Identification of Growth-Related SNPs and Genes in the Genome of the Pearl Oyster (Pinctada fucata) Using GWAS
Round 1
Reviewer 1 Report
General comment
The manuscript presents a potential interesting study focusing on pearl oyster, an important aquaculture species that has been scarcely investigated so far. In particular, the study tries to uncover genetic markers, candidate genes and biological pathways associated with growth-related traits in pearl oyster.
The most important comments are the following:
1. It is not clear why authors have decided to use so many softwares; GCTA, for example, has been used just for PCA, but this software can compute also LD scores, estimate the genetic relationship matrix between individuals and perform GWAS analysis (in this study performed using three different softwares); again, if TASSEL is used for some reasons, I do not understand why the K matrix has been constructed with another software; the same for statistical analyses: it is declared that R has been used, but then SPSS is mentioned.
2. Introduction section should be expanded and better focused; please take into account the point-to-point comments below and try to provide a comprehensive background.
3. Please, avoid to repeat or report information belonging to the Materials and Methods section in the Results section.
3. I see a serious lack of consistency between Materials and Methods and Results sections. At the moment, some results are reported but not explained properly in the materials and methods. Moreover, GWAS is reported in the M&M as performed using TASSEL (L123 forward), but then in the results it is reported to be performed using GEMMA (L191).
4. In the Results section there is no mention of the annotation of the SNPs; I suggest to report also that information, in particular how many chromosomes the SNPs have been assigned, since it is important to understand GWAS results.
5. In the Results, I was expecting a detailed paragraph focusing on the detected candidate genes and pathways, but there is just a small and difficult-to-read figure, which has not been commented at all.
6. Discussion section should be completely revised, deepened and reorganized following a logical flow; at the moment, results are not properly discussed.
7. Pay attention to cite the relevant bibliographic reference; I refer in particular to the Materials and methods section, where several softwares are reported without reference (fastp, annovar, SPSS, …) or with a wrong reference (R software, which is R Core Team).
Point-to-point revision
L13-14: “to obtain more genetic information” does not sound good; the beginning of sentence should be rephrased
L17: is it “studied population” maybe?
L17-18: At this stage, the reader has no idea about the PCA analysis that has been performed. I suggest a re-organization of this part in the abstract
L32: please, clarify what “decreased farming” means
L28-38: From this first paragraph, I understand that growth-related traits are the most important traits for pearl oyster culture, but there is no mention of which traits are taken into account (I guess shell length/height/width?); please, expand this part. Furthermore, authors should clarify if growth-related traits are the final objectives of genetic selection, or whether those traits are used because associated with an increased production of pearls.
L46-47: What does “regarding the better growth performance” mean? Please rephrase and clarify
L49-50: Maybe “hindering the application of selective breeding”? “hindering the advancement of selective breeding”?
L53-54: please, be clear: GWAS exploits linkage disequilibrium between genetic markers and causative mutations, or tests genetic variants (=markers) to find those statistically associated with a specific trait
L56-57: high density SNP arrays
L64-65: this should be better clarified; also, several studies are now focusing on the improvement of genomic prediction accuracy for complex traits integrating functional information; this means that SNPs are pre-selected or prioritized on the basis of such information (gene expression, function, open chromatin regions)
L65-66: MAS is effective only when traits are controlled by one or few genes with huge effects, but complex traits are commonly polygenic; this should be expanded
L76-77: the sentence should be rephrased
L96: please clarify in the text what “Phred score” means
L110-112: this sentence seems not appropriate as it is; please, if authors want to maintain it, it should be rephrased, clearly explaining why the kinship matrix has been constructed in this way and proposing relevant references (thus avoiding “GEMMA manual explains that...”)
L112: acronyms should be defined at their first appearance in the text and not in the subtitle
L114: what are D’ and r2? Please add definitions to the text
L119: normally it is “mean values ± standard error”; please rephrase
L120: experimental groups are group L and S? It should be better clarified
L121: why ANOVA has been performed with SPSS? Above it is reported that the analyses were performed with R
L129: the model is weirdly written; it is a mixed linear model, so you can simply write it as Bradbury et al. (2007); please, revise this part
L137-140: I understood what authors mean, but the form is not optimal
L139: how the plots (Manhattan and Q-Q) have been built?
