Comparative Study of the Genetic Diversity of Local Steppe Cattle Breeds from Russia, Kazakhstan and Kyrgyzstan by Microsatellite Analysis of Museum and Modern Samples^†

Round 1

Reviewer 1 Report

The authors present a study comparing measures of genetic diversity across historical and modern samples of three different cattle breeds. While diversity seems to have been preserved for two of the breeds, the Kazakh breed seems to have lost some of its past diversity, and also seem to be more related to the Hereford breed.

I find valuable the overall finding that all breeds retain a significant fraction of the past diversity, encouraging their use in conservation programs. However, there are important limitations that put in question these findings.

- Sample sizes for some of the historical samples are very low (eg. n = 3 for Kazakh). This is extremely limiting to establish accurate diversity change for this breed, and also when measuring allelic diversity for all breeds.

- Lines 256-258, following, and Table 2. Does these values refer to rarefacted allelic diversity? I feel they don't (values in Table 3 do). I don't understand the use of non-rarefacted allelic diversity here. Given the disparity in population sample sizes (and the low value of historic Kazakh samples), total number of alleles found are meaningless.

- In terms of gene diversity measures based of the allele frequencies (e.g. expected heterozygosity), results are sometimes expressed in terms of "significance", but no actual statistical tests is reported here, and no P values or anything to support these claims. Is uHe = 0.73 in modern Kazakh significantly lower than uHe = 0.77 in historical samples? This seems to be an important result, but is presented weakly, with no statistical support, and small sample sizes. It also looks challenging to find significance for rarefacted allelic diversity.

Minor comments:

- Another major result is the finding that the Kazakh breed clusters with Hereford. It is discussed (lines 401-403) that the use of this breed could have contributed to lower diversity in the Kazakh cattle. I'd hope this to be discussed in terms of the allelic diversity shared by them, since it is usually expected that different breeds carry different alleles resulting in more diverse and heterozygote progeny. This could not be the case (agreeing with author's conclusion), but I find difficult to consolidate it, again mostly because of the limited sample size.

- What does represent the "drift parameter" in Figure 4? This is never mentioned in the text.

Author Response

The authors present a study comparing measures of genetic diversity across historical and modern samples of three different cattle breeds. While diversity seems to have been preserved for two of the breeds, the Kazakh breed seems to have lost some of its past diversity, and also seem to be more related to the Hereford breed.

Reply: Dear Reviewer, we found all your comments extremely helpful and we have revised our paper accordingly.

I find valuable the overall finding that all breeds retain a significant fraction of the past diversity, encouraging their use in conservation programs. However, there are important limitations that put in question these findings.

- Sample sizes for some of the historical samples are very low (eg. n = 3 for Kazakh). This is extremely limiting to establish accurate diversity change for this breed, and also when measuring allelic diversity for all breeds.

Reply: We agree with the Reviewer that the small sample size for museum Kazakh cattle is very low that could cause the biases in the estimating the accurate diversity changes for this breed.  Unfortunately, we were able to estimate the reliable consensus genotypes only for three museum specimens of Kazakh cattle. We noted in the text of the manuscript the possible biases in allelic pattern of museum samples due to the limited number of samples.

- Lines 256-258, following, and Table 2. Does these values refer to rarefacted allelic diversity? I feel they don't (values in Table 3 do). I don't understand the use of non-rarefacted allelic diversity here. Given the disparity in population sample sizes (and the low value of historic Kazakh samples), total number of alleles found are meaningless.

Reply: the Table 3 contains the rarefied numbers of allelic richness (Ar), which were calculated according Keenan et al. [2013] using R packages diversity. We mentioned it in the Material and Methods section.

- In terms of gene diversity measures based of the allele frequencies (e.g. expected heterozygosity), results are sometimes expressed in terms of "significance", but no actual statistical tests is reported here, and no P values or anything to support these claims. Is uHe = 0.73 in modern Kazakh significantly lower than uHe = 0.77 in historical samples? This seems to be an important result, but is presented weakly, with no statistical support, and small sample sizes. It also looks challenging to find significance for rarefacted allelic diversity.

Reply: We applied the Wilcoxon rank-sum test (p >0.05) to estimate the p-value of the differences for genetic diversity estimates among studied populations (we mentioned it in the Materials and Methods section). No significant differences among museum and modern populations of studied breeds were found. Significant differences in expected heterozygosity and allelic richness estimates were observed only among Herefords and several other studied breeds. We indicated it in the Table 3 and made corrections in the text of the manuscript.

