Genetic diversity and population structure of Aberdeen Angus cattle in Kazakhstan based on microsatellites and the TG5 and LEP genes

Under conditions of intensive beef cattle industry development in Kazakhstan, evaluating the genetic diversity and population structure of Aberdeen Angus cattle, as well as the distribution of alleles and genotypes of functional TG5 and LEP SNPs associated with meat quality, is an urgent task. The study aimed to perform an integrated population and functional genetic analysis of Aberdeen Angus cattle using neutral STR markers and TG5 and LEP SNPs to simultaneously assess genetic diversity, population structure, and the distribution of alleles potentially associated with meat productivity traits and beef quality. A total of 122 bulls from three farms were analyzed; genotyping at 15 microsatellite loci was performed using capillary electrophoresis, while the SNP loci of the TG5 and LEP genes were investigated via PCR-RFLP with validation by Sanger sequencing; statistical processing included the calculation of genetic diversity indices and F-statistics, and PCoA and STRUCTURE analyses. A moderate level of genetic diversity was established, with close heterozygosity values and no signs of inbreeding; interpopulation differentiation was low (mean Fst≈0.016) with a high gene flow, while cluster analysis revealed the absence of a distinct population structure and a high degree of admixture. For TG5, a predominance of the C allele and the CC genotype was noted, whereas for the LEP gene, a more uniform distribution of genotypes with a high proportion of heterozygotes was observed. The obtained results indicate that the studied populations are characterized by moderate genetic diversity and low interpopulation differentiation, which can be applied to genetic monitoring and the enhancement of breeding programs.

Cattle farming plays a key role in the meat sector of Kazakhstan’s agriculture industry, providing a significant share of domestic beef production. In recent years, the industry has demonstrated steady growth, reflected in an increased volume of beef production, a sustained positive trend, and a simultaneous expansion of the cattle population (Ministry of Agriculture of the Republic of Kazakhstan, 2026). The Aberdeen Angus breed holds a leading position due to its early maturity, efficient feed conversion, pronounced marbling, and excellent adaptability to the climatic and environmental conditions of Kazakhstan (Shevchenkо et al., 2024b). However, the intensive use of a limited number of sires and the rapid expansion of the breeding base can lead to a reduction in genetic diversity, an increase in inbreeding levels, and, consequently, a decline in the adaptive potential and breeding value of populations. In this regard, regular genetic monitoring becomes an essential element for the sustainable development of the beef cattle industry.

Molecular genetic methods are widely used to assess the status of the gene pool, among which microsatellite markers (STR, short tandem repeats) play a particularly prominent role. Due to their high degree of polymorphism and codominant inheritance, STR markers serve as an effective tool for assessing genetic diversity, analyzing population structure, detecting signs of inbreeding, and determining the level of genetic differentiation and interpopulation gene flow (Cortes et al., 2022; Bilginer et al., 2022). Their informativeness has been confirmed by numerous studies on various cattle breeds, including Aberdeen Angus (Kale et al., 2010; Kramarenko et al., 2018; Matyukov et al., 2025; Gamarra et al., 2020; Márton et al., 2024; Shevchenko et al., 2024a; Moreno-Sierra et al., 2020).

In recent years, high-density SNP arrays and whole-genome sequencing have been increasingly utilized to study genetic diversity, population structure, genomic relatedness, and signatures of selection, providing higher resolution compared to traditional molecular markers (Ogunbawo et al., 2024; Fan et al., 2010; Song et al., 2024; Hu et al., 2024). These approaches have been successfully applied to characterize the genetic structure and diversity of numerous beef cattle breeds, including Aberdeen Angus and other commercial populations (Bhowmik et al., 2023; Kolpakov et al., 2024; Kasarda et al., 2020). At the same time, microsatellite markers retain their relevance as a reliable, cost-effective, and internationally standardized tool for routine genetic monitoring, comparative population studies, and the management of breeding resources (Ajmone-Marsan et al., 2023; FAO, 2011; Cortes et al., 2022).

Despite the high informativeness of modern genome-wide approaches, they are not designed for the direct assessment of the distribution of functionally significant alleles associated with economically valuable traits. In turn, STR markers are selectively neutral and primarily reflect demographic and population processes; therefore, they do not allow for a direct evaluation of the distribution of alleles associated with economically valuable traits, including meat quality.

To characterize functional genetic diversity, single-nucleotide polymorphisms (SNPs) of candidate genes influencing animal productive traits are used. The TG5 and LEP SNPs were selected for this study as they are among the most thoroughly investigated functional candidate genes associated with cattle productivity traits (Bеkseitov et al., 2017; Safronova et al., 2017; Moldakarimov and Buralkhiev, 2015; Panier et al., 2010; Van Dung et al., 2024; Gao et al., 2024). The TG5 gene encodes thyroglobulin, a precursor of thyroid hormones involved in regulating energy expenditure, lipid metabolism, and intramuscular fat deposition (Kostusiak et al., 2023), whereas the LEP gene encodes leptin, a key regulator of energy balance, adipose tissue accumulation, and animal growth (Almeida et al., 2008; Wylie, 2011).

TG5 polymorphisms are considered potential quantitative trait markers associated with meat marbling and lipid profile characteristics (Ardıçlı et al., 2019; Shin and Chung, 2007; Dolmatova et al., 2020; Konjaria, 2023; Tyshchenko et al., 2020; Zhang et al., 2015; Barendse et al., 2004), whereas LEP gene variability is linked to live weight and the chemical composition of the meat (Almeida et al., 2008; Wylie, 2011; López-Rojas et al., 2017; Sycheva et al., 2023; Buchanan et al., 2002). Thus, due to their well-characterized nature and confirmed associations with meat performance traits, the TG5 and LEP polymorphisms represent promising markers for evaluating the distribution of functionally significant alleles in beef cattle populations.

