A PCR-RFLP Method for Distinguishing Closely Related Common Quail (Coturnix coturnix) and Japanese Quail (Coturnix japonica): Forensics and Conservation Implications
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsA brief summary
Coturnix coturnix (Common quail) and Coturnix japonica (Japanese quail) are to high extent similar morphologically which poses challenges in species identification, leading to illegal trade of wild C. coturnix in the name of farmed C. japonica. This study investigated two approaches; 1) mined species specific short sequence repeats (SSRs) and 2) designed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay targeting the COX1 gene to distinguish these species. The SSRs proved unreliable in this study, the PCR-RFLP assay successfully distinguished between C. coturnix (Common quail) and C. japonica (Japanese quail) based on the unique BsaBI restriction site in the C. coturnix COX1 gene located at the 637th position, which is absent in C. japonica. This single nucleotide difference allows BsaBI to cleave the C. coturnix COX1 amplicon into two distinct fragments.
General concept comments
The generalization of the results is not good as one study investigated 2-8 samples from each species, does not confirm the intraspecific variation within each tested species. So, this study must declare study limitations (small number examined) because they could not generalize this data so further studies are needed on larger samples from same species. It must be correct in abstract and conclusion sections.
The introduction and knowledge gap were very clear and identified.
The material and method section was well-described. Figure 1 is very representative and illustrative to the readers.
The results and discussion were covered with smooth flow and well organized for the readers. In discussion, the author mentioned very important challenge too this method of investigation which in the form of hybridization of this 2 species.
The references were appropriate.
Specific comments
Page 3 line 96-97
The specimens identified to the species level in the field 96 using a field guide, Birds of the Indian subcontinent [25]. Please rewrite this sentence.
Page 3 Figure 3
If the authors did optimization, it must do for all tested species not one species only. Thus, figure 3B is repetition to part of figure 3A. To avoid any confusion to the readers, the author should delete figure 3B. The author should also delete the comment [(B) Under optimised conditions (7 units of BsaBI enzyme, 50 ng of PCR 242 product, and a 220-minute incubation at 60°C), restriction digestion of C. coturnix jqcqre_1 PCR am- plicon (samples 9-10) shows complete digestion of 850 bp amplicon into 637 bp and 213 bp fragments], because it is already mentioned in result section.
Comments on the Quality of English LanguageThere is few editing mistakes within the manuscript to be corrected.
Author Response
Response to Reviewer #1
Comment 1: General concept comments “The generalization … conclusion sections.”
Response: We are thankful to Reviewer #1 for your considerate review and praise of our work. We have added text in the Discussion part (line 321-327), about our study caveat following Dr. Jukka Jokimäki and your review. These additional lines aim to discourage generalizations and highlight the limitations of our study.
Comment 2: Specific comments “Page 3 … rewrite this sentence.”
Response: Sentence edited as advised (line 105-107).
Comment 3: Sp “Page 3 Figure 3. If the authors … in result section.”
Response: We respectfully insist to keep the figure as it is. The optimization was conducted on all samples (n = 10) regardless of species. Figure 3B serves a distinct purpose: since Japanese Quail samples show no digestion by BsaBI (as expected), Figure 3B specifically demonstrates the complete digestion pattern of Common Quail amplicons under optimized conditions. Figure 3A shows the comparative results across all samples, while Figure 3B provides clear documentation of the near complete restriction digestion with better clarity. Both panels are necessary to fully demonstrate the assay's functionality and diagnostic capability. The detailed caption ensures readers can interpret the results without extensive reference to the main text.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study presented has substantial implications for the management of the quail. The ability to discriminate quickly between Coturnix Coturnix and Coturnix Japonica is crucial. I believe it is very well written and presented exhaustively in all paragraphs. I suggest publication in the present form.
Author Response
Response to Reviewer #2
Comment: The study presented has substantial implications for the management of the quail. The ability to discriminate quickly between Coturnix Coturnix and Coturnix Japonica is crucial. I believe it is very well written and presented exhaustively in all paragraphs. I suggest publication in the present form.
Response: We are thankful to Reviewer #2 for such kind and considerate review.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors in this manuscript analyzed 2 specimens of common quails and 8 specimens of Japanese quails. They used mined species-specific SSR markers and also designed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay targeting the COX1 gene to distinguish the two species. They report that SSR markers failed to distinguish the two species whereas the PCR-RFLP provide a reliable, efficient, and accessible technique for it. The manuscript is well structured, clearly written and matches to the scope of the journal. There needs some improvements before further processing of the manuscript.
