*3.4. Estimation of Genomic Inbreeding Coefficient Based on Runs of Homozygosity in Russian Local Sheep Breeds*

Except for the Romanov, Kuchugur, and Russian longhaired breeds, studied sheep populations regardless of the wool type had low to moderate FROH values. Thus, the FROH values varied from 0.033 (MONG) to 0.072 (ANDB) in coarse wool group (Figure 4A), and from 0.033 (ALTM) to 0.069 (NCSN) in the semi-fine wool group (Figure 4B), and from 0.035 (BKFF) to 0.066 (VOLG) in fine wool group (Figure 4C).

**Figure 4.** *Cont.*

**Figure 4.** The genomic inbreeding coefficient based on ROH (FROH) in Russian sheep breeds: (**A**) FROH values within the group of coarse wool breeds; (**B**) FROH values within the group of semi-fine wool breeds; (**C**) FROH values within the group of fine wool breeds.

The mean genomic inbreeding coefficient values were the highest in the Romanov (FROH = 0.106), Russian longhaired (FROH = 0.097), and Kuchugur breeds (FROH = 0.084).

Among all studied animals, the maximum individual FROH value was calculated in the Kuchugur breed (FROH = 0.33) from the coarse wool group. The highest individual FROH values were found in animals from the Stavropol breed from fine wool group (FROH = 0.16), from the Russian longhaired breed from semi-fine wool group (FROH = 0.16) and from the Romanov breed from coarse wool group (FROH = 0.17).

#### **4. Discussion**

Estimation of genetic diversity in local livestock species is of special priority to prevent the steady inbreeding increase which leads to negative consequences of inbreeding depression and to endangered status. There are several approaches to address biodiversity and its dynamics in the populations of livestock species: effective population size, heterozygosity and runs of homozygosity [10].

In our previous study, we calculated and analyzed effective population sizes and heterozygosity to unlock the current state of genetic diversity in Russian local sheep breeds [24]. Nonetheless, estimation of runs of homozygosity is a useful tool to reveal the presence of long-term inbreeding in livestock populations [2,5].

A strong primary subdivision of the Russian local sheep populations according to their wool type (fine wool, semi-fine wool, and coarse wool) was reported based on using the medium density DNA arrays [24]. Therefore, in the present study, we divided Russian sheep populations corresponding to their wool type to analyze the specific patterns of distribution of the runs of homozygosity in their genomes.

The breeds in the fine wool group demonstrated a high consistency in the ROH distribution. Thus, most individuals from these breeds fit into «90 ROH number and 200 Mb sum of ROH length» pattern. These findings might be occurred because of similarities in the developmental history and of long-term underling of strong positive selection for wool production [22]. Besides Dzomba et al. [17] reported that the Merino-type breeds had similarities in ROH distribution. These results are agreed with our findings on Russian fine wool breeds which also belong to the group of Merino-derived breeds.

A common trend was not established in semi-fine wool breeds which might be divided into three groups based on ROH distribution. The first group included Kuibyshev, Altai Mountain, and Tsigai breeds. Most individuals of these breeds had 55–60 ROH numbers and 150 Mb sum ROH length. The second group represented by sheep from the North Caucasian breed was characterized by 60–80 ROH numbers and 150–220 (250) Mb sum ROH length per individual. In addition, the individuals from the Russian longhaired had the greatest ROH numbers (≥75) and ROH length (200–350 Mb) as well as the highest genomic inbreeding coefficients.

The coarse wool group included fourteen populations collected from thirteen breeds. Based on the PCA results, animals from the Buubei breed clearly separated into two groups. This pattern was not explained by the sampling locations. The history of the Buubei breed provides a tragic lesson for future generations because the valuable gene pool of this ancient native breed was lost in the Republic of Buryatia. The contemporary Buubei breed was re-introduced into the territory of Russia from a small group of animals that had been previously imported to China and which escaped the extinction in the homeland habitat [31]. However, it might be hypothesized that some re-introduced sheep were of admixed origin which resulted in establishment of a few genetic strains within the contemporary gene pool. Nonetheless, there were no significant differences between two Buubei populations in the ROH distribution.

Most sheep in the coarse wool group had similar ROH numbers and ROH lengths which were up 105 and 250 Mb, which corresponded to previously detected patterns based on high-density genotypes [32].

However, three breeds did not fit into the genetic patterns, which were characteristic for coarse wool (Romanov, and Kuchugur) and semi-fine wool groups (Russian longhaired) and displayed the highest estimates of mean ROH length (282 Mb in Romanov; 257 Mb in Russian longhaired; 223 Mb in Kuchugur).

The contemporary gene pools of studied coarse wool breeds were formed by folk selection (somehow or other) and by required adaptions to survive in severe natural environments. Nonetheless, the Romanov breed was underling a stronger selective pressure by selection individuals, which had the best pelt traits and the highest prolificacy [22]. This could result in fixing definite genome regions that related to desirable traits and might overlap with the ROH segment. Nevertheless, this assumption should be addressed more fully with a larger sample.

However, the severity of the consequences of the recent autozygosity's events on the gene pool of these three breeds is different. Due to higher resilience to feeding and keeping conditions, the Romanov breed is reared in 26 regions. Besides, the pedigree base for the Romanov breed includes twenty breeding enterprises and multipliers [33]. Thus, a rising of the genomic inbreeding might be prevented in the future by smart choices for the unrelated rams and rotation of the founder's lines within the breed.