However, the distribution of functional gene alleles cannot be accurately interpreted without accounting for the overall genetic structure of the population. Allele frequencies, homozygosity levels, and the distribution of genetic variants are shaped by the population’s demographic history, genetic drift, migration, and selection pressure. Therefore, relying solely on functional SNPs fails to assess the overall genetic status of the population, whereas using exclusively neutral STR markers provides no information on the prevalence of economically valuable genetic variants. Consequently, the combined use of neutral STR markers and functional SNPs provides a more comprehensive population characterization, integrating population genetic analysis with the evaluation of genetic factors influencing product quality.

Despite the active development of Aberdeen Angus cattle breeding in the Republic of Kazakhstan, comprehensive studies integrating the analysis of neutral STR markers and functionally significant TG5 and LEP SNPs remain absent to date. Existing works generally examine either population structure (Shevchenko et al., 2024a) or individual candidate genes (Dushayeva et al., 2021), without integrating these data into a unified analytical model.

The aim of this study is to conduct an integrated population and functional genetic analysis of Aberdeen Angus cattle in the Republic of Kazakhstan using neutral STR markers and TG5 and LEP polymorphisms to simultaneously evaluate genetic diversity, population structure, and the distribution of alleles potentially associated with meat productivity and beef quality.

Sample collection and DNA extraction

A total of 122 Aberdeen Angus bulls, aged 18–24 months, obtained from private farms in the Republic of Kazakhstan, were analyzed in the study (Table 1, Figure 1). The animals were kept under similar feeding, housing, and veterinary care conditions and were considered clinically healthy at the time of sampling. Sampling was performed randomly from breeding herds.

Hair follicles collected from the tail switch (20–30 hairs per animal) were used as biological material. The samples were placed in paper envelopes labeled with the animal identification number and the collection date.

DNA extraction was performed using a commercial kit, DNA-Extran-2 (SYNTOL, Russia). DNA concentration and quality were evaluated using an Agilent Cary 60 spectrophotometer (Agilent Technologies, USA), as well as by 1.5% agarose gel electrophoresis in 1× TBE buffer. DNA samples were stored at -20 °C until further analysis.

STR genotyping

Genotyping was performed using the COrDIS Cattle kit (GORDIZ, Russia), which includes 15 microsatellite loci. Among these, 12 loci belong to the standard marker panel recommended by the International Society for Animal Genetics (ISAG): ETH3, INRA023, TGLA227, TGLA126, TGLA122, SPS115, ETH225, TGLA53, BM2113, BM1824, ETH10, and BM1818. Additionally, three highly polymorphic microsatellite loci recommended by the Food and Agriculture Organization of the United Nations (FAO) were included in the analysis: CSRM60, CSSM66, and ILSTS006.

DNA amplification was performed using a ProFlex™ PCR System thermal cycler (Applied Biosystems, USA) according to the manufacturer’s instructions. The separation of amplified fragments was carried out by capillary electrophoresis in a polymer gel on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA) using the S550 internal size standard (GORDIZ, Russia). Allele sizing and profile interpretation were conducted using GeneMapper v6.0 software (Applied Biosystems, USA).

To ensure genotyping quality control, approximately 10% of the DNA samples were reanalyzed, resulting in a 100% concordance rate between the obtained genotypes.

SNP genotyping of TG5 and LEP

The study analyzed two functionally significant single-nucleotide polymorphisms (SNPs) associated with lipid metabolism and carcass quality traits in cattle: TG5: c.-422C>T and LEP: c.73T>C (p.Arg25Cys). Genotyping was performed using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method, with subsequent validation of the results by Sanger sequencing.

PCR amplification

Amplification of the TG5 (548 bp) and LEP (239 bp) gene fragments was performed using a ProFlex™ PCR System thermal cycler (Applied Biosystems, USA). Each reaction contained 1× PCR buffer (1.5 mM MgCl2), 0.25 mM of each dNTP, 0.5 µM of each primer, 1 U of Taq DNA polymerase (SibEnzyme, Russia), and approximately 50 ng of genomic DNA. The total reaction volume was adjusted to 20 µL with nuclease-free water. A negative control (no DNA template) was included in all amplification runs to monitor for contamination.

Amplification of the TG5 gene fragment (548 bp) consisted of the following steps: initial denaturation at 94 °C for 4 minutes, followed by 40 cycles of denaturation at 94 °C for 30 seconds, annealing at 63 °C for 30 seconds, elongation at 72 °C for 20 seconds, and a final elongation at 72 °C for 5 minutes.

The PCR program for the amplification of the LEP gene fragment (239 bp) consisted of an initial denaturation at 94 °C for 4 minutes, followed by 40 cycles of denaturation at 94 °C for 10 seconds, annealing at 68 °C for 10 seconds, elongation at 72 °C for 10 seconds, and a final elongation at 72 °C for 5 minutes.

PCR products were verified by horizontal agarose gel electrophoresis in Tris-borate-EDTA (TBE) buffer using SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific, USA). The TG5 (548 bp) and LEP (239 bp) amplicons were resolved on a 2.5% agarose gel alongside a 50 bp DNA Ladder (Thermo Fisher Scientific, USA). PCR products were visualized under UV light.

The primer sequences and annealing temperatures for each locus are presented in Table 2.