L141: the title of the sub-chapter is “Selection of candidate genes” but the paragraph just explains how resequencing have been performed and there is no mention of how candidate genes have been detected/analyzed, which, in turn, is reported in the Results section
L143: are they new individuals from the same lines? Please specify that
L149-151: these were not reported in the materials and methods section
L159: again, correlations were not reported in the materials and methods section. I guess they are Pearson correlations? What are the significances of the correlations?
L164: these are descriptive statistics
L178-179: this sentence is more appropriate for the materials and methods section
L179: shower?
L181-182: How can you say that? I see a clear overlapping and, in fact, in L183-185, authors contradict themselves reporting “a high degree of overlapping”
L191: I am confused: in the materials and methods is reported that GWAS was performed using TASSEL
L194: so all the chromosomes?
L197, L201, L203: what does “bate” mean?
Figure 3: the acronym of the trait should be placed in a better position
L208-210: this sentence is not appropriate for the Results section
L212-221: this is Materials and Methods
L233: also compared to aquaculture species (salmonids for example)
L238-240: this is not what has been done in the study
L240: the six growth traits should be defined
L243: GWAS results have not been discussed yet
L244-255: this is appropriate for the Introduction section
L279-288: no one of these genes have been reported in the Results section
The manuscript is generally adequately written, but the form can be improved further. In particular, please avoid contracted forms (country’s, L30; China’s, L31; rows’, L168…).
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Journal: Fishes (ISSN 2410-3888)
Manuscript ID: fishes-2355658
Type: Article
Title: Identification of growth-related SNPs and genes in the genome of the pearl oyster (Pinctada fucata) using GWAS
In this research, the authors have conducted whole genome resequencing of 60 pearl oyster individuals from two breeding lines, obtaining millions of SNPs and measuring six growth traits. This research is interesting, and the results obtained could be used to design genomic tools to implement in the improvement of breeding programs. Nevertheless, I have some questions about the methodology, and more information is required from the authors (from methodology to accessibility of raw data in an Open Access journal). I have found many typos that must be corrected. The pending issues that must be addressed before the manuscript can be considered for publication are listed in the attached pdf in two sections: (1) Content issues and (2) Formatting issues.
Comments for author File: Comments.pdf
I have recommended minor modifications about the English language.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
General comments
1) Authors have answered to my comment on the use of many different softwares saying that they always use this process in the lab. I totally understand that normally researchers try and test different softwares/protocols/…, but at the end a method has to be chosen and clearly explained in a scientific paper, without reporting all the tests performed (it is unnecessary) and reporting only the results obtained with the chosen method. In the specific case, reporting in Materials and Methods all the softwares you have tested, and then reporting results obtained using just one software, creates confusion to the readers and makes the manuscript not consistent. Authors also answered that they chose GCTA because of its advantages in performing PCA analysis, it is capable of computing LD scores, estimating the genetic relationship matrix between individuals, and performing GWAS analysis, but, in the manuscript, it is declared that GCTA is used just for PCA analysis; this remains very unclear to me. Again, I understand that testing different sowftares and protocols is a normal research process, but I suggest to choose the results you want to show, write them down, and then explain in Materials and Methods how did you obtain them.
2) I found again information belonging to the Materials and Methods section in the Results section (L184-185; L222-223; L233-234; L258-260). Please, remove them.
Point-by-point revision
L64: I would say “At present, selective breeding in aquaculture principally focuses on growth traits [15] and disease resistance [16].”
L67-73: these two sentences are not clear; please try to rephrase or specify better
L75-76: this is a sentence that should be included in the conclusions section; here it is not appropriate
L94: cannot be just said that the value is between 1.8 and 2?
