample and seven for the total number of belugas studied. R. Matache [50] focuses on the genotyping of 29 beluga whales caught in the Caspian Sea, where 10 alleles were found at the LS19 locus.

N.A. Nebesikhina et al. [51] analyzed 439 brood fish of Russian sturgeon from the repair and breeding stock of the Azdonrybvod and 167 specimens caught during the spawning period in the Sea of Azov. The study was conducted on five STR markers (An20, Afug41, Afug51, AoxD161, AoxD165), the number of alleles at matching loci was not much lower than the values we obtained, except the AGa sample; the Ho and He indicators were significantly higher than ours  and approached 1. According to Nebesikhina et al., the indicators of genetic diversity of samples from the natural environment were lower than the corresponding values for samples from artificial populations, although the opposite was observed in our work.

From our study, we obtained data that suggest species with high mean Ne and He and low genetic distance (AGn1, AGn3) should be implemented in RAS to increase genetic diversity, although, It is believed that RAS systems might mitigate inbreeding; the data received from our study suggest otherwise, and this may result from inadequate inbreeding or mismanagement. This finding calls for further research, a reassessment of the current RAS system, and more stringent monitoring

The obtained data on the genetic polymorphism of natural groups and those grown in RAS can be used to preserve the gene pools of Russian sturgeon and beluga typical for a region. The Ho and F values indicate a deficiency of heterozygotes in most of the studied samples as a result of closely related interbreeding within populations, which indicates a decrease in genetic diversity and a shortage of brood fish. This work should be carried out annually, covering a larger number of specimens and species of sturgeon, to monitor the state of indicators of genetic diversity, develop crossing schemes, and prevent the formation of parental pairs with a high level of genetic kinship.

Funding

The work was carried out within the framework of a grant funding project for scientific and (or) scientific and technical projects of the Ministry of Science and Higher Education of the Republic of Kazakhstan for 2022-2024 “Study of the specific features of the genetic structure of sturgeon fish and their hybrids grown in closed water supply installations” IRN AP14870980, state registration No. 0122RK00243.

Author Contributions

Nurbek Ginayatov: Conceptualization, Methodology, Data Analysis, Writing – Review & Editing. Vadim Ulyanov: Methodology, Investigation, Writing – Original Draft. Indira Beishova: Project Administration, Supervision, Writing – Review & Editing. Tatyana Ulyanova: Data Curation, Investigation, Writing – Original Draft. Aziza Sidarova: Investigation, Data Analysis, Writing – Review & Editing. Alexandr Kovalchuk: Software, Validation, Data Visualization. Bekbol Sariyev: Formal Analysis, Writing – Review & Editing. Ulbolsyn Kuzhebayeva: Investigation, Data Curation, Writing – Original Draft. Anna Bakhareva: Investigation, Resources, Writing – Review & Editing. Kuantar Alikhanov: Supervision, Funding Acquisition, Writing – Review & Editing.

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

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