- Simple summary has use of several scientific terms which should have been avoided.
- The authors have used scientific names of the two research species throughout the manuscript. I suggest the authors to write the scientific names once in the main text and then use only the common names in the entire manuscript.
- There are inconsistencies in naming the common name. common vs. Common
- Keywords are too few and repeated from the title.
- Figure 1: At which position of the mitogenome is the mutation shown in the bottom panel of the figure B? It would be more informative to mention nucleotide position of this oligonucleotide chain presented here. In the top panel, 0 bp should be 1 bp.
- Subheadings 2.1, 2.2 are not written in proper format
- Table 1: Adding the position in mitogenome where these newly designed primer sequences align would give more information.
- Line 144: Was the annealing temperature used for all six pairs of primers the same- 63.8°C? And why was it run for 90S? Any specific reason for using such a long time for primer annealing? Detailed description would benefit future research.
I have concern to this because too long annealing time can have negative effects including nonspecific amplification, primer dimerization, false positive amplifications, etc. - Figure 3: I suggest the authors to use the symbol of approx for the values in the bands since they did not sequence those amplicons. ~850 bp, ~637 bp, .....
I have highlighted several other areas in the annotated PDF.
All the best!
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer #3
Comment 1: “The authors … the manuscript.”
Response: We are thankful to Reviewer #3 for the constructive remarks and support to the manuscript. We have revised the manuscript as advised.
Comment 2: “Simple summary … avoided”
Response: Revised as per suggestion.
Comment 3: “The authors … entire manuscript.”
Response: The scientific names of the species are mentioned only once; revision is done as per suggestion.
Comment 4: “There are .... Common”
Response: We apologize for the error; appropriate changes has been made.
Comment 5: “Keywords … the title”
Response: Additional keywords have been added.
Comment 6: “Figure 1: At which … be 1 bp.”
Response: We thank the reviewer for this suggestion. The nucleotide polymorphism highlighted in the bottom panel of Figure 1B is located at position 7200 in the Common Quail mitogenome (16,696 bp total length) and at position 7201 in the Japanese Quail mitogenome (16,698 bp total length). The oligonucleotide sequence shown in this panel spans position 7192-7201 in Common Quail and 7193-7202 in Japanese Quail. We have revised the top panel to correctly indicate the nucleotide numbering starting from position 1 bp rather than 0 bp.
Changes made, have been incorporated in Figure 1 caption (line 101 -106)
Comment 7: “Subheadings 2.1, 2.2 are not written in proper format”
Response: Subheadings have been edited in the revised manuscript as advised.
Comment 8: “Table 1: Adding … more information.”
Response: We agree. We have added positions on the respective mitogenomes where newly designed primer sequences would align have been mentioned in the revised manuscript (Table 1).
Comment 9: “Line 144: Was the annealing … amplifications, etc.”
Response: To standardize annealing temperatures, we conducted gradient PCR across a range of 52°C to 65°C, guided by the thermal profiles provided in the primer manufacturer's data sheets. All six primers (CQ1, CQ2, CQ3, JQ1, JQ2, and JQ3) demonstrated efficient amplification across multiple temperatures within this range. We selected 63.8°C as the optimal annealing temperature because all primers showed consistent, robust amplification at this temperature, and importantly, this temperature above 60°C minimizes the chances of non-specific binding. The 90-second annealing time was based on the modified manufacturer's protocol for the Qiagen Type-it Multiplex PCR Master Mix, which recommends extended annealing time of 90s. Similarly, for the jqcqre_1 primer set we used this extended annealing time of 90s. Such a method was adopted to ensure adequate primer binding and balanced amplification of all target loci. We acknowledge the reviewer's valid concerns about potential negative effects of prolonged annealing times. However, we did not observe evidence of non-specific amplification, primer dimerization, or false positives under these conditions, as confirmed by our gel electrophoresis results and the specificity of our downstream restriction digestion assay.
Respectfully, we have already mentioned the detailed methodology in the Methods section.
Comment 10: “I suggest the authors … amplicons. ~850 bp, ~637 bp, .....”
Response: Apologies for the error, we have added all amplicon sizes with “approx.” symbol throughout the manuscript for better accuracy.
Comment 11: “I have highlighted several other areas in the annotated PDF”
Response: All changes have been incorporated in the revised manuscript as advised in the PDF document.