Minor comments:

- Another major result is the finding that the Kazakh breed clusters with Hereford. It is discussed (lines 401-403) that the use of this breed could have contributed to lower diversity in the Kazakh cattle. I'd hope this to be discussed in terms of the allelic diversity shared by them, since it is usually expected that different breeds carry different alleles resulting in more diverse and heterozygote progeny. This could not be the case (agreeing with author's conclusion), but I find difficult to consolidate it, again mostly because of the limited sample size.

Reply: Thank you very much for your suggestion. We expanded the discussion of the data concerning contribution of Hereford cattle in development of Kazakh white-headed breed.

- What does represent the "drift parameter" in Figure 4? This is never mentioned in the text.

Reply: Thank you very much for your suggestion. We discussed the results of the ThreeMix analysis in the Discussion section: “The contribution of Herefords is confirmed … by observed gene flow from ancestors of modern Hereford cattle to the ancestral population of the Kazakh White-Headed breed (Fig. 4a). The positive SE value observed between Hereford and Kazakh White-headed cattle at residual matrix plotted from a TreeMix analysis (Fig. 4b) indicates that these two breeds even more closely related to each other than in the modeled tree with migration event”.

Reviewer 2 Report

An important study on genetic diversity of native cattle breeds based on museum and modern samples. This will no doubt contribute to our understanding of diversity, characterization and sustainable use of native cattle breeds. I have made few suggestions in the edited attached text to help the authors improve on the quality of the manuscript. Finally the authors have indicated some findings which should be well discussed in relation to the sustainable use and conservation of native cattle breeds in the study area.

Comments for author File: Comments.pdf

Author Response

An important study on genetic diversity of native cattle breeds based on museum and modern samples. This will no doubt contribute to our understanding of diversity, characterization and sustainable use of native cattle breeds. I have made few suggestions in the edited attached text to help the authors improve on the quality of the manuscript.

Reply: We are very grateful to the reviewer for suggestions for editing our manuscript and believe that they have significantly improved our manuscript. We made the necessary corrections according to the suggestions of the reviewer. We have corrected the list of keywords, removing some of the words that are already present in the manuscript title, and added new ones.

Finally, the authors have indicated some findings, which should be well discussed in relation to the sustainable use, and conservation of native cattle breeds in the study area.

Based on the findings it will be useful to make some recommendations on sustainable use and conservation of the native cattle studied.

Reply: Thank you very much for your suggestions. We expanded the discussion section by adding the following: “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 re-sources of these breeds may consist at least of preventing mass crossbreeding with trans-boundary 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”.

Reviewer 3 Report

This work could advance the knowledge of the bovine research community on the lost and current genetic diversity of local steppe cattle breeds, and has broad implication for conserving genomic resources, with great contribution on global biodiversity. I really enjoyed reading the manuscript, and here I have few comments for the authors that hopefully improve the quality of the work.

There are two points that should be addressed in the manuscript:

- Sample size: The sample size for KZKH_H (n=3) is much smaller than the other historic and modern breeds, which could cause biases. For example, the emergence of 34 novel microsatellite alleles in KZWH_M (N=30) and the absence of them from other populations could be simply because these alleles were not sampled in other ones and specially in KZKH_H due to the smaller sample size. However, considering the museum samples are limited in number, and scientist should work with what is available it’s important that the results will be discussed in the frame of the limitations and the conclusion will be made considering it.

-Missing data:

The 131 alleles called in historical samples are shown in Table S2, and Table 1, but no such detailed information available for modern populations. This information should be added as well, unless publicly available data are used. If the publicly available data were used, why the authors did DNA extraction from modern samples! Please clarify as it seems some results are not presented in the manuscript, but were discussed in results.

Comments:

Grammar checking should be made before publication

Line 34: replace “having undergone the greatest changes” to “has undergone great changes”

Line 35: replace “research results” to “results”

Line 60: replace “description their properties” to “description of their properties”

Line 99: replace “the most of native breeds” to “most of the native breeds”

Line 101: replace “losses” to “loss”

Line 106: replace “a breed’s origin” to “breed’s origin”

Line 106-107: replace “tracing the maintenance of ancestral” to “maintaining the ancestral”

Line 108-111: here, it should be described how mtDNA can overcome the problem of “small amount” and “high degree of degradation” in DNA derived from museum samples. please re-write the paragraph to something like this:

Over the last decades, the field of ancient DNA (aDNA) heavily relied on Mitochondrial DNA (mtDNA) marker. This is due to the low copy number and general difficulties associated with the recovery of nuclear DNA from archaeo- zoological material.

