The Landscape of Genome-Wide and Gender-Specific Microsatellites in Indo-Pacific Humpback Dolphin and Potential Applications in Cetacean Resource Investigation
Round 1
Reviewer 1 Report
The manuscript by Ming et al. describes the distribution of microsatellite loci across the genome of Indo-Pacific humpback dolphin. The manuscript is well written, presents important new data and provides important resources for the scientific community. I only have a few minor comments:
- The manuscript misses an important reference: Srivastava, S., Avvaru, A. K., Sowpati, D. T., & Mishra, R. K. (2019). Patterns of microsatellite distribution across eukaryotic genomes. BMC Genomics, 20(1), 1-14. Please cite this study and compare the patterns you report for the species studied with the patterns reported there for a broad range of taxa.
- Line 116 – please be precise regarding what you mean by “the gene coding regions” – do you mean exons, entire genes (regions within open reading frames) or still something different? Also, it would be very interesting to see how those microsatellites that are not located within “the gene coding regions” are distributed across the genome. Is this distribution random, or are some particular genomic regions overrepresented?
- Line 205 – “was” is redundant
- Line 231 – should be either “microsatellite-containing genes” or “genes containing microsatellites”
- Line 341 – should be “ecologically important”
Author Response
Dear reviewer, thank you very much for your valuable suggestions. We make a reply point by point as follows:
Point 1: The manuscript misses an important reference: Srivastava, S., Avvaru, A. K., Sowpati, D. T., & Mishra, R. K. (2019). Patterns of microsatellite distribution across eukaryotic genomes. BMC Genomics, 20(1), 1-14. Please cite this study and compare the patterns you report for the species studied with the patterns reported there for a broad range of taxa.
Response 1: Thanks for your kind advice. The literature you recommended identified microsatellites and analyzed their genomic trends from protists to mammals. It is a systematic and conprehensive study that has good implications for our study. We have added this important reference in the progress summary of this manuscript. Lines 61-63 of the manuscript.
Point 2: Line 116 – please be precise regarding what you mean by “the gene coding regions” – do you mean exons, entire genes (regions within open reading frames) or still something different? Also, it would be very interesting to see how those microsatellites that are not located within “the gene coding regions” are distributed across the genome. Is this distribution random, or are some particular genomic regions overrepresented?
Response 2: The gene coding regions we counted in this study were exactly the exon regions. Our result showed that the most amount of markers distributed in non-coding regions of the genome. This trend was also found in other species, such as higher plants and vertebrates (Wang et al., 1994; Jurka and Pethiyagoda, 1995; Van Lith and Van Zutphen, 1996; Metzgar et al., 2000). SSR genomic distribution is nonrandom across coding and noncoding regions (Li et al., 2002). Due to the high microsatellite mutation rate, it is to be expected that coding regions have a low microsatellite density because if they do not these regions would be significantly altered, possibly leading to loss of functionality (Oliveira et al., 2006).
Reference:
Wang, Z., Weber, J.L., Zhong, G., and Tanksley, S. (1994). Survey of plant short tandem DNA repeats. Theoretical and applied genetics 88(1), 1-6.
Jurka, J., and Pethiyagoda, C. (1995). Simple repetitive DNA sequences from primates: compilation and analysis. Journal of molecular evolution 40(2), 120-126.
Van Lith, H., and Van Zutphen, L. (1996). Characterization of rabbit DNA micros extracted from the EMBL nucleotide sequence database. Animal genetics 27(6), 387-395.
Metzgar, D., Bytof, J., & Wills, C. (2000). Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome research, 10(1), 72-80.
Li, Y. C., Korol, A. B., Fahima, T., Beiles, A., & Nevo, E. (2002). Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Molecular ecology, 11(12), 2453-2465.
Oliveira, E. J., Pádua, J. G., Zucchi, M. I., Vencovsky, R., & Vieira, M. L. C. (2006). Origin, evolution and genome distribution of microsatellites. Genetics and Molecular Biology, 29(2), 294-307.
Point 3: Line 205 – “was” is redundant
Response 3: Thanks for the grammatical correction of this sentence. We have deleted “was” and revised the sentence here to “The total amount of microsatellites in cetaceans ranged from 70,000 to 120,000”. Line 201 of the manuscript.
Point 4: Line 231 – should be either “microsatellite-containing genes” or “genes containing microsatellites”
Response 4: We have revised the “microsatellites containing genes” to “genes containing microsatellites” in the manuscript.
