**4. Discussion**

Conservation of the genetic diversity of animal genetic resources is important for food security and the ability of agricultural systems to adapt to possible future changes in production environments, including climate, market requirements, and diseases [58,59]. Predominantly using highly productive transboundary breeds in agricultural production and the associated decline in the population size of the local breeds has led to a drastic decrease in the genetic diversity of farm animal species, including cattle [59,60]. To ensure the sustainability of agricultural production, programs for the conservation of farm animal genetic resources should be developed [59,61].

One of the important points in developing such programs is the selection of the individuals to be used for conservation. The most valuable resources are individuals who are carriers of breed-specific genetic components and would allow reconstitution of that breed in case of its extinction [62]. However, in modern populations of local cattle breeds, the complex genetic backgrounds and active crossbreeding make it difficult to estimate the ancestral genetic components and infer the origin of breeds. The absence of such information would negatively affect their future development and breeding management. The study of museum and archeological samples can help in the identification of such components in the genome of modern representatives of breeds [29,63,64].

During the century-long history of the territories in the south steppe of Russia and the neighboring republics of the former Soviet Union, cattle breeds developed were well adapted to the harsh environment of this region and able to survive under poor foraging conditions [8,9]. In the present study, we were able to obtain valid genotypes for 11 microsatellite loci for the museum specimens of the native steppe Kalmyk, Kyrgyz, and Kazakh cattle dated to the first quarter of 20th century.

The level of genetic diversity of the modern Kalmyk and Kyrgyz cattle (uHe = 0.771–0.778) was similar to that determined in previous studies of these breeds (He = 0.76–0.78) [65] and comparable to those observed in the museum samples (uHe = 0.772–0.776) (Table 3). The modern populations of both of these breeds were less affected by crossing with transboundary breeds and were not subjected to artificial selection with high selection pressure [66,67]. On the contrary, we detected a visible decrease in the genetic variability of the modern Kazakh white-headed breed compared to the museum Kazakh cattle (uHe = 0.726 and 0.767; AR = 3.727 and 3.336, respectively) (Table 3). A possible explanation is that only a part of the Kazakh cattle inhabiting the northern and western territories of Kazakhstan were used for developing the modern population of Kazakh white-headed cattle [17]. Higher selection pressure for the limited number of traits and the use of a limited number of Hereford sires for breed improvement can be considered as additional factors leading to the decline in genetic diversity in the modern population of this breed. Meanwhile, the genetic diversity of the Kazakh white-headed cattle was higher than in Herefords (uHe = 0.726 vs. 0.653; AR = 3.336 vs. 2.994, *p* < 0.05), which is in general agreement with other studies [65,68–70] showing a lower level of genetic diversity in transboundary commercial breeds compared to local ones; He = 0.76–0.78 in Kalmyk and Kyrgyz cattle comparing to 0.70–0.71 in transboundary Brown Swiss and Holstein breeds [65]; He = 0.679–0.802 in twenty-seven Chinese indigenous yellow cattle breeds comparing to 0.661–0.697 in three commercial breeds [68]; uHe = 0.791–0.804 in local Vietnamese pigs comparing to 0.606–0.632 in commercial Yorkshire and Landrace pig breeds [69]; He = 0.72–0.81 in twenty-two Vietnamese pig breeds comparing to 0.56–0.65 in commercial Duroc, Yorkshire and Landrace pig breeds [70].

We observed a significant deviation from the Hardy-Weinberg equilibrium in heterozygote number for museum samples of the Kalmyk breed (heterozygote deficiency), which has been indicated by a positive value of u*F*IS (0.131). Interestingly, we found the heterozygote excess beyond the expected number of heterozygotes in the modern Kyrgyz cattle (u*F*IS = −0.085) that has the lowest population size among studied breeds. This observation is in agreement with the results of previous studies [65], indicated the heterozygote excess in two studied populations of Kyrgyz cattle (*F*IS = 0.132–0.135). The possible

explanation could be the low pressure of the artificial selection, extremely long productive life (up to 10 lactations) of the cows, and the calves' exchanges between owners.