Line 111: replace “mitochondrial DNA” to “mtDNA”

Line 125: add three sub-headings to the material and methods section, something like below, in order to have a clear section for each part.

2.1 Sample collection

2.2 DNA extraction

2.3 Microsatellite profiling or Autosomal STR Analysis

Line 151-157: This paragraph should be moved up to the sample collection

Line 200: reference after R software (http://cran.r-project.org)

Line 225: replace “museum samples.” to “museum samples”

Line 230: add the sample ID of the four and two samples that were only genotyped for 10, and 9 loci respectively. This information is important in terms of knowing missing loci are observed in which historic population.

Line 254: The 131 alleles called in historical samples are shown in Table S2, and Table 1, but no such detailed information available for modern populations. This information should be added as well, unless publicly available data are used. If that is the case, why the authors did DNA extraction on modern samples!

Line 254-256: please mention that this numbers are referring to the number of alleles only in historic samples – the data for modern is missing.

Line 257: From which table/figure this 124 vs. 96 alleles has been extracted? Based on Table 1, 131 alleles are reported for historic samples, the corresponding numbers for modern samples are missing.

Line 278: Please clarify if the abcense of the novel 64 alleles in historic populations is not the result of smaller historic sample size, and simply the alleles are absent because the individuals are not sampled.

Line 278; In Table S4, only 61 novel alleles are reported but in the text 64 – please clarify.

Line 293-296: please use some statistical tests such as Bonferroni correction or Wilcoxon rank-sum test (p >0.05) to statistically backup that there is no significant differences.

Line 296: Almost half of the novel alleles (34 alleles) are emerging in Kazakh white-headed population, and 4 are missed from the historic correspondence. Here I am confused that how this high number of novel alleles are not contributing to high genetic diversity.

Line 303: replace “between” to “among”

Line 319: remove “equaled” and replace with “is”.

Line 327: the values are reported wrongly in comparison to Table S5.

Fst between modern kalmyk and historic kalmyk = 0.018

Fst between modern Kyrgyz and historic Kyrgyz = 0.025

Line 328: The value below is missing in the text as the Kazakh white headed was only compared to historic kalmyk and historic Kazakh white headed, and not with historic Kyrgyz. Please add the value below: Fst between Kazakh white headed to historic Kyrgyz = 0.082

Line 330-332: what about KZKH-H and MONG_M with Jost’s D of 0.019?

Line 361: replace “red” with “orange”

Line 364: replace “closer” to “more closely”

Line 327; remove “and as a result of diseases” or complete the sentences in a way that make sense.

Line 379: remove “these”

Line 381-384: rephrase suggestion:

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 inferr the origin of breeds. The absence of such information would negatively affect their future development and breeding managements.

Line 410: replace “the Kazakh white headed” to “the modern Kazakh white headed’

Line 411-412: remove “in the modern population”

Tables and Figures:

Figure S2: replace [Ivanova O.A. et al., 1934] to [16].

Table S1: replace the heading “Figure S1” to “Table S1”.

In addition, add the DNA extraction info for modern samples as well – although that is not very important but should be kept uniform – either show it for both historic and modern samples or not represent.

Table S2: replace the heading “Figure S2” to “Table S2”. In addition in the Table caption add that the information is for historical samples.

The genotyping information for modern samples should be added to Table S2 as well.

Table S3: replace “quality indices is describes” to “quality indices is described”

What the “paragraph 2.2” refers in the caption of table S3?

The same information on quality of microsatellite genotyping for modern samples should be added.

Table S4: replace the heading “Figure S4” to “Table S4”.

Author Response

This work could advance the knowledge of the bovine research community on the lost and current genetic diversity of local steppe cattle breeds, and has broad implication for conserving genomic resources, with great contribution on global biodiversity. I really enjoyed reading the manuscript, and here I have few comments for the authors that hopefully improve the quality of the work.

Reply: We appreciate so positive evaluation of our manuscript. Thank you.