L170-172: this is Materials and Methods, please do not provide background information here, just explain how analyses have been performed and to obtain what
L199-203: this is more appropriate for Discussion
L324-327: please revise these two sentences
L329-335: this is just a copy of the results, please avoid it and rather focus on their discussion
The form can be improved further. Please avoid to start the sentences with “And”; revise completely the manuscript and rephrase all those sentences using appropriate conjunctions.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 2 Report
Journal: Fishes (ISSN 2410-3888)
Manuscript ID: fishes-2355658
Type: Article
Title: Identification of growth-related SNPs and genes in the genome of the pearl oyster (Pinctada fucata) using GWAS
The authors have answered almost all the questions and issues in this new research version. The quality of the manuscript has improved. Nevertheless, I have found some typos and issues that must be solved (e.g., inconsistencies between the Material and Methods and Results sections). The main concern would be related to the material of Supplementary Table S2. I did BLAST with these SNP-flanking sequences at NCBI, and the chromosomes and positions were different with respect to those indicated in Table S2. The differences could be caused by using different reference genomes (i.e., Du et al. 2017 vs Takeuchi et al. 2022), having the last one (according to assembly statistics) the highest quality. This could also imply changes in the candidate SNPs’ functional annotation. I have attached the NCBI-BLAST results and the FASTA file I generated from Table S2. I recommend reviewing it carefully.
The pending issues that must be addressed before the manuscript can be published are listed in the attached PDF (within zip) in two sections: (1) Content issues and (2) Formatting issues.
Comments for author File: Comments.zip
Minor corrections were performed.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
The paper has been improved since the first version, but there are still some issues and it seems that the manuscript has been partially revised.
Comments:
L77: this is not exactly the aim of a GWAS, I suggest to revise this sentence trying to specify better what you would like to transmit
L82-84: are there any relevant references here? If not, remove this sentence
L123-124: what does it mean "citing the bibliographic reference"? Please find a better format
L157: for PopLDecay software the citation is missing
L164: Better "Statistical analyses" or "Descriptive statistics", but anyway please try to standardize the titles of Materials and Methods and Results
L165: R is not just a statistical software. I suggest to remove this from here and then specify that you used the R package stats
L196: for MATLAB the citation is missing
L246: I suggest "the correlation, measured as the Pearson correlation coefficient,..."
L253: descriptive statistics, not statistical details
L256, L281-282, L306-308: Please find a better caption
L323: maybe "drastically" rather than precipitously
I suggest again moderate editing of English language, as some sentences are very bad or wrong; again, I found some sentences starting with "And" (L174) and also abbreviations (L257). Some examples:
- L14-15: the sentence has to be rephrased
- L63: "to" followed by "identifying" is grammatically wrong
- L174 the sentence is starting with "And". Please, avoid that and revise carefully the manuscript to remove any similar sentences.
- L216: "calculating formula"?
- L221-222: Is the sentence complete?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Journal: Fishes (ISSN 2410-3888)
Manuscript ID: fishes-2355658
Type: Article
Title: Identification of growth-related SNPs and genes in the genome of the pearl oyster (Pinctada fucata) using GWAS
The authors have answered almost all the questions and issues in this research version. The quality of the manuscript has improved adequately. The pending issues that must be addressed before the manuscript can be published are listed below:
Lines 123-124: Please remove "citing the bibliographic reference".
Line 137: Please change "And only" to "Uniquely".
Line 198: Please change "The related" to "The relationship".
Line 266: Please change "sequencing depth for each sample and SNP was 9.5 and 6.5" to "sequencing depth for each sample and SNP was 9.5x and 6.5x, respectively".
Line 267: Remove "And".
Line 268: Please change "8.2 G± 0.6 G" to "8.2 Gb ± 0.6 Gb"
Line 282: "P. fucata" must be in italics.
Line 378: Please always add the bibliographic citation between square brackets. For instance, please change "Brondum and his associates" to "Brondum and associates [15]".
Line 383: Please change "Bentley" to "Bentley [39]".
Lines 501-503: Again, please change "Date Availability Statement".
Line 619: "Pinctada fucata martensii" must be in italics.
Line 621: "Crassostrea virginica" must be in italics. Please review carefully the References format.
Minor modifications were suggested.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