PCR-RFLP analysis

For SNP genotyping, the PCR products were subjected to restriction fragment length polymorphism (PCR-RFLP) analysis. The amplified fragments of the TG5 and LEP genes were digested using the restriction endonucleases B st X2I and BspAC I, respectively, according to the manufacturer’s instructions (SibEnzyme, Russia). The reaction mixtures contained 2.5 U of BstX2 I and 1.25 U of BspAC I in their respective reaction buffers. Restriction digestion was performed at the optimal temperatures for each enzyme: 60 °C for the TG5 locus and 37 °C for the LEP locus.

The restriction products were resolved by horizontal electrophoresis on a 2.5–3.0% agarose gel containing SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific, USA) for 90 minutes, after which the DNA fragments were visualized under a UV transilluminator. The fragment sizes for the TG5 and LEP loci were determined using a 50 bp DNA Ladder (Thermo Fisher Scientific, USA). Genotype interpretation was performed based on the expected fragment sizes (Table 2).

Sanger sequencing validation

To confirm genotyping accuracy, 10% of randomly selected samples were reamplified and analyzed by Sanger sequencing. To increase the accuracy of nucleotide sequence determination, sequencing was performed in both directions (forward and reverse).

Prior to sequencing, PCR products were purified from primers, primer-dimers, and non-specific products using the ExoSAP-IT™ PCR Product Cleanup kit (Applied Biosystems, USA) according to the manufacturer’s instructions. Sequencing reactions were performed using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). For the TG5 (548 bp) and LEP (239 bp) loci, the same primers as for PCR were used.

Following the sequencing reaction, the products were purified using the BigDye XTerminator Purification Kit (Applied Biosystems, USA) and analyzed on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA).

Chromatograms were analyzed using Sequencing Analysis Software v7.0 (Applied Biosystems, USA). The obtained sequences were then aligned and analyzed using Geneious Prime ® v2026.0.2 software to identify single-nucleotide polymorphisms (Geneious, 2026).

A genotype was considered confirmed upon 100% concordance between the PCR-RFLP and sequencing results.

Statistical analysis

STR-based population analysis

The genetic diversity of the populations was analyzed using the GenAlEx 6.51 software package (Peakall and Smouse, 2006). The following parameters were calculated for each locus and population: allele frequencies, number of alleles per locus (Na), effective number of alleles (Ne), Shannon’s information index (I), observed (Ho) and expected (He) heterozygosity, and unbiased expected heterozygosity (uHe).

Population differentiation was assessed using Wright’s F-statistics (Fis, Fit, and Fst). Based on the Fst values, the level of gene flow between populations (Nm) was calculated. Genetic distances between populations were determined according to Nei (Nei, 1972). The spatial genetic structure of the populations was visualized by Principal Coordinate Analysis (PCoA) based on the genetic distance matrix.

The hidden population structure was assessed using STRUCTURE v.2.3.4 software (Pritchard et al., 2000) based on the Bayesian Markov Chain Monte Carlo (MCMC) algorithm. The analysis was performed for a number of clusters ranging from K = 1 to 10, with a burn-in period of 100, 000 iterations followed by 1, 000, 000 MCMC iterations. For each K value, 10 independent runs were executed.

The optimal number of clusters was determined by the Puechmaille method (Puechmaille, 2016) using the StructureSelector online service (StructureSelector, 2026), which is designed for the analysis and visualization of genetic clusters based on various methods (LnP(K), DeltaK, Choose K, MedMedK, MedMeaK, MaxMedK, and MaxMeaK) (Li and Liu, 2018; Kopelman et al., 2015).

The STR locus allele frequencies were visualized in the RStudio environment v2026.01.0 + 392 (R Core Team, 2025) using the following packages: tidyverse v2.0.0 (Wickham et al., 2019) and viridis v0.6.5 (Garnier et al., 2024). The results are presented as heatmaps.

SNP analysis

The investigation of the genetic structure of Aberdeen Angus cattle groups based on the TG5 and LEP polymorphisms included the analysis of allele and genotype frequencies, as well as an assessment of the conformity of genotype distribution to the Hardy–Weinberg equilibrium (HWE).

The genotype frequencies for the TG5 c.-422C>T and LEP c.73T>C (p.Arg25Cys) polymorphisms were determined by direct counting. The allele frequencies were calculated based on the observed genotypes (Merkuryeva and Shangin-Berezovsky, 1983).

The conformity of the genotype distribution to the Hardy–Weinberg equilibrium (HWE) was tested using the chi-square test with one degree of freedom (df = 1) (Ayala and Kiger, 1984).

The χ2 test was also used to compare the distributions of allele and genotype frequencies between populations. In cases where the expected values in individual classes were less than 5, the calculation was performed using Yates’s correction.

Statistical significance of differences was set at p < 0.05. Statistical data processing was performed using Microsoft Excel software (Microsoft, USA).

Integration analysis

To compare the results obtained from the analysis of neutral STR markers and functional SNPs, an integrated analysis of the population structure was performed.

Population clustering was assessed separately based on STR markers and TG5 and LEP SNPs using STRUCTURE software. Given the absence of a distinct cluster structure and the high degree of admixture revealed by the STRUCTURE analysis, an additional analysis of SNP distribution across clusters was not considered methodologically justified.

To evaluate the consistency of genetic differentiation obtained by different marker types, Fst coefficients were calculated for both STR and SNP data, and a comparative analysis was performed. Additionally, the results of the Principal Coordinate Analysis (PCoA) based on STR and SNP markers were compared to assess the consistency of the identified genetic structure patterns. Statistical calculations were performed using Microsoft Excel software (Microsoft, USA).

Genetic diversity based on STR markers

During the study, complete genotypes for all 15 microsatellite loci were obtained for all analyzed Aberdeen Angus cattle individuals. A total of 94 alleles were identified across all loci, with 81 alleles found in the KB population, 85 in SHUK, and 81 in TIM, indicating a moderately high level of allelic diversity in the studied groups.