In comparison with the Romanov breed, the state of genetic resources of the Kuchugur and Russian longhaired breeds are more unstable. The Kuchugur breed was created by intense folk selection in the Voronezh region in the second half of the 19th century [22]. An identification of highly inbred animals (FROH = 0.33) in the Kuchugur breed was expected because this breed is in endangered status (no official census recordings are available) and most likely only a few sires are used to multiply the last existing flocks which are kept by the smallholder farmers in the Voronezh and Kursk regions. Considering Russian longhaired breeds, a rising demand for mutton has contributed to revived interest in raising breeds to produce meat (Kuibyshev, Altai Mountain, North Caucasian breed and Tsigai). However, the Russian longhaired breed has long crossbred wool (in Lincoln type), which is not in high demand currently. Thus, higher inbreeding level in this breed might correspond to the small population size (1400 heads at the end of 2019, Supplementary Material Table S1) [33] and to using of a limited number of rams. Thus, the Kuchugur and Russian longhaired breeds without proper management might be extinct in the nearest future.

Nevertheless, common genetic patterns were found in all studied Russian breeds as well. A prevalence of short ROH segments detected in all Russian local sheep populations are compatible with the relevant patterns identified in other local and cosmopolitan sheep breeds. Thus, Border Leicester, and Poll Dorset breeds predominantly had short ROH segments (1 to 5 Mb) [10] as well as Italian local sheep breeds were characterized by the highest numbers of short ROH segments (<10 Mb) [13]. Analyzing the ROH distribution in genomes of South African sheep breeds, Dzomba et al. [17] showed that 88.2% of identified ROH were in the short (1–6 Mb) category [17]. A similar pattern was reported in five Chinese sheep breeds [16] and six commercial meat breeds including Suffolk, and Texel [11].

Nonetheless, chromosome coverage in ROH varied in different sheep populations. Thus, Abied et al. [16] showed the highest coverage rate on OAR2 in Chinese sheep populations, which corresponded with our findings. Purfield et al. [11] reported the highest and the lowest percentage of the autosome residing in a ROH on OAR15 and on OAR24 in the Charollais and Suffolk populations. Dzomba et al. [17] found that South African sheep breeds were characterized by even ROH distribution amongst chromosomes.

Considering genome coverage in ROH, Russian sheep populations displayed greater mean ROH length (86.77–282.15 Mb) and higher mean ROH number (37.64–123.14) in comparison with those estimated in Italian (3.85–5.51 Mb and 10.58–44.54) [13] and in eight Swiss sheep breeds (1.88–103.25 Mb and 6.58–29.14) [14]. In addition, mean ROH length calculated in our study was a bit higher than those obtained in commercial sheep breeds (92.61–128.31 Mb [11] and 94.88–126.06 Mb [10]). However, larger variation was observed in Chinese sheep breeds for which ROH number ranged from 259 to 796, and mean ROH length varied from 15.23 Mb to 46.8 Mb with individuals values up to 273 and 984 Mb [16].

Variation of ROH lengths within the studied breeds was from moderate to high. In addition, animals with large ROH coverage were found in several breeds including Kuchugur (872.75 Mb), Romanov (457.36 Mb), Ossetin (463.41 Mb), and Buubei (2) (483.11 Mb). However, in general, maximum individual ROH length values estimated in our study were close to those obtained in Australian populations of Border Leicester, Merino, and Poll Dorset breeds (427.2, 410.5 and 396.45 Mb) [10]. Besides, the presence of several individuals, which had experienced recent autozygosity events, is typical in livestock species [13].

Although standard deviation values revealed high variability in autozygosity levels within each population, genomic inbreeding coefficients estimated per breed predominantly demonstrated a pattern of low to moderate inbreeding in Russian sheep populations (FROH from 0.033 to 0.106). Comparable results were observed in Italian (FROH from 0.016 to 0.099) [13] and Swiss local sheep breeds (FROH from 0.021 to 0.102) [14], while most South African breeds exhibited much more high inbreeding levels (FROH from 0.10 to 0.31) [17].

### **5. Conclusions**

Here, we presented a detailed analysis of the pattern of the runs of homozygosity distribution in twenty-seven Russian local sheep breeds based on SNP profiles. The results corresponded to breed history and used production system under which populations are reared. The calculated levels of ROH reflect the inbreeding history of the studied sheep populations. Our findings provide evidence of a low to moderate genomic inbreeding in major local sheep populations. The results suggest that several animals from Kuchugur, Romanov, Ossetin, and Buubei breeds have experienced recent autozygosity events. The study results provide useful information and might contribute to designing conservation programs for local genetic resources of sheep in Russia.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/d13080360/s1, Table S1: Population numbers of breeds in breeding farm (by the end of 2019).

**Author Contributions:** Conceptualization, N.Z., G.B. and T.D.; methodology, A.D. and T.D.; software, A.D.; validation, T.D., A.D. and N.Z.; formal analysis, A.D.; investigation, T.D. and A.D.; resources, N.Z. and M.S.; data curation, A.D.; writing—original draft preparation, T.D.; writing—review and editing, T.D.; visualization, A.D.; supervision, N.Z.; project administration, T.D.; funding acquisition, T.D. and A.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** The 50k SNP genotypes of the studied breeds were obtained within the framework of the Ministry of Science and Higher Education of Russia theme No. 0445-2019-0024. The 600k SNP genotypes were obtained with financial supported by the Russian Scientific Foundation (RSF) within Project No. 19-16-00070.

**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. 4 from the 19 January 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The SNP-genotypes presented in this study are available on request from the corresponding author.

**Acknowledgments:** The ovine HD BeadChip was developed under the auspices of the International Sheep Genomics Consortium in work underwritten by FarmIQ [34], a joint New Zealand government and industry Primary Growth Partnership programme.

**Conflicts of Interest:** The authors declare no conflict of interest. The Russian Scientific Foundation (RSF) and Ministry of Science and Higher Education of the Russian Federation 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.
