Genetic Characterization of the Arabian Horse Population in Tunisia Using Microsatellites

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper described the divergence in a population of Arabian horses in Turkey using the standard set of STR markers as currently in use.

line 51-57, can you be more specific about what you exactly analyze? You speak of Tunisian Arabian, Eastern and Western lineages. what are Eastern an Western lineages? Are they geographical? Are the genetic? If just geographical, do they have similar genetics?

2.1 Please get more description on the samples. Itis only listed there were 130 samples, but in the results text they are supposedly two different groups. But these groups add up only to 99 samples (Eastern 36 and Western 63 based on your results in section 3.1.2. Specify here how many per group and also where they are from if applicable.

Tabel 1 shows only 16 STR markers. Missing LEX3 I presume?

section 2.3 please include DNA extraction protocol.

section 2.4.1.3 last part of sentence, should that be " frequency of the most common allele is equal to or LESS than 0.95"?

section 2.4.1.1 lines 90-96 are hard to understand what you want to say here. "number one price that paperwork ...."? Can you look it over and make sure it says what you want it to say.

2.4.1.2 Please check statement in 108-109. I thought it was supposed to be within one locus and then you can average. Not between two loci and then average.

2.4.1.3 sentence 123, at the end you say greater than 95%. I think it should be less than 95% here.

 

2.4.1.4 Please check your assumptions. It cannot be that there is heterozygote excess unless FIS = 1.  I assume 0<F<1 is homozygote excess? or Het shortage?

2.4.2.1 line 166 where does FIT come from (inbreeding coefficient) is this a typo? If not, please add more info for FIT like for all other parameters.

2.4.2.4 why is this so simple written, no explanation of parameters and symbols?

results lines 192-196, where is this data coming from? You only genotyped Tunisian horses so there has to be a reference publication for this data. Please list that here and in your references

I also calculate there were 36 Eastern Arabians (line 193), and 63 Western Arabians (line 202). This does not add up to the 130 animals. Where i the rest? Please add more clarity to the section describing sample sin M&M

All tables, make sure you use periods and not commas for decimals.

Tabel 2 and Tabel 4 have the same name and thus unclear what they represent.

Table 2, 3 and 4 also need clarification, e.g. SB2 has two alleles in Tabel 3 as least abundant at 0.8% but in table 5 for private alleles we see different alleles at different frequencies. Something is off here.

Table 4. 0.8% and 1.4% are private alleles, how come the vales in this table are different, and how come there is 2.4 and 2.5 %, as this would be a difference of  less than 1 allele. Same for 0.8% and 0.9%.

Tabel 7 which samples are form previous studies? Only the Syrian non-registered? Please update table to show source for all literature samples, each lien their own [25] if that is the source. Also why are Tunisian horses now analyzed together and not as Eastern vs western?

Table 8, Na and Ne are undefined in the M&M section. Are they related to 2.4.1.5 where they are labeled A and Ae?

lines 293-294 and 299-300, did you test whether the horses within the group were unrelated? This as if they were related it is very likely they are not in HW equilibrium and therefore this statement might be overreach.

Table 9, typo for eastern and western, missing "n"

line309 where do thoroughbreds come from? They were not listed as samples in sample section. Please fix.

 

line 328 you did not study any genes, only STR. SO this statement is invalid.

Fig 2. what does OC, OR, AN mean in the graph? It is better to be also included in the figure legend than several lines down in text only. It took me a while to figure this out it was down in the text. Also the naming seems not intuitive. Why OR, OC and AN instead of EA, WA, TB? Coloring by breed would also help.

section 3.2.3 Nm is undefined in M&M

lines 356 does the data from Syrian and Polish horses come from reference 25?

line 366, i thought 1.4% and 0.8% were the minimal values. Anything below is not existent or not present.

 

I have skipped most of the discussion as I am not certain what data is from this paper and what is from literature, too many different types of Arabian horses that I cannot account for in the sample section of the M&M.

Line 438 etc when I look at the PA plot, to me they seem highly overlapping for the Arabians, the only different group to me could be the thoroughbreds, as presented.

 

Please also address/discuss why you used 17 STR markers and not a SNP chip with say 65,000 SNPs (equine 70K SNP chip from Illumina) as currently is used in a lot of research projects or another method that would generate larger amounts of data and could provide a deeper dice into the genetics and allow you to do more follow up studies.