The PCA plot (Figure 2a), STRUCTURE clustering (Figure 2b), *FST*-based tree (Figure S3), and Jost's D network (Figure 3) showed strong maintenance of the historical genetic backgrounds in the modern populations of the Kalmyk and Kyrgyz cattle. The allele pool of the modern Kazakh white-headed cattle has undergone the greatest changes compared to the museum Kazakh cattle, which is associated with multiple backcrossing with Hereford bulls [8,9]. This revealed in more distant localization of Kazakh white-headed breed in relation to museum samples at PCA plot (Figure 2a); occurrence of the large part of Hereford specific genetic components in the cluster structure of Kazakh white-headed cattle (Figure 2b); formation by Kazakh white-headed and Hereford breeds of the joined branch on Neighbor-Net trees (Figure 3, Supplemental materials, Figure S3). The contribution of Herefords is confirmed by more than a threefold decrease of the genetic distance with Kazakh white-headed cattle (*FST* = 0.049) comparing to museum Kazakh cattle (*FST* = 0.154) (Supplemental materials, Table S5), as well as by observed gene flow from Hereford ancestors to the ancestral population of the Kazakh white-headed breed at TreeMix tree (Figure 4a). The positive SE value observed between Hereford and Kazakh white-headed cattle at residual matrix plotted from a TreeMix (Figure 4b) indicates that these two breeds are even more closely related to each other than in the modeled tree. However, individuals that have retained a visible portion of their historical genetic components are still present among modern Kazakh white-headed cattle (Figure 2b). Including such individuals in conservation programs has great value for maintenance of the genetic diversity of the local animal genetic resources.

Thus, our research results have demonstrated that the studied cattle breeds have still retained their historical genetic background, which allows them to this day not only to successfully compete with transboundary breeds, but also to perform important functions ranging from providing foods to socio-economic, cultural and ecological roles in their breeding areas. In our opinion, the strategy for the further preservation of the genetic resources of these breeds may consist at least of preventing mass crossbreeding with transboundary breeds. In the ongoing breeding policy, special attention should be paid to the preservation of historical allele pool, since it composes the genetic uniqueness of breeds, formed by combining the ecological processes, gene flow, local breeding practices, and geographical features.

Considering the development of new powerful molecular genetics tools that enable high-throughput analysis of cattle at the genome level [18,19,22,23,71,72], including analysis of museum and archeological specimens [73,74], additional studies are required to identify and trace more precisely the historical genomic components in the modern representatives of breeds.

#### **5. Conclusions**

Using the genotypes for 11 microsatellite loci, we compared the genetic diversity and established the genetic relationships between the museum (dated to the first part of 20th century) and modern populations of Kalmyk, Kyrgyz, and Kazakh cattle, distributed in the south steppe region of the European part of Russia, Kazakhstan, Kyrgyzstan, and West Siberia. We showed that the allele pool of modern cattle populations has undergone changes that have manifested visibly, which are most significant in the Kazakh whiteheaded cattle. At the same time, we were able to clearly show that modern representatives of all of the studied breeds retained a portion of their historical genetic components, making them valuable national genetic resources. These research results can be used for developing sustainable programs for conservation of the genetic diversity of native cattle breeds.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/d13080351/s1, Figure S1: Representatives of Kalmyk (a) and Kyrgyz (b) cattle demonstrated on the first agricultural exhibition of breeding cattle in Moscow 1896, Figure S2: Schematic map of the zoning of cattle breeds on the territory of the USSR in 1934, Figure S3: Neighbor-Net graphs based on *F*ST genetic distances characterizing genetic relationships between studied museum and modern cattle populations, Table S1: Quantitative and qualitative characteristics of DNA extracted from the museum specimens and number of replicates used for estimating the consensus genotypes, Table S2: Microsatellite genotypes of museum and modern samples used for the studies, Table S3: Quality of microsatellite genotyping and distribution of genotyping errors among microsatellite loci in museum samples of studied cattle populations, Table S4: Alleles of microsatellites, which were occurred in museum samples, but were lost in modern populations and novel alleles, which are appeared in modern populations, Table S5: Genetic distances between the studied populations based on FST and Jost's D indices.

**Author Contributions:** Conceptualization, N.A.Z.; methodology, A.S.A. and N.A.Z.; software, A.S.A. and A.V.D.; validation, A.S.A., A.V.D. and A.A.S.; investigation, A.S.A., V.R.K., V.V.V., A.A.S. and R.Y.C.; resources, O.I.B. and E.M.L.; data curation, A.S.A. and N.A.Z.; writing—original draft preparation, A.S.A. and N.A.Z.; writing—review and editing, A.S.A., A.A.S., O.I.B., J.S., G.B. and N.A.Z.; supervision, N.A.Z.; visualization, A.S.A.; supervision, N.A.Z.; project administration, N.A.Z.; funding acquisition, N.A.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation within project No. 19-76- 20012 (study of museum samples) and the Russian Ministry of Science and Higher Education within theme No. 0445-2019-0024 (study of modern samples).

**Institutional Review Board Statement:** The study was approved by the Ethics Commission of the L.K. Ernst Federal Research Center for Animal Husbandry (protocol No. 3 from the 19 January 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in Supplementary Materials, Table S2.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