There are two points that should be addressed in the manuscript:

- Sample size: The sample size for KZKH_H (n=3) is much smaller than the other historic and modern breeds, which could cause biases. For example, the emergence of 34 novel microsatellite alleles in KZWH_M (N=30) and the absence of them from other populations could be simply because these alleles were not sampled in other ones and specially in KZKH_H due to the smaller sample size. However, considering the museum samples are limited in number, and scientist should work with what is available it’s important that the results will be discussed in the frame of the limitations and the conclusion will be made considering it.

Reply: We agree that the small sample size of museum Kazakh cattle can cause the biases, especially in genetic diversity indices. Unfortunately, we were able to estimate the reliable consensus genotypes only for three specimens. We noted in the text of the manuscript the possible biases in allelic pattern of museum samples that could be caused by the limited number of samples.

-Missing data:

The 131 alleles called in historical samples are shown in Table S2, and Table 1, but no such detailed information available for modern populations. This information should be added as well, unless publicly available data are used. If the publicly available data were used, why the authors did DNA extraction from modern samples! Please clarify as it seems some results are not presented in the manuscript, but were discussed in results.

Reply: We provided the genotypes of modern samples in Table S2. We restructured the Table 1 and provided the allele ranges and the number of identified alleles by loci for entire data set as well as for each of museum and modern samples.

Comments:

Grammar checking should be made before publication.

Reply: Thank you for your suggestion. We used MDPI English Editing Service before we submitted the first draft of our manuscript to the Diversity. We included the English Edition Certificate issued by MDPI as the non-published materials to our manuscript.

Line 34: replace “having undergone the greatest changes” to “has undergone great changes”

Line 35: replace “research results” to “results”

Line 60: replace “description their properties” to “description of their properties”

Line 99: replace “the most of native breeds” to “most of the native breeds”

Line 101: replace “losses” to “loss”

Line 106: replace “a breed’s origin” to “breed’s origin”

Line 106-107: replace “tracing the maintenance of ancestral” to “maintaining the ancestral”

Reply: Thank you for your suggestions. We made corrections accordingly.

Line 108-111: here, it should be described how mtDNA can overcome the problem of “small amount” and “high degree of degradation” in DNA derived from museum samples. please re-write the paragraph to something like this:

Over the last decades, the field of ancient DNA (aDNA) heavily relied on Mitochondrial DNA (mtDNA) marker. This is due to the low copy number and general difficulties associated with the recovery of nuclear DNA from archaeo- zoological material.

Reply: Thank you very much for your suggestions. We made the correction in the text of the manuscript.

Line 111: replace “mitochondrial DNA” to “mtDNA”

Reply: We made corrections.

Line 125: add three sub-headings to the material and methods section, something like below, in order to have a clear section for each part.

2.1 Sample collection

2.2 DNA extraction

2.3 Microsatellite profiling or Autosomal STR Analysis

Line 151-157: This paragraph should be moved up to the sample collection

Reply: According suggestions of the Reviewer, we divided the Material and Methods section to the sub-sections and moved the above-mentioned paragraph to the sample collection subsection.

Line 200: reference after R software (http://cran.r-project.org)

Line 225: replace “museum samples.” to “museum samples”

Line 230: add the sample ID of the four and two samples that were only genotyped for 10, and 9 loci respectively. This information is important in terms of knowing missing loci are observed in which historic population.

Reply: We made corrections.

Line 254: The 131 alleles called in historical samples are shown in Table S2, and Table 1, but no such detailed information available for modern populations. This information should be added as well, unless publicly available data are used. If that is the case, why the authors did DNA extraction on modern samples!

Reply: We provided the genotypic data for modern samples in Supplemental materials - Table S2b. Additionally, we restructured Table 1. We included in Table 1 the allele ranges and the number of identified alleles for entire data set as well as for museum and modern samples.

Line 254-256: please mention that this numbers are referring to the number of alleles only in historic samples – the data for modern is missing.

Reply: We restructured the Table 1 and provided the allele ranges and the number of identified alleles by loci for entire data set as well as for each of museum and modern samples. We moved the data concerning amplification failure and genotyping errors of museum samples from Table 1 to the Supplemental materials and added this to the Table S3. We corrected the capture of Table 1 and Table S3 in accordance with the novel content of these Tables.

Line 257: From which table/figure this 124 vs. 96 alleles has been extracted? Based on Table 1, 131 alleles are reported for historic samples, the corresponding numbers for modern samples are missing.