Author Response

Response to Reviewer 1 Comments

 

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Are all figures and tables clear and well-presented?	( )		(x)		( )		( )

 

Comments and Suggestions for Authors

 

This paper described the divergence in a population of Arabian horses in Turkey (in Tunisia) using the standard set of STR markers as currently in use.

 

Comment 1: line 51-57, can you be more specific about what you exactly analyze? You speak of Tunisian Arabian, Eastern and Western lineages. what are Eastern an Western lineages? Are they geographical? Are the genetic? If just geographical, do they have similar genetics?

Response 1: Eastern and Western Arabian lineages refer to long-standing breeding traditions, not regions within Tunisia. Eastern horses descend from original Bedouin strains of the Arabian Peninsula, while Western lines were developed in Europe and the USA through more open breeding programs. These historical differences also reflect slight but real genetic distinctions, as supported by our FST and PCA results.

 

Comment 2: 2.1 Please get more description on the samples. It is only listed there were 130 samples, but in the results text, they are supposedly two different groups. But these groups add up only to 99 samples (Eastern 36 and Western 63 based on your results in section 3.1.2. Specify here how many per group and also where they are from if applicable.

Response 2: The study analyzed a total of 130 horses: 36 belonging to Eastern lineages and 63 to Western lineages, as detailed in the Results, along with 31 Thoroughbreds included as an outgroup for comparison.

 

Comment 3 : Tabel 1 shows only 16 STR markers. Missing LEX3 I presume?

Response 3: Yes, I I add the LEX

 

Comment 4: section 2.3 please include  DNA extraction protocol.

Response 4: Genomic DNA was extracted using the PureLink Genomic DNA Extraction Kit (Invitrogen), which enables rapid and efficient purification of genomic DNA. This kit is specifically designed to isolate high-quality DNA by separating it from other cellular and tissue components.

 

Comment 5: section 2.4.1.3 last part of sentence, should that be " frequency of the most common allele is equal to or LESS than 0.95"?

Response 5: common allele is at a frequency of less than 0.95

Comment 6: section 2.4.1.1 lines 90-96 are hard to understand what you want to say here. "number one price that paperwork ...."? Can you look it over and make sure it says what you want it to say.

Response 6: Allelic frequency is the fundamental parameter used to describe and analyze genetic variability within a population

Comment 7: Section 2.4.1.1 lines 90-96 are hard to understand what you want to say here. "Number one price that paperwork ...."? Can you look it over and make sure it says what you want it to say.

Response 7: The sentence has now been rewritten to accurately convey that allelic frequency is the fundamental parameter used to describe and analyze genetic variability within a population.

Comment 8: 2.4.1.2 Please check statement in 108-109. I thought it was supposed to be within one locus and then you can average. Not between two loci and then average.

 

Response 8:

The heterozygosity rate corresponds to the proportion of heterozygous individuals at a given locus, and the overall heterozygosity of a population is obtained by averaging these values across all loci studied.

Comment 9:  2.4.1.3 sentence 123, at the end you say greater than 95%. I think it should be less than 95% here.

Response 9:  the frequency of the most common allele is equal to or less than 0.95

Comment 10: 2.4.1.4 Please check your assumptions. It cannot be that there is heterozygote excess unless FIS = 1.  I assume 0<F<1 is homozygote excess? or Het shortage?

Response 10: A positive FIS value (0 < FIS < 1) indicates a heterozygote deficit (i.e., homozygote excess), not heterozygote excess. Conversely, negative FIS values indicate heterozygote excess.

Comment 11: 2.4.2.1 line 166 where does FIT come from (inbreeding coefficient) is this a typo? If not, please add more info for FIT like for all other parameters.

Response 11: FIT is not a typo. FIT is the overall fixation index, representing the reduction of heterozygosity in individuals relative to the total population, considering both within- and between-subpopulation effects. It integrates the contributions of FIS (inbreeding within subpopulations) and FST (differentiation among subpopulations) according to the relationship:

 

A positive FIT indicates an overall deficit of heterozygotes in the global population, whereas negative values reflect an excess of heterozygosity. FIT therefore provides a global assessment of deviation from Hardy–Weinberg equilibrium across all subpopulations combined.

Comment 12: 2.4.2.4 why is this so simple written, no explanation of parameters and symbols?