The distribution of allele frequencies across the STR loci in the studied populations is presented as a heatmap (Supplementary Figure 1). The visualization demonstrates moderate differences among the populations regarding the polymorphism levels of individual loci. A detailed analysis of allele frequencies showed that the highest number of alleles per locus was observed in the SHUK population (up to 9), whereas in the KB and TIM populations, this parameter ranged from 4 to 7 and from 4 to 8, respectively. The TGLA122 and SPS115 loci were characterized by a relatively low level of polymorphism, while CSSM66, TGLA227, and BM2113 demonstrated high allelic variability. The presence of unique alleles in certain populations may reflect early signs of genetic differentiation; however, their contribution to the overall population structure appears to be limited.

Genetic diversity parameters (Supplementary Table 1) demonstrate a comparable level of variability among the groups. The number of alleles per locus (Na) ranged from 4 to 7 in KB, from 4 to 9 in SHUK, and from 4 to 8 in TIM. The effective number of alleles (Ne), which accounts for frequency distribution, showed slightly higher values in the SHUK population (2.322–5.088) compared to KB (1.838–4.880) and TIM (2.046–5.007), reflecting a more uniform distribution of allele frequencies and, consequently, a higher level of effective genetic diversity. A similar trend was observed for Shannon’s index (I), the values of which were slightly higher in SHUK. In most cases, observed heterozygosity (Ho) marginally exceeded expected heterozygosity (He), indicating a mild excess of heterozygotes and an absence of inbreeding signs. The unbiased expected heterozygosity (uHe) values were close to He, indicating that sample size had a minimal impact on the estimation of this parameter. The fixation index (F) for most loci took negative values, which further indicates the absence of inbreeding at the level of the analyzed loci.

The summary parameters averaged across all loci (Table 3) confirm the identified trends. The mean number of alleles (Na) was 5.400 in KB, 5.667 in SHUK, and 5.400 in TIM, whereas the effective number of alleles (Ne) varied within a narrow range (3.149–3.333). Shannon’s index (I) also showed close values (1.300–1.339), reflecting a moderate level of genetic variability in all populations. The standard errors (SEs) for all parameters were relatively low, indicating the stability of the estimates and limited variability among the loci. The higher Na values in the SHUK population may be attributed to its larger sample size (n = 55). At the same time, the elevated Ne and I values, which are less dependent on sample size, indicate that the observed increase in genetic diversity in this population reflects actual features of its genetic structure, likely associated with its history of formation or breeding management practices. Overall, the obtained results indicate a moderate level of genetic diversity in all studied populations of Aberdeen Angus cattle, accompanied by poorly defined interpopulation differences.

Population differentiation and genetic structure

Estimates of the intrapopulation fixation index (FIS) for most of the studied loci were negative, ranging from -0.016 (TGLA227) to -0.073 (INRA23), with a mean FIS value of -0.022 ± 0.010 (Supplementary Table 2А). Positive FIS values were observed only for a few loci (CSSM66, TGLA126, TGLA122, and SPS115). This pattern is consistent with the results of the heterozygosity analysis and indicates the absence of pronounced inbreeding. The mean value of the total fixation index (FIT) was -0.006 ± 0.011, which demonstrates a low level of genetic structure within the aggregate of the studied populations. The values of the intersubpopulation fixation index (Fst) ranged from 0.003 to 0.029, with a mean value of 0.016, which corresponds to a low level of interpopulation differen-tiation according to Wright’s classification (Wright, 1978). Gene flow values ranged from 8.399 to 95.159 (with a mean value of 23.145), significantly exceeding the threshold of Nm greater than 1, which is required to prevent genetic differentiation. Low Fst values can be attributed to high gene flow, a shared history of population formation, and similar breeding practices, all of which promote genetic homogenization. This high level of gene flow is likely due to the intensive use of artificial insemination, the exchange of breeding material between farms, and the potential use of imported genetic material. A significant role may also be played by centralized breeding support systems, which allow for the use of the same sires across different farms, thereby increasing the degree of genetic similarity between them. The extremely high Nm value for the TGLA122 locus is likely due to its low informativeness resulting from a limited number of alleles and, consequently, a low Fst value, which reduces the resolution power of this locus when assessing interpopulation differentiation.

Estimates of genetic distances (Supplementary Table 2B) also indicate a very low level of differentiation among the studied groups. The highest value was observed between the TIM and SHUK populations (0.050), and the minimum was between TIM and KB (0.046), with the differences between all population pairs remaining negligible. This demonstrates a weak influence of the geographical factor on the formation of the genetic structure. Principal Coordinate Analysis (PCoA) based on Nei’s genetic distances demonstrates the spatial arrangement of the studied Aberdeen Angus cattle populations (Figure 2). The populations form closely situated groups in the space of the first two coordinates without distinct clusters, indicating a low level of genetic differentiation. The SHUK population is shifted along the first coordinate axis, whereas TIM and KB differ predominantly along the second coordinate axis. Overall, the obtained results are consistent with the low Fst values and evidence the genetic proximity of the studied populations.