Reply: We added the data concerning the distribution of alleles by loci for entire data set as well as for museum and modern samples (Table 1). We brought the text into accordance with the data presented in Table 1.

Line 278: Please clarify if the absence of the novel 64 alleles in historic populations is not the result of smaller historic sample size, and simply the alleles are absent because the individuals are not sampled.

Reply: We agree with the Reviewer that the small sample size could cause the biases, especially in genetic diversity estimates. Unfortunately, we were able to extract the reliable consensus genotypes only for three specimens. We noted in the text of the manuscript the possible biases in allelic pattern of museum samples that could be caused by the limited number of samples.

Line 278; In Table S4, only 61 novel alleles are reported but in the text 64 – please clarify.

Reply: We carefully checked the number of novel alleles. We fixed the occurred typographical error and clarified the number of alleles.

Line 293-296: please use some statistical tests such as Bonferroni correction or Wilcoxon rank-sum test (p >0.05) to statistically backup that there is no significant differences.

Reply: We applied the Wilcoxon rank-sum test (p >0.05) to estimate the p-value of the differences for genetic diversity estimates among studied populations and indicated it in Materials and Methods section. We statistically backup that there is no significant differences among museum and modern populations. We found statistically significant differences only among Herefords and several other studied breeds. We indicated it in the Table 3.

Line 296: Almost half of the novel alleles (34 alleles) are emerging in Kazakh white-headed population, and 4 are missed from the historic correspondence. Here I am confused that how this high number of novel alleles are not contributing to high genetic diversity.

Reply: We believe that such high number of the novel alleles emerged in Kazakh white-headed breed was the result of multiple backcrossing with Hereford bulls. Using the limited number of bulls could lead to the increase of homozygosity. We mentioned it in the text of the manuscript.

Line 303: replace “between” to “among”

Line 319: remove “equaled” and replace with “is”.

Reply: We made corrections.

Line 327: the values are reported wrongly in comparison to Table S5.

Fst between modern kalmyk and historic kalmyk = 0.018

Fst between modern Kyrgyz and historic Kyrgyz = 0.025

Reply: We made corrections in the text according to the values in Table S5.

Line 328: The value below is missing in the text as the Kazakh white headed was only compared to historic kalmyk and historic Kazakh white headed, and not with historic Kyrgyz. Please add the value below: Fst between Kazakh white headed to historic Kyrgyz = 0.082

Reply: Thank you very much for your suggestions. We made the correction in the text of the manuscript.

Line 330-332: what about KZKH-H and MONG_M with Jost’s D of 0.019?

Reply: We mentioned it in the text.

Line 361: replace “red” with “orange”

Line 364: replace “closer” to “more closely”

Line 327; remove “and as a result of diseases” or complete the sentences in a way that make sense.

Line 379: remove “these”

Reply: We made corrections.

Line 381-384: rephrase suggestion:

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 inferr the origin of breeds. The absence of such information would negatively affect their future development and breeding managements.

Reply: Thank you very much. We made corrections.

Line 410: replace “the Kazakh white headed” to “the modern Kazakh white headed’

Line 411-412: remove “in the modern population”

Reply: We made corrections.

Tables and Figures:

Figure S2: replace [Ivanova O.A. et al., 1934] to [16].

Reply: We made corrections.

Table S1: replace the heading “Figure S1” to “Table S1”.

Reply: We corrected the Table caption

In addition, add the DNA extraction info for modern samples as well – although that is not very important but should be kept uniform – either show it for both historic and modern samples or not represent.

Reply: For museum samples, we measured the concentrations of double-stranded DNA by Qubit, because the low amount of high-degraded DNA is one of the main factors affected the efficiency of genotyping for historic samples. Besides, the low amount of high-degraded DNA used for PCR can increase the rate of failure amplification as well as the rate of genotyping errors. As the control of DNA concentration and quality are important for reliable genotyping museum samples, we provided the detailed data on DNA concentration and quality for museum samples. The DNA extraction from modern samples is the standard laboratory praxis. Using the DNA extraction kits according to the recommendations of the manufacture allows producing enough amount of DNA for PCR analysis. The DNA concentrations are routinely checked for the part of samples by measurement of absorbance at 260 nm. These values are not comparable with dsDNA concentrations, measured for museum samples by Qubit. For this reason, we did not provide the data on DNA concentration for modern samples.