Response 12: Gene flow (Nm) represents the number of migrants exchanged between populations per generation. It is estimated from Wright’s fixation index using the formula: Nm = (1 – FST) / (4FST). A high Nm value indicates substantial gene exchange and low genetic differentiation, whereas a low Nm value suggests restricted migration and stronger population subdivision. In this study, Nm was calculated for each pair of populations using their corresponding FST values to assess the level of connectivity among groups.

Comment 13: results lines 192-196, where is this data coming from? You only genotyped Tunisian horses so there has to be a reference publication for this data. Please list that here and in your references

Response 13:

The comparative allelic richness values mentioned in lines 192–196 were derived from previously published studies, not from our own genotyping. The revised section explicitly states that the values for Syrian, Iranian, Saudi, Polish, Shagya, USA-Egyptian, USA-Saudi, and Davenport Arabian horses originate from Khanshour et al. (2013) = reference 25

Comment 14: I also calculate there were 36 Eastern Arabians (line 193), and 63 Western Arabians (line 202). This does not add up to the 130 animals. Where i the rest? Please add more clarity to the section describing sample sin M&M

Response 14:

A total of 130 horses were included in this study. Among them, 99 were Tunisian Arabian horses registered in the national studbook and classified into two lineages based on pedigree information: 36 Eastern (Oriental-type) Arabians and 63 Western (Occidental-type) Arabians. In addition, 31 English Thoroughbred horses were included as an outgroup to assess inter-population genetic differentiation.

Comment 15: All tables, make sure you use periods and not commas for decimals

Response 15: Thank you for pointing this out. We corrected all decimal formats throughout the manuscript. All tables have now been standardized to use periods instead of commas for decimal values, in accordance with international scientific formatting conventions.

Comment 16: Tabel 2 and Tabel 4 have the same name and thus unclear what they represent.

Response 16: Table 2. Private alleles and their frequencies in Eastern Arabian horses

                          Table 4. Private alleles and their frequencies in Western Arabian horses.

Comment 17: Table 2, 3 and 4 also need clarification, e.g. SB2 has two alleles in Tabel 3 as least abundant at 0.8% but in table 5 for private alleles we see different alleles at different frequencies. Something is off here.

Response 17: Table 2 lists only private alleles detected in Eastern Arabian horses.

Table 3 lists the lowest-frequency alleles for Western Arabians.

Table 4 lists private alleles detected in Western Arabian horses.

To avoid confusion, alleles with the lowest frequencies in each population are presented separately from private alleles. Tables 2 and 3 list the least frequent alleles detected in Eastern and Western populations, respectively, whereas Table 4 includes only private alleles, i.e., alleles detected exclusively in a single population.

 

Comment 18: Table 4. 0.8% and 1.4% are private alleles, how come the vales in this table are different, and how come there is 2.4 and 2.5 %, as this would be a difference of less than 1 allele. Same for 0.8% and 0.9%.

Response 18: these values are the means of the studied populations .0.8 % and 0.9% indicate low private frequencies

Low frequencies of private alleles do not reflect substantial dissimilarity; rather, they indicate the similarity that exists between the populations.

Comment 19: Table 7 which samples are form previous studies? Only the Syrian non-registered? Please update table to show source for all literature samples, each lien their own [25] if that is the source. Also why are Tunisian horses now analyzed together and not as Eastern vs western?

Response 19: Table 7, several heterozygosity values were indeed extracted from previously published studies, not only the Syrian non-registered horses. i updated the table to clearly indicate the specific reference corresponding to each population (Saudi Arabian, Syrian registered, Syrian non-registered, Iranian Arabian), with each line including its proper citation.
For the Tunisian horses, the values presented originally reflected the overall mean heterozygosity reported in our previous national study.

Comment 20 Table 8, Na and Ne are undefined in the M&M section. Are they related to 2.4.1.5 where they are labeled A and Ae?

Response 20 : In Section 2.4.1.5, the parameters “A” (allelic richness) and “Ae” (effective number of alleles) correspond respectively to “Na” and “Ne” reported in Table 8.

I changed this section

Comment 21: lines 293-294 and 299-300, did you test whether the horses within the group were unrelated? This as if they were related it is very likely they are not in HW equilibrium and therefore this statement might be overreach.

Response 21: We did not perform a formal relatedness test (exemple : pedigree-based kinship coefficient or molecular relatedness estimation) on the sampled horses. Although the animals were registered in the studbook and selected to avoid obvious close relatives, we acknowledge that some level of relatedness may still exist within each lineage. This could indeed contribute to deviations from Hardy Weinberg equilibrium and may influence FIS estimates.