The optimal number of genetic clusters (K) was determined based on the Puechmaille methods (MedMed K, MedMean K, MaxMed K, and MaxMean K) (Puechmaille, 2016). All utilized criteria demonstrate consistent results, indicating an optimal value of K = 2 (indicated by the red line) (Supplementary Figure 2). The obtained data indicate the presence of two main genetic clusters in the studied population; however, their weak differentiation may point to a low level of population structuring. Despite the identification of two clusters (K = 2), a significant mixing of genetic components among the populations is observed, indicating a high level of admixture and the absence of a distinct population structure (Figure 3). With an increase in the number of clusters, the situation remains unchanged, and all three populations demonstrate a mixed genetic composition with a predominance of one of the clusters (Supplementary Figure 3). This points to a common origin of the populations alongside early-stage differentiation. Differences among the populations are poorly defined, and intrapopulation variability is comparable to interpopulation variability. Taken together, the PCoA and STRUCTURE results are consistent with the FST and Nm estimates, indicating weak genetic differentiation and a high degree of genetic consolidation among the studied populations.

Distribution of SNP genotypes

PCR-RFLP analysis of the TG5 and LEP polymorphisms revealed distinct electrophoretic profiles corresponding to different genotypes (Figures 4A, 5A). For the TG5 (c.-422C>T) and LEP (c.73T>C) loci, the genotypes were clearly distinguished by restriction fragment lengths after digestion with the enzymes BstX2 I and BspAC I, respectively. The obtained profiles completely matched the expected fragment sizes. The accuracy of SNP genotyping was confirmed by Sanger sequencing. Representative chromatograms (Figures 4B–D, 5B–D) demonstrated unambiguous identification of homozygous and heterozygous states, and multiple sequence alignment confirmed the presence of the target nucleotide substitutions at the loci. The complete agreement between the PCR-RFLP and sequencing results (100% concordance) demonstrates the high reliabil-ity of genotyping and rules out the possibility of systematic errors.

The distribution of genotypes and allele frequencies for the TG5 and LEP polymorphisms in the studied populations (KB, SHUK, and TIM) is presented in Figure 6. For the investigated TG5 SNP, a pronounced predominance of the C allele was observed in all populations, with its frequency consistently increasing from KB (0.671) to SHUK (0.727) and TIM (0.813). Accordingly, the proportion of the homozygous CC genotype increased in the same direction, reaching its maximum value in the TIM population (71.9%). In contrast, the heterozygous CT genotype predominated in the KB population (54.3%), which may reflect a higher degree of genetic variability or the specific features of this group’s formation. The low frequency of the TT genotype (5.7–9.4%) in all populations indicates a limited distribution of the alternative T allele. For the LEP SNP, the genotype distribution was characterized by a more balanced structure. A high proportion of heterozygotes (CT: 37.1–54.5%) was observed across all populations, whereas the frequencies of the homozygous CC and TT genotypes were comparable. The T allele predominated in all groups (0.563–0.586); however, its frequency range was narrow, indicating genetic homogeneity of the populations at this locus. The absence of a pronounced bias toward any single genotype indicates the maintenance of genetic polymorphism at the locus under study.

Comparative analysis showed that differences among the populations were more pronounced for the TG5 locus than for LEP. The observed trend toward an increase in the frequency of the C allele and the CC genotype from the KB to the TIM population may be attributed to the specific demographic and historical formation of the studied populations, including genetic drift, the founder effect, or differences in the breeding histories of the farms. At the same time, the differences among the populations for LEP were minimal, which is consistent with the results of STR markers and indicates a high degree of genetic consolidation among the studied groups. The obtained results demonstrate varying degrees of variability for the studied SNPs: the TG5 locus shows signs of directional changes in allele frequencies among the populations, whereas the LEP locus is characterized by a stable distribution and higher heterozygosity. These differences may be associated with the specific formation of the populations under study, including their origin, the exchange of breeding stock, and the breeding system employed.

Hardy–Weinberg equilibrium

Table 4 presents the results of the Hardy–Weinberg equilibrium analysis for the TG5 (c.-422C>T) and LEP (c.73T>C) polymorphisms in the three studied populations (KB, SHUK, and TIM), as well as in the pooled sample. For the TG5 locus, observed heterozygosity (Ho) ranged from 0.188 (TIM) to 0.543 (KB), whereas expected heterozygosity (He) ranged from 0.305 to 0.441. The χ² values were within the range of 0.38–2.85, with all p-values exceeding 0.05 (0.17–0.54), indicating no statistically significant deviations from the Hardy–Weinberg equilibrium. In the pooled sample, Ho was 0.365 and He was 0.381 (χ²= 0.39; p = 0.53), which also confirms compliance with the equilibrium. For the LEP locus, observed heterozygosity ranged from 0.371 (KB) to 0.545 (SHUK), while expected heterozygosity ranged from 0.485 to 0.492. The χ² values remained low across all groups (0.01–1.93), and the p-values were high (0.16–0.93), further confirming the Hardy–Weinberg equilibrium in all populations. In the total sample, Ho and He were 0.472 and 0.488, respectively (χ²= 0.01; p = 0.92). Thus, for both studied loci, no significant deviations from the Hardy–Weinberg equilibrium were detected in any populations or in the pooled sample (p > 0.05). The obtained results indicate the absence of pronounced disruptions in random mating and are consistent with the overall low level of population differentiation revealed by the STR marker analysis. At the same time, the absence of significant deviations from the HWE should be interpreted with caution, as the power of the χ²-test is limited by the sample size, and the identified differences in allele frequencies, particularly for the TG5 locus, may reflect the formation patterns of the population.

Integration of STR and SNP results

The distribution of the TG5 and LEP polymorphisms was compared with the population genetic structure revealed by SNP data using the STRUCTURE software. It should be noted that this analysis was exclusively supplementary in nature, as the use of only two SNPs significantly limits the resolving power compared to analyses based on STR markers or high-density SNP panels. Therefore, the obtained results were not used as a standalone assessment of the population structure, but rather served to verify whether the distribution of functional alleles was consistent with the overall genetic structure established using neutral STR markers.