Table S2: replace the heading “Figure S2” to “Table S2”. In addition in the Table caption add that the information is for historical samples.

Reply: We replaced the heading “Figure S2” to “Table S2” and corrected the Table caption as «Consensus genotypes and efficiency of genotyping of museum specimens used for the studies»

The genotyping information for modern samples should be added to Table S2 as well.

Reply: We added the genotyping information for modern samples to Table S2.

Table S3: replace “quality indices is describes” to “quality indices is described”

Reply: We made corrections.

What the “paragraph 2.2” refers in the caption of table S3?

Reply: We replaces “paragraph 2.2” to the “section 2.2 of the main text”.

The same information on quality of microsatellite genotyping for modern samples should be added.

Reply: the multiple-tube approach was used for estimating the consensus genotypes for museum samples only. For this reason, the calculation of quality indices and genotyping errors was performed only for museum samples. The genotyping of modern samples was carried out using standard single-tube approach. We provided the genotypes of modern samples in Table S2.

Table S4: replace the heading “Figure S4” to “Table S4”.

Reply: We made corrections.

Reviewer 4 Report

This is an interesting paper, and an important one because linking present populations to past populations is a high priority for meaningful breed conservation. The introduction could benefit from splitting the huge paragraph into several shorter ones.

line 85: "Holland" breed should likely be further designated.

line 93: It would be interesting to specifically note if these few "native Kyrgyz" cattle show external influence of introgression or not. Given the introgression from transboundary breeds in most of the populations, a specific note should be added as to whether these have that influence or not. It is also important to note whether this modern "native" population is the one that was specifically sampled for the study.

line 267 is a bit confusing as to whether the Mongolian cattle share extensive Hereford introgression with Kazakh cattle.

line 301: the excess of heterozygous Kyrgyz cattle is interesting, and it sould be interesting to speculate on reasons for this in the discussion.

The discussion could benefit from splitting it up into shorter paragraphs.

 

Author Response

This is an interesting paper, and an important one because linking present populations to past populations is a high priority for meaningful breed conservation.

Reply: We appreciate the encouraging comments. Thank you.

The introduction could benefit from splitting the huge paragraph into several shorter ones.

line 85: "Holland" breed should likely be further designated.

Reply: We clarified in the text of the manuscript the contribution of Brown Swiss and Holland cattle in the development of the Kyrzyz cattle population: “In Kyrgyzstan, the greatest part of the local cattle population was crossed with Brown Swiss and Holland breeds that resulted in developing in the Soviet time the novel synthetic breeds [8, 9]. The small remaining part of native Kyrgyz cattle was further developed without contribution of other breeds”. Because, Brown Swiss and Holland cattle breeds did not affected the native Kyrgyz cattle we did not consider these in our study.

line 93: It would be interesting to specifically note if these few "native Kyrgyz" cattle show external influence of introgression or not. Given the introgression from transboundary breeds in most of the populations, a specific note should be added as to whether these have that influence or not. It is also important to note whether this modern "native" population is the one that was specifically sampled for the study.

Reply: Thank you very much for your comments. Yes, Eugenia M. Lutshikhina from the Kyrgyz Institute for Animal Husbandry (one of the co-authors of our manuscript) collected the samples especially for the study of genetic diversity of Kyrgyz cattle. Because no pedigree records are available for the native Kyrgyz cattle, the selection of animals for sampling was done based on the exterior characteristics and verbal information got from the owners. We mentioned this in the text of the manuscript.

line 267 is a bit confusing as to whether the Mongolian cattle share extensive Hereford introgression with Kazakh cattle.

Reply: Thank you very much for your comments. We rephrased the sentence.

line 301: the excess of heterozygous Kyrgyz cattle is interesting, and it should be interesting to speculate on reasons for this in the discussion.

Reply: Thank you very much for your suggestion. We discuss the possible reason of the heterozygote excess in Kyrgyz cattle in the Discussion section of the manuscript: “Interestingly, we observed the heterozygote excess in the modern Kyrgyz cattle (F_IS = -0.085) that has the lowest population size comparing to other studied breeds. The possible explanation could be the practically lacking the pressure of the artificial selection, extremely long cows’ productive life (up to 10 lactations), and the calves' exchanges between owners”.

The discussion could benefit from splitting it up into shorter paragraphs.

Reply: We made corrections. Thank you.