Comment 22 Table 9, typo for eastern and western, missing "n"

Response 22: Fixation index (FIS) values for Eastern and Western Arabian horses.

Comment 23 line 309 where do thoroughbreds come from? They were not listed as samples in sample section. Please fix.

Response 23: i have explicitly mentionned that 31 Thoroughbred horses were included as an outgroup and describe where these samples came from.

Comment 24: line 328 you did not study any genes, only STR. SO this statement is invalid.

Response 24: yes I replaced gene with microsatellite markers (STRs)

Comment 25: Fig 2. what does OC, OR, AN mean in the graph? It is better to be also included in the figure legend than several lines down in text only. It took me a while to figure this out it was down in the text. Also the naming seems not intuitive. Why OR, OC and AN instead of EA, WA, TB? Coloring by breed would also help.

Response 25:

 The abbreviations are explained directly in the figure legend: the original paper iti is in french we keep the original pot than
OR= Eastern Arabian; OC=Western Arabian; TB= Thoroughbred.
In addition, the group labels in the figure have been changed to EA, WA, and TB for intuitive understanding.

Comment 26: section 3.2.3 Nm is undefined in M&M

Response 26: A definition of Nm and the method of calculation have been added to the Materials and Methods section.

Comment 27: lines 356 does the data from Syrian and Polish horses come from reference 25?

Response 27:: Comparative data for Syrian and Polish horse populations were taken from previously published work [25].”

Comment 28: line 366, i thought 1.4% and 0.8% were the minimal values. Anything below is not existent or not present.

Response 28 Rare alleles (<1.4% in Eastern, <0.8% in Western populations) indicate the presence of low genetic variants specific to each population, thereby contributing to overall genetic diversity

Comment 29: I have skipped most of the discussion as I am not certain what data is from this paper and what is from literature, too many different types of Arabian horses that I cannot account for in the sample section of the M&M.

 Response 29: A total of 130 horses were included in this study. Among them, 99 were Tunisian Arabian horses registered in the national studbook and classified into two lineages based on pedigree information: 36 Eastern (Oriental-type) Arabians and 63 Western (Occidental-type) Arabians. In addition, 31 English Thoroughbred horses were included as an outgroup to assess inter-population genetic differentiation.

Comment 30: Line 438 etc when I look at the PA plot, to me they seem highly overlapping for the Arabians, the only different group to me could be the thoroughbreds, as presented.

Response 30: yes but we discuss two subpopulation from the arabian breed (OR: orientale =Eastern arabian horse) ;OC : occidental =western arabian horse)

OR, OC reflect the acronyme in French

Comment 31: Please also address/discuss why you used 17 STR markers and not a SNP chip with say 65,000 SNPs (equine 70K SNP chip from Illumina) as currently is used in a lot of research projects or another method that would generate larger amounts of data and could provide a deeper dice into the genetics and allow you to do more follow up studies

 Response 31: We used 17 STR markers because they are highly polymorphic, cost-effective, and widely used in horse population genetic studies. While high-density SNP chips, such as the 70K Equine SNP chip, provide more detailed genomic information, STR markers remain a valid tool to assess genetic diversity, population structure, and relatedness.

Hopping in the future when we have the possibility for collaboration under an international project, we do this work with SNP

 

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of the paper is interesting. The chosen method is not really new, but the application to the given horses breed might be interesting. 
The articles 10.3390/vetsci9070333, 10.3390/d16050281 and 10.3390/vetsci12080776 are relevant to this paper, so inclusion of them might be relevant in the literature overview and also in the discussion part of the manuscript.
The Materials and Methods detailly describes the workflow and the parameters of genetic variability. This part might increase the similarity index, but necessary for readers.

There are data in Results section (3.1.2.) about Eastern Arabian, Syrian, Western Arabian and Polish horses but no info in the Materials and Methods section about these horses. Detailled information about these horses should be included in the manuscript.

Figure 1a and 2b in 3.1.3. must be improved as captions cannot be read. Decimals should be also corrected. Mixing of decimal signs is really confusing. Decimal signs should be also corrected in tables: 2, 4, 5, 6, 8, 9, 10, 11, 12 and 13. 

Authors use the term 'Thoroughbreds'. I think, 'Thoroughbreds' means here 'Arabian Thoroughbreds', but it must be clarified.  To avoid confusing of Arabian and English Thoroughbreds, the term Arabian Thoroughbred might be a better solution. 