The analysis showed that at the optimal number of clusters (K = 2), all studied populations are characterized by a similar distribution of genetic components and a high degree of admixture (Supplementary Figure 4A). A clear separation of animals into genetically distinct groups is absent. With an increase in the number of clusters to K = 3, the population structure maintains its mixed character without the formation of stable and genetically differentiated clusters (Supplementary Figure 4B). The observed distribution of genetic components points to a common origin of the studied animals and intensive genetic exchange among the populations. Due to the absence of distinct genetic clusters, an additional analysis of the TG5 and LEP genotype distributions within individual clusters would not yield biologically interpretable results and was therefore not considered. The obtained results are consistent with the low Fst values for the TG5 and LEP SNPs and do not indicate a pronounced differentiation of the investigated functional loci between the populations.

Table 5 presents the values of the genetic differentiation coefficient (Fst) calculated based on STR markers and TG5 and LEP polymorphisms for pairs of the studied populations. For STR markers, the Fst values varied within a narrow range from 0.011 to 0.012 for all population pairs (KB–TIM, KB–SHUK, and SHUK–TIM), indicating a consistently low level of interpopulation differentiation. A similar pattern was observed for SNPs, for which the Fst values remained low (0.002–0.013) despite slightly higher variability between individual population pairs. Comparison of the results obtained using STR and SNP markers demonstrated the consistency of the estimates: in both cases, a weak level of genetic differentiation among the populations was revealed. Minor differences in Fst values may reflect the different nature of the markers, as STRs characterize neutral genomic variability, whereas TG5 and LEP SNPs are associated with functionally significant traits. Thus, the analysis results confirm that both neutral and functional markers demonstrate a similar pattern of population structure, indicating limited interpopulation differentiation.

Figure 7 presents the results of the Principal Coordinate Analysis (PCoA) performed on the basis of the TG5 and LEP gene SNPs. The spatial distribution of the populations along the first two coordinates demonstrates their genetic relationships based on functional markers. The KB and TIM populations exhibit the greatest divergence along the first coordinate (Coord. 1), whereas SHUK occupies an intermediate position, shifting along the second coordinate (Coord. 2). Despite the observed spatial separation, no clear clustering of the populations was detected, indicating weak genetic structuring based on SNP markers.

Taken together, the results obtained using STR markers and TG5 and LEP polymorphisms demonstrate a consistent pattern of population structure among the studied groups of Aberdeen Angus cattle. According to the STR marker data, a low level of interpopulation differentiation was revealed, which is supported by low Fst values, the results of the STRUCTURE analysis, and the spatial distribution of the populations in the PCoA. A similar pattern is observed in the analysis of the TG5 and LEP SNPs. The Fst values remain low, and the PCoA results reveal no clear clustering of the populations, indicating weak genetic structuring at the level of functional markers. Comparison of the results obtained using neutral and functional markers demonstrated their consistency, despite differences in the nature of the analyzed loci. STR markers reflect overall genomic variability, whereas TG5 and LEP polymorphisms are associated with lipid metabolism and meat productivity traits. At the same time, the SNP analysis revealed certain differences in allele distribution among the populations, which were most pronounced for the TG5 locus. The observed increase in the C allele frequency along the KB–SHUK–TIM gradient may be associated with both demographic processes (genetic drift and the founder effect) and the specific formation history of the populations under study; however, since genomic signatures of selection were not analyzed in the present study, these differences should not be interpreted as direct evidence of selection acting at the TG5 locus. Thus, the integrated analysis confirms that the genetic variability of the studied populations is predominantly intrapopulation in nature, with weak interpopulation differentiation, and demonstrates that functionally significant polymorphisms can exhibit local differences against the background of overall genetic homogeneity.

Genetic diversity and population structure of Aberdeen Angus cattle in Kazakhstan

The present study provides an integrated assessment of the genetic diversity, population structure, and distribution of the investigated TG5 and LEP polymorphisms among Aberdeen Angus cattle populations in Kazakhstan. The combined use of neutral STR markers and candidate genes allowed for the simultaneous characterization of both the overall genetic population structure and the distribution of alleles associated with economically important traits.

The results of the STR marker analysis indicate a combination of a moderate level of genetic diversity with a low degree of interpopulation differentiation and pronounced genetic admixture. The observed and expected heterozygosity values for the STR markers (Ho = 0.682; He = 0.666) correspond to the range characteristic of commercial beef cattle populations.

Similar indicators have been reported for Aberdeen Angus herds in Europe (Márton et al., 2024) and South America (Montoya et al., 2010), as well as in other populations within Kazakhstan (Shevchenko et al., 2024a), indicating that the identified level of genetic variability is typical. Maintaining this level of diversity is an important prerequisite for preserving adaptive potential and population resilience to environmental changes. From a biological perspective, the observed level of heterozygosity indicates that the studied populations retain sufficient evolutionary potential to respond to future selection and environmental pressures. This is particularly important for commercial beef cattle breeds, as long-term selection for a limited number of production traits can be accompanied by a gradual reduction in genetic diversity and a decrease in the adaptive capacity of the animals. The obtained results indicate that this risk currently remains low in the investigated Aberdeen Angus cattle populations.

Negative FIS values and the absence of heterozygote deficit indicate the lack of pronounced inbreeding. This is consistent with data obtained for other Angus populations, where the application of modern breeding technologies contributes to the maintenance of genetic material (Márton et al., 2024; Shevchenko et al., 2024a). Thus, the existing approaches to breeding management in the studied farms can be considered effective in terms of preventing inbreeding.

