*3.2. Allelic Variability and Genetic Diversity of Studied Breeds*

We identified 130 microsatellite alleles in eight cattle populations including 124 alleles in modern samples and 95 alleles in museum samples. The most polymorphic were the TGLA122 and TGLA53 loci (21 and 19 alleles, respectively), while the least number of alleles (7 alleles) was found in the BM1824 locus (Table 1).


**Table 1.** Number of identified alleles among microsatellite loci in studied cattle populations (%).

Notes: locus—microsatellite locus; observed allele ranges—the limits of allelic lengths of studied microsatellite loci in historical samples analysed (a allele sizes were standardised according to International Society of Animal Genetics (ISAG) International Bovine (Bos Taurus) short tandem repeat (STR) typing comparison test 2018–2019).

> Of the alleles, 83.33% found in the museum Kalmyk cattle and 81.16% in the museum Kyrgyz cattle were present in the modern representatives of these breeds. Despite longterm crossbreeding of native Kazakh cattle with Herefords, 82.93% of the alleles of museum Kazakh cattle appeared in the modern Kazakh white-headed breed. Meanwhile, 92.98% of alleles observed in Herefords appeared in Kazakh white-headed cattle. The great contribution of Mongolian cattle to the development of the gene pool of the studied breeds of steppe cattle was reflected in the high rate of common alleles with Mongolian cattle: 87.50%, 88.41%, and 92.68% in the museum Kalmyk, Kyrgyz, and Kazakh cattle, respectively. These rates decreased to 81.61% and 81.82% in the modern populations of Kalmyk and Kyrgyz cattle, respectively. The Kazakh white-headed cattle retained among other modern breeds the highest rate of alleles in common with the Mongolian cattle (93.15%) (Table 2).


**Table 2.** Allelic variability and number of common alleles in studied breeds.

Notes: museum populations: KALM\_H—Kalmyk, KRGZ\_H—Kyrgyz, KZKH\_H—Kazakh, modern populations: KALM\_M—Kalmyk, KRGZ\_M—Kyrgyz, KZWH\_M—Kazakh White-Headed, HRFD\_M—Hereford, MONG\_M—Mongolian cattle; number of alleles identified in studied breeds is shown at diagonal; the number of alleles, which are common for two breeds are shown below the diagonal.

> Comparing the museum and modern Kalmyk, Kyrgyz, and Kazakh cattle showed that 15 alleles, which were distributed in the museum populations, were lost in the modern cattle. By contrast, 60 novel alleles appeared in the modern populations. The greatest number of novel alleles (*n* = 33) was found in the Kazakh white-headed breed, reflecting the gene flow from Hereford breed. The TGLA122 and TGLA53 loci were the most altered: Three and two ancestral alleles were lost, while 11 and 9 novel alleles appeared, respectively (Supplementary Materials, Table S4). Considering the differences in sample size between museum and modern populations, it should be noted that the absence of part of alleles in historic populations could be the result of their smaller sample size.

Estimations of the genetic diversity calculated based on the genotypes for 11 microsatellite loci are summarized in Table 3.

**Table 3.** Summary statistics for museum and modern populations of studied breeds based on genotypes of 11 microsatellites.


Notes: *n*, number of individuals; Ho, observed heterozygosity; uHe, unbiased expected heterozygosity; AR, rarefied allele richness; u*F*IS, unbiased inbreeding coefficient; M, mean value; SE, standard error; CI 95%, range variation coefficient of u*F* is at a confidence interval of 95%; museum populations: KALM\_H—Kalmyk, KRGZ\_H—Kyrgyz, KZKH\_H—Kazakh, modern populations: KALM\_M—Kalmyk, KRGZ\_M—Kyrgyz, KZWH\_M—Kazakh White-Headed, HRFD\_M—Hereford, MONG\_M—Mongolian cattle; \* *p* < 0.05 comparing to Hereford breed according to pairwise comparing Wilcoxon rank-sum test.

> We did not observe significant differences in the genetic diversity between the museum and modern populations of all studied breeds. For Kalmyk and Kyrgyz cattle, the values of the unbiased expected heterozygosity and rarefied allelic richness were similar (uHe = 0.772–0.776 for the museum samples and 0.771–0.778 for the modern samples; AR = 3.635–3.693 and 3.664–3.704, respectively). The Kazakh white-headed cattle were characterized by decreased genetic diversity compared to the museum Kazakh cattle (uHe = 0.726 vs. 0.767; AR = 3.336 vs. 3.727). This could be associated with lower level of genetic diversity of Hereford cattle and may reflect the contribution of the limited number of Hereford bulls in formation of the Kazakh white-headed breed. A significant deficiency of heterozygotes was found in the museum Kalmyk cattle (u*F*IS = 0.131), as well as in the modern Mongolian cattle (u*F*IS = 0.115), while the modern Kyrgyz cattle were characterized by an excess of heterozygotes (u*F*IS = −0.085) (Table 3).