The Conclusions section is extremly long, it should be shortened.

 

Author Response

Response to Reviewer 2 Comments

 

Review Report Form

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

Quality of English Language

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	( )	(x)	( )	( )
Are the methods adequately described?	( )	(x)	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	( )	(x)	( )	( )
Are all figures and tables clear and well-presented?	( )	( )	(x)	( )

 ( ) The English could be improved to more clearly express the research.
(x) The English is fine and does not require any improvement.

 

Comments and Suggestions for Authors

Comment 1: The aim of the paper is interesting. The chosen method is not really new, but the application to the given horses breed might be interesting. 
The articles 10.3390/vetsci9070333, 10.3390/d16050281 and 10.3390/vetsci12080776 are relevant to this paper, so inclusion of them might be relevant in the literature overview and also in the discussion part of the manuscript.

Reponse 1 The suggested articles have now been incorporated into the Introduction to strengthen the background on the use of molecular markers in horse genetic studies and to contextualize our approach. They have also been integrated into the Discussion to compare our findings with recent research addressing genetic diversity, population structure, and conservation strategies in Arabian and other horse breeds

Comment 2: The Materials and Methods detailly describes the workflow and the parameters of genetic variability. This part might increase the similarity index, but necessary for readers.

Comment 3There are data in Results section (3.1.2.) about Eastern Arabian, Syrian, Western Arabian and Polish horses but no info in the Materials and Methods section about these horses. Detailled information about these horses should be included in the manuscript.

Response 3 : done: The study analyzed a total of 130 Arabian horses: 36 belonging to Eastern lineages and 63 to Western lineages, as detailed in the Results, along with 31 Thoroughbreds included as an outgroup for comparison.

Comment 4: Figure 1a and 2b in 3.1.3. must be improved as captions cannot be read. Decimals should be also corrected. Mixing of decimal signs is really confusing. Decimal signs should be also corrected in tables: 2, 4, 5, 6, 8, 9, 10, 11, 12 and 13. 

Reponse 4: done

Comment 5: Authors use the term 'Thoroughbreds'. I think, 'Thoroughbreds' means here 'Arabian Thoroughbreds', but it must be clarified.  To avoid confusing of Arabian and English Thoroughbreds, the term Arabian Thoroughbred might be a better solution. 

 Response 5: Thoroughbreds:  English; Arabian horses: eastern and western horses

Comment 6: The Conclusions section is extremly long, it should be shortened.

Response 6: Done

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

My apologies for mixing up countries (Turkey - Tunisia) in my first review.

All my comments were addressed appropriately, except where noted below..

Just a few minor edits based on reading the new version.

Line 69: Start sentence with a capital.

Line 70, please check your tubes but I assume you mean 5 ml vacuum tubes that contain EDTA. Most EDTA tubes contain dried down K2 EDTA or may contain 20 uL of K3 EDTA. 5 ml of EDTA solution is a lot. 

Section 2.3 how much DNA was used in the reactions?

page 7, why Fig 1a and Fig 2 b?  Should just be Fig 1 and Fig 2, no added letters.

(If possible it might help visualize if within each of the graph, the alleles by marker would alternate between blue and red, e.g., VHL20 - red, HTG4 - blue, AHT4 -red, etc)

Line 267, marker AHMS1 should be HMS1

Figure 2, it is hard to see the 2 separate groups of Arabians, would a 3D plot make it more clear? Esp as the first two represent ~50%, and adding the 3rd makes it 64.29%. 2nd and 3rd are very similar. I could see adding the 3rd one shows the overlap is in a different plane, and not in the same plane.

Lines 423-425, why in italics?

For the discussion point on page16, line 448 and 459 on LEX3 on heterozygote deficits (and FIT), did you take into account this is a chrX marker and therefor all males are hemizygous (not homozygous)?

Lines 496-499, what do you mean with the studied genes? You only analyzed STR markers. Similar as previous comment 24.

Author Response

Response to Reviewer 1 Comments

Review Report Form

Open Review

( ) I would not like to sign my review report
(x) I would like to sign my review report

Quality of English Language

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	(x)	( )	( )	( )
Are the conclusions supported by the results?	(x)	( )	( )	( )
Are all figures and tables clear and well-presented?	(x)	( )	( )	( )

 ( ) The English could be improved to more clearly express the research.
(x) The English is fine and does not require any improvement.