Population differentiation and gene flow

Low Fst values (averaging 0.016) indicate weak genetic differentiation among the populations, which corresponds to Wright’s classification (Wright, 1978). Such values are characteristic of populations with intensive genetic exchange and a unified direction of selection, and are significantly lower than the levels typically observed between genetically distinct livestock breeds, where FST values can reach 0.05–0.15 or higher (Hall, 2022). The practical implication of the revealed weak genetic differentiation is that a high level of genetic material exchange is maintained between the farms. Such a scenario contributes to maintaining the genetic integrity of the population and reduces the likelihood of local inbreeding accumulation. At the same time, excessive genetic homogeneity could eventually lead to a reduction in differences between breeding herds and diminish the potential for further intra-breed selection. Therefore, an optimal breeding strategy must maintain a balance between the exchange of breeding stock and the preservation of the genetic distinctiveness of individual farms.

High gene flow values (Nm significantly exceeding the threshold value of 1) confirm an active exchange of genetic material among the farms, likely driven by the use of common sire lines and centralized breeding resources. The obtained results indicate the formation of a genetically consolidated population in which interpopulation differences are smoothed out due to intensive gene flow. Under such conditions, a relatively homogeneous distribution of alleles is expected for both neutral and functionally significant loci.

The results of the STRUCTURE and PCoA analyses are consistent with a low level of genetic differentiation among the studied populations, indicating the absence of a pronounced population structure, as evidenced by significant cluster overlap and a high degree of admixture. At the same time, given the low Fst values, the results of these methods should not be interpreted as evidence for the presence or absence of genetically distinct groups but rather as reflecting limited genetic differentiation and a high degree of genetic similarity among the studied herds. This is consistent with current concepts regarding the structure of commercial cattle populations, where geographical isolation plays a lesser role compared to selection practices (Tenhunen et al., 2024; Erdogan et al., 2024).

Functional polymorphisms of TG5 and LEP genes

The analysis of functional polymorphisms revealed the predominance of the TG5 C allele across all populations. This finding is consistent with previously published data showing that this allele is widespread and associated with meat quality traits, including marbling (Shin and Chung, 2007; Surundayeva, 2016). At the same time, it has been demonstrated that the TG5 polymorphism may differentially affect intramuscular fat levels depending on the genotype and feeding conditions, with the TT genotype being associated with higher fat content in several studies (Anton et al., 2011). This indicates the complex nature of the influence of this locus and the potential context dependence of its effects. The increased frequency of the CC homozygous genotype in certain populations is of interest from the perspective of the distribution of functional alleles. However, this finding should be interpreted with caution, as the observed differences may reflect the demographic history and formation of the studied herds, as well as stochastic demographic processes, rather than necessarily indicating the action of directional selection.

Overall, the obtained results are consistent with published data for other Aberdeen Angus populations. In the Hungarian Aberdeen Angus population, the frequency of the TG5 C allele was 0.666, whereas the T allele frequency was 0.334, with the CC (45.7%) and CT (41.0%) genotypes predominating (Anton et al., 2011). In Russian and Belarusian Aberdeen Angus populations, the frequency of the T allele ranged from 0.122 to 0.136, while the proportion of CC homozygotes reached 75.8–77.8%, and the heterozygous CT genotype occurred in approximately 20% of animals (Surundayeva, 2016; Guminskaya et al., 2023). Thus, the TG5 allele distribution observed in the studied Kazakhstan Aberdeen Angus populations falls within the range of variation previously reported for the breed. However, the differences detected among populations likely reflect variation in animal origin, herd formation history, and breeding strategies, which have contributed to shaping the current genetic structure of these populations.

For the investigated LEP SNP, the predominance of the T allele was established. The high frequency of the heterozygous CT genotype indicates the preservation of genetic variability at the investigated locus in the analyzed populations. Concurrently, several studies have shown that the LEP SNP is associated with morphological and productive traits in Aberdeen Angus cattle, with heterozygous genotypes being considered the most preferable in breeding work (Ruchay et al., 2021; Dzhulamanov et al., 2022).

Comparison with published data indicates that the distribution of LEP alleles varies considerably among different Aberdeen Angus populations. In the Hungarian Aberdeen Angus population, the C allele predominated, with a frequency of 0.753, whereas the T allele frequency was 0.247 (Anton et al., 2011). In contrast, the Belarusian population studied by Guminskaya et al. (2023) was characterized by a predominance of the T allele (0.600), with the heterozygous CT genotype being the most frequent (48.9%) (Guminskaya et al., 2023). A similar predominance of the T allele was reported by Selionova and Evstafyeva (2024) in a Russian Aberdeen Angus population, where the published genotype frequencies (CC = 21.9%, CT = 42.2%, TT = 35.9%) correspond to an estimated T allele frequency of 0.57 (Selionova and Evstafyeva, 2024). The results obtained in the present study are consistent with those reported for Belarusian and Russian Aberdeen Angus populations and further confirm the maintenance of LEP locus polymorphism in the breed.

The distribution of the TG5 and LEP alleles indicates the maintenance of polymorphism at both loci across all studied populations. From a practical perspective, the maintenance of polymorphism is of interest for future research and the development of marker-assisted selection programs, as it ensures the presence of alternative alleles for assessing their associations with economically important traits under local conditions.

Because the analysis was limited to two functional loci (TG5 and LEP), the obtained results do not allow the effects of selection to be reliably distinguished from the consequences of demographic processes. Therefore, the present study provides a characterization of the distribution of functional alleles but was not designed to detect signatures of selection or to assess the intensity of selective breeding. As demonstrated in recent studies, the reliable identification of selection signatures requires the analysis of a large number of markers distributed across the genome, since allele frequency differences at individual candidate genes may result from either selection or neutral evolutionary processes (Gutiérrez-Gil et al., 2015).

