Assessment of Heterozygosity in European Local and Cosmopolitan Pig Populations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, authors aimed to identify genomic signatures of heterozygous, known as Heterozygosity-Rich regions (HRRs), from 20 local and 3 cosmopolitan pig breeds. Authors assessed and compared the HRRs, and found Large White with the highest number of HRRs and Alentejana with lowest number. Then they performed the gene enrichment analysis of HRRs and tried to characterize the biological functions. The materials are comprehensive, and the methods are supported clearly. The results sound technical, and the written is scientific. 

1. The INTRODUCTION section of the article seems somewhat inadequate in summarizing previous research efforts in this area. Additional relevant research background should be supplemented in the INTRODUCTION.

2. The article investigates the trends and distribution of HRRs (Heterozygosity-Rich Regions) across different pig breeds, emphasizing their role and providing a definition. However, it appears that the article does not explicitly define homozygosity (ROH) nor clearly differentiate the functional differences between these two concepts.

3. Is there an issue with the description of SNP chip data in line 73 of the article? Additionally, the descriptions of Whole Genome Sequencing data appear inconsistent between lines 74 and 75.

4. Lines 95-101 of the article provide a detailed definition and explanation of the scenarios established. However, the rationale behind these definitions, the choice to include multiple scenarios, and the advantages of such an approach are not explicitly addressed.

5. The article compares PCA results using complete SNP chip data and SNP data from HRRs, providing a thorough interpretation of the findings. However, the number of SNPs contained within the HRR islands is only 6,228. The article does not clearly explain the potential impact of this limited SNP count on the PCA results. For instance, could the observation in line 203, “The within-breed variability was extended in almost all the other pig breeds,” be attributed to the reduced dataset size?

6. The DISCUSSION section provides a comprehensive exploration of the study's results and their potential implications. However, in line 310, there seems to be a lack of explanation as to why a lower occurrence of HRRs affects the p-value.

7. The conclusion in lines 321-330 appears somewhat lacking in summarizing the study. The presentation of the key findings is neither sufficiently concise nor fully articulated.

Author Response

Rev 1

In this manuscript, authors aimed to identify genomic signatures of heterozygous, known as Heterozygosity-Rich regions (HRRs), from 20 local and 3 cosmopolitan pig breeds. Authors assessed and compared the HRRs, and found Large White with the highest number of HRRs and Alentejana with lowest number. Then they performed the gene enrichment analysis of HRRs and tried to characterize the biological functions. The materials are comprehensive, and the methods are supported clearly. The results sound technical, and the written is scientific.

 

1. The INTRODUCTION section of the article seems somewhat inadequate in summarizing previous research efforts in this area. Additional relevant research background should be supplemented in the INTRODUCTION.

Introduction has been improved (L65, L72)

2. The article investigates the trends and distribution of HRRs (Heterozygosity-Rich Regions) across different pig breeds, emphasizing their role and providing a definition. However, it appears that the article does not explicitly define homozygosity (ROH) nor clearly differentiate the functional differences between these two concepts.

Authors decided to analyze heterozygosity because it is an aspect often treated superficially, and only as a small part of a more comprehensive study. Heterozygosity islands become an important topic to explore, especially in local breeds. ROHs are investigated in other papers as reported in L75, and for this reason, they are not analyzed again.

 

3. Is there an issue with the description of SNP chip data in line 73 of the article? Additionally, the descriptions of Whole Genome Sequencing data appear inconsistent between lines 74 and 75.

Modified L82.

4. Lines 95-101 of the article provide a detailed definition and explanation of the scenarios established. However, the rationale behind these definitions, the choice to include multiple scenarios, and the advantages of such an approach are not explicitly addressed.

The explanation has been addressed (L102-L107)

5. The article compares PCA results using complete SNP chip data and SNP data from HRRs, providing a thorough interpretation of the findings. However, the number of SNPs contained within the HRR islands is only 6,228. The article does not clearly explain the potential impact of this limited SNP count on the PCA results. For instance, could the observation in line 203, “The within-breed variability was extended in almost all the other pig breeds,” be attributed to the reduced dataset size?

A subset of SNPs was used to verify if heterozygous regions could be able to discriminate breeds better or equal to the entire SNP data. If the clustering is maintained, heterozygosity (as evolutionary force) could be considered a satisfactory discriminant factor.  A sentence has been added at L128.