 

 

Comments and Suggestions for Authors

My apologies for mixing up countries (Turkey - Tunisia) in my first review.

All my comments were addressed appropriately, except where noted below..:

Just a few minor edits based on reading the new version.

Comment 1: Line 69: Start sentence with a capital.

Reponse 1: Done

Comment 2: Line 70, please check your tubes but I assume you mean 5 ml vacuum tubes that contain EDTA. Most EDTA tubes contain dried down K2 EDTA or may contain 20 uL of K3 EDTA. 5 ml of EDTA solution is a lot. 

Reponse 2: Done, 5 mL vacuum tubes containing 20 uL of K3 EDTA

Comment 3: Section 2.3 how much DNA was used in the reactions?

Reponse 3: Each Pcr reaction contained 50 ng of genomic DNA

Comment 4 : page 7, why Fig 1a and Fig 2 b?  Should just be Fig 1 and Fig 2, no added letters.

(If possible it might help visualize if within each of the graph, the alleles by marker would alternate between blue and red, e.g., VHL20 - red, HTG4 - blue, AHT4 -red, etc)

Reponse 4: Done, I have therefore revised the figure labels from Fig. 1a and Fig. 2b to simply Fig. 1 and Fig. 2.

Regarding your suggestion to alternate colors (e.g., red/blue) for the alleles within each marker, we appreciate this recommendation as it indeed improves visual clarity but unfortunately i can’t switch the colors.

Comment 5: Line 267, marker AHMS1 should be HMS1

Reponse 5: done

 

Comment 6: Figure 2, it is hard to see the 2 separate groups of Arabians, would a 3D plot make it more clear? Esp as the first two represent ~50%, and adding the 3rd makes it 64.29%. 2nd and 3rd are very similar. I could see adding the 3rd one shows the overlap is in a different plane, and not in the same plane.

Reponse 6: I agree that the separation between the two Arabian groups is not fully clear in the current 2D plot. Since the first two components explain only about 50% of the variance and the third increases this to 64.29%, we acknowledge that adding the third axis could improve visualization.

We use the PCA methods so we can’t plot it 3D, hope it will be acceptable

 

Comment 7: Lines 423-425, why in italics?

Reponse 7: Done

Comment 8: For the discussion point on page16, line 448 and 459 on LEX3 on heterozygote deficits (and FIT), did you take into account this is a chrX marker and therefor all males are hemizygous (not homozygous)?

Reponse 8: Yes, LEX3 is an X-linked marker and therefore males are hemizygous rather than homozygous. We acknowledge that this characteristic may influence heterozygosity estimates and related fixation indices (FIS and FIT), which are traditionally computed assuming autosomal loci. I have added a clarification regarding the X-linked nature of LEX3 and its implications, and I interpret the observed heterozygote deficit and high FIT values with appropriate caution. I also specify that sex information was not incorporated in the computation of diversity indices for this locus, which may partly explain the apparent heterozygote deficit.

Comment 9: Lines 496-499, what do you mean with the studied genes? You only analyzed STR markers. Similar as previous comment 24.

Reponse 9: Genetic distance results indicate that 94.8% of the studied microsatellite loci show similarity between the Eastern Arabian and Western Arabian populations, while 62.6% of the loci analyzed demonstrate similarity between the Eastern Arabian and English Thoroughbred populations, and 75.7% of the studied loci exhibit similarity between the Western Arabian and English Thoroughbred populations.”

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is significantly improved. Authors taken into account and/or answered correctly my comments, so I accept the manuscript. Only a few typo check is necessary before the final publication of the manuscript.

Author Response

Response to Reviewer 2 Comments

Review Report Form

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

 

Quality of English Language

( ) The English could be improved to more clearly express the research.
(x) The English is fine and does not require any improvement.

 

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	(x)	( )	( )	( )
Are the conclusions supported by the results?	(x)	( )	( )	( )
Are all figures and tables clear and well-presented?	(x)	( )	( )	( )

 

Comments and Suggestions for Authors

 

Comment 1:

The manuscript is significantly improved. Authors taken into account and/or answered correctly my comments, so I accept the manuscript. Only a few typo check is necessary before the final publication of the manuscript.

Reponse 1 Thank you very much for your positive evaluation and for acknowledging the improvements made in the manuscript. We appreciate your careful review and constructive comments throughout the revision process. We have performed another thorough proofreading to correct the remaining typographical errors before the final submission.

 

Author Response File: Author Response.docx