Accordingly, the observed differences in TG5 and LEP allele frequencies should be interpreted as reflecting the distribution of functional variants within the studied populations rather than as evidence of selection. A more robust assessment of the role of selective breeding will require the analysis of a substantially larger number of functional loci or the application of genome-wide approaches capable of detecting selection signatures across the entire genome.

Integration of neutral and functional genetic markers

The combined use of neutral STR markers and functional SNPs provides a more comprehensive assessment of population status than the use of either marker type alone. STR markers reflect the level of genetic diversity and the degree of population differentiation, whereas candidate gene polymorphisms characterize the distribution of alleles with potential economic importance. This integrated approach provides a more comprehensive characterization of the gene pool of the studied populations and may be useful for the development of conservation and breeding programs.

It is important to note that differences in the allele distribution of functional genes are observed against the background of minimal differentiation at STR markers. This indicates distinct dynamics between neutral and functional genetic variability. Similar patterns have been previously described in studies demonstrating that SNP markers are less susceptible to the effects of relatedness and population structure compared to STR markers (Fernández et al., 2013).

The fact that an integrated approach combining the analysis of neutral STR markers and functional SNPs of candidate genes (TG5 and LEP) has previously been virtually unapplied to Aberdeen Angus cattle populations under the conditions of the Republic of Kazakhstan deserves special attention. Unlike prior studies that focused either on population structure (Shevchenkо et al., 2024b) or on individual candidate genes (Dushayeva et al., 2021; Nametov et al., 2022), this approach allows for the simultaneous assessment of the genetic diversity level and the distribution of functionally significant alleles within the context of the population structure, thereby expanding the interpretative capacity of the obtained data.

The obtained results have practical implications for improving Aberdeen Angus cattle breeding programs in Kazakhstan. The absence of signs of pronounced inbreeding and the maintenance of polymorphism in functionally important genes indicate the potential for further genetic improvement of the animals without a substantial risk of rapid loss of genetic variability. At the same time, the application of molecular genetic monitoring can provide regular assessment of the state of the gene pool and serve as an additional tool in the planning of breeding programs.

From the perspective of genetic resource conservation, the obtained results may serve as a baseline for the long-term monitoring of Aberdeen Angus cattle populations in Kazakhstan. Regular repetition of such studies will make it possible to monitor changes in heterozygosity levels, the frequencies of economically important alleles, and the degree of genetic differentiation between herds, thereby ensuring timely decision-making aimed at preserving genetic diversity. This approach aligns with modern principles of sustainable management of animal genetic resources.

Study limitations

The present study has several limitations that should be considered when interpreting the results. First, the analysis was conducted on a relatively small sample of animals (122 bulls) sourced from three breeding enterprises in the Republic of Kazakhstan. Although the studied herds are among the largest producers of Aberdeen Angus cattle in the country, the findings may not fully reflect the genetic diversity of the entire breed population in Kazakhstan.

Second, the study lacked detailed pedigree information, which precluded the correlation of molecular genetic indicators with kinship coefficients and limited the ability to assess the impact of individual sires on the observed population structure.

Third, the analysis of functional genetic variation was based solely on the evaluation of TG5 and LEP polymorphisms. Despite the well-established association of these genes with meat production traits, employing only two candidate gene SNPs does not provide sufficient resolution for a detailed investigation of population structure, nor is it comparable in informativeness to multi-locus STR panels, high-density SNP arrays, or whole-genome sequencing. Therefore, the results of the TG5 and LEP analysis should be considered as a complementary characterization of the distribution of functionally significant alleles, rather than a standalone tool for population genetic analysis.

Moreover, the limited number of functional loci examined does not permit comprehensive conclusions regarding selection processes or genome-wide signatures of selection across the entire genome. Consequently, the results of the present study should be interpreted in the context of the overall population structure established using neutral STR markers.

Despite these limitations, the use of an internationally standardized STR marker panel in combination with the analysis of functionally important SNPs enabled, for the first time, a comprehensive assessment of genetic diversity, population structure, and the distribution of TG5 and LEP gene alleles in the Aberdeen Angus cattle population of the Republic of Kazakhstan. In the future, expanding the animal sample size and the number of studied farms, incorporating detailed pedigree information, and utilizing high-density SNP genotyping and whole-genome sequencing will significantly broaden our understanding of the genetic diversity, genomic structure, and potential selection signatures in Kazakhstani Aberdeen Angus cattle populations, while also enabling a direct comparison of the studied animals with Aberdeen Angus populations from other countries.

As a result of the conducted study, it was established that Aberdeen Angus cattle populations in Kazakhstan are characterized by a moderate level of genetic diversity along with a low degree of interpopulation differentiation and high genetic admixture. The population structure was evaluated based on the analysis of 15 STR markers, whereas TG5 and LEP polymorphisms were utilized for a complementary characterization of the distribution of functionally significant alleles associated with economically important traits. The absence of signs of inbreeding and the presence of intensive gene flow among the populations indicate genetic consolidation and active exchange of breeding material. The predominance of the TG5 C allele and LEP T allele is consistent with literature data and may be linked to the genetic history of the studied populations.

The present study provides the first comprehensive characterization of the genetic diversity and population structure of Aberdeen Angus cattle in the Republic of Kazakhstan using neutral STR markers and functional TG5 and LEP polymorphisms. The obtained results provide a basis for further genetic monitoring and may be used to improve breeding programs. In future studies, the application of high-density SNP genotyping and whole-genome sequencing in an expanded animal sample will provide a more comprehensive understanding of the genetic structure of populations, the level of genomic diversity, and the distribution of functionally important variants.