6. The DISCUSSION section provides a comprehensive exploration of the study's results and their potential implications. However, in line 310, there seems to be a lack of explanation as to why a lower occurrence of HRRs affects the p-value.

The sentence has been removed from the manuscript.

7. The conclusion in lines 321-330 appears somewhat lacking in summarizing the study. The presentation of the key findings is neither sufficiently concise nor fully articulated.

The conclusion has been improved (L347-352)

Reviewer 2 Report

Comments and Suggestions for Authors

This study identified the Heterozygosity-Rich regions in 23 pig breeds and revealed the highest and lowest number of HRRs in the specific pig breeds. In addition, several biologically important genes including TRIM28 and CDC27 in the common HRRs were also detected. The results depicted the heterozygosity pattern in European local pig breeds, providing new insight into the genetic background related to population structure and breeding history of these pig breeds. Several issues need to be further addressed.

1. Whether figure 4 displayed all the genes found in HRRs island? If not, a supplementary table was recommended to list all gene information.
2. Many HRRs island and involved genes were identified in the pig breeds analyzed in this study. Whether these HRRs have similarities or differences with those identified in pig breeds from other continents. These can be further discussed.

Author Response

This study identified the Heterozygosity-Rich regions in 23 pig breeds and revealed the highest and lowest number of HRRs in the specific pig breeds. In addition, several biologically important genes including TRIM28 and CDC27 in the common HRRs were also detected. The results depicted the heterozygosity pattern in European local pig breeds, providing new insight into the genetic background related to population structure and breeding history of these pig breeds. Several issues need to be further addressed.

 

Whether figure 4 displayed all the genes found in HRRs island? If not, a supplementary table was recommended to list all gene information.

Figure 4 shows genes found in HRRs islands shared by at least 10 breeds. The complete list will be added as Supplementary Table 1.

Many HRRs island and involved genes were identified in the pig breeds analyzed in this study. Whether these HRRs have similarities or differences with those identified in pig breeds from other continents. These can be further discussed.

L302 and L305 have been added. Few studies have been found in bibliography using the same methodology to allow a comprehensive comparison.

Reviewer 3 Report

Comments and Suggestions for Authors

The data are provocative and raise interesting questions.  It would be helpful if there were more attention to mechanisms and function of the Conserved Polymorphic Sequences.  It would be expected that such heterozygosity would lead to recombination suppression and might include regulatory sequences resulting in quantitative phenotypic differences between the breeds.  The emphasis on genes might give the impression that the authors are only interested in coding regions and the number of genes.  Additional discussion of fig 3 might address the possibility that the distributions of HRR islands are rather similar although the extent of each is breed specific.  Why?  Do recombination rates differ?  

Is it possible the include a phylogenetic tree?

Is it possible to include Berkshires and some comment on meat quality and other commercial outcomes?

Author Response

The data are provocative and raise interesting questions.  It would be helpful if there were more attention to mechanisms and function of the Conserved Polymorphic Sequences.  It would be expected that such heterozygosity would lead to recombination suppression and might include regulatory sequences resulting in quantitative phenotypic differences between the breeds.  The emphasis on genes might give the impression that the authors are only interested in coding regions and the number of genes.  Additional discussion of fig 3 might address the possibility that the distributions of HRR islands are rather similar although the extent of each is breed specific.  Why?  Do recombination rates differ? 

The authors thank the reviewer for the point raised.  Relating the general HRRs similarities, Figure 6 answers the question. Figure 3 describes the extent of HRRs across chromosomes, which is related to the length of chromosomes and surely also to the recombination rate which is breed specific as reported by Brekke et al. (https://doi.org/10.1186/s12711-022-00723-9). Unfortunately, these insights were not evaluated in the study, so authors prefer to not formulate hypotheses.

 

Is it possible the include a phylogenetic tree?

The phylogenetic tree was previously reported by Poklukar et al. (doi: 10.1186/s12711-023-00858-3).

Is it possible to include Berkshires and some comment on meat quality and other commercial outcomes?

Berkshire samples were not collected during TREASURE project, and consequently, for the uniformity of sampling and SNP chip, we used the 23 breeds included in the project, without downloading other data from websites. This number of breeds allows us to perform a very comprehensive study on heterozygosity in the European context.