Point 5: Line 341 – should be “ecologically important”
Response 5: We have revised the conclusion paragraph. The sentences were rephrased here. “Given that many marine mammals are species at risk of extinction, there is an urgent need to develop diverse conservation tools. The extensive and reliable genome-wide microsatellites information we identified in this study is expected to expand to other cetacean species and will play an important role in their population genetics studies and conservation”. Line 333-337 in the manuscript.
Reviewer 2 Report
In this manuscript, Ming et al have identified microsatellites markers through genome-wide mining for the species Sousa chinensis. Majority of markers identified are of dinucleotide motifs followed by tetranucleotide motifs, are associated with genes that are functionally enriched in methylation process. Further the authors claims that newly identified gender specific microsatellites markers would help in better conservation of species.
CRITIQUE:
A separate section as 'Discussion' in addition to already presented conclusion can be included. This would help readers to summarize all findings and connect to others work.
Author Response
Dear reviewer, thank you very much for your valuable suggestions. We make a reply point by point as follows:
Point 1: A separate section as 'Discussion' in addition to already presented conclusion can be included. This would help readers to summarize all findings and connect to others work.
Response 1: Thanks for the suggestion of including ‘Discussion’ as a separate section in the manuscript, which, as you mentioned, has its unique advantages. As we noticed that JMSE does’t have strict formatting requirements for manuscripts before our submission, we wrote the Results and Discussion sections together. This manuscript structure also has its advantages, such as readers can access our findings and understand the relevant discussion directly at the same time and in the same position, without having to search for content links.
Reviewer 3 Report
The maunscript is a technical paper that depicts the results of a study aimed at developing a latge panel of microsatelite merkes via NGS. In addition it offers also some interesting views on the distribution, composition and potential role of microsatellite loci across the genome of ceteceans. As a plus, it also shed a light for the first time on potential microsatellite loci linked to the Y chromosome of the investigated species. However, before being accepted for publication, the manuscript would benefit by a thorough revision of English language carried out by a native English speaker. Indeed, some parts are not very clear and may need to be rephrased to improve their clarity, Below, I have listed some non exhaustive examples of statemnets that vcan be improved.
Lines 72-78: in particular authors should check the use of articles and improve the meaning of this part
Lines 89-90: improve for clarity, please
Lines 110-111: rephrase for clarity, please
Lines 137-138: see the comment above
In addition, section 4 of the manuscript (Conclusions) would benefit from a rephrasing. For instance, what does mean the staements between lines 342-347? Authors should also consider that mining microsatellites does not mean they could also useful for population genetic studies. They need to be further tested.
Author Response
Dear reviewer, thanks very much for your valuable suggestions. We make a response point by point as follows:
Point 1: However, before being accepted for publication, the manuscript would benefit by a thorough revision of English language carried out by a native English speaker.
Response 1: Thanks for your suggestion. We have invited a native English speaker to help with the language editing of the manuscript. We have also carefully revised the manuscript in accordance with the suggestions of the reviewers and the editor, and we believe that with your help, the revised manuscript has been greatly improved.
Point 2: Lines 72-78: in particular authors should check the use of articles and improve the meaning of this part
Response 2: We thank you for your valuable suggestions. We have rephrased lines 72-78 to read
“However, currently few SSR markers have been reported for S. chinensis (Chen and Yang, 2008; Gui et al., 2013; Dai et al., 2021). Genome-wide characterization of microsatellites is imperative for better species identification and conservation research, particularly for the threatened S. chinensis. Identification and characterization of microsatellites, and thus the establishment of a database, will contribute to the study of genetic diversity and population structure of S. chinensis and other cetaceans”. Line 74-79 in the manuscript.
Point 3: Lines 89-90: improve for clarity, please
Response 3: Thanks for your suggestion. We have rephrased lines 89-90 to read “Besides, common features and composition trends of microsatellites among the cetacean species will be explored. It also has important implications for genetic diversity surveys and conservation of cetaceans”. Line 88-90 in the manuscript.
Point 4: Lines 110-111: rephrase for clarity, please
Response 4: We have modified the line 110-111 to “The motif types of microsatellites in eight cetaceans (S. chinensis, B. mysticetus, B. acutorostrata, O. orca, T. truncates, D. leucas, L. vexillifer and N. asiaeorientalis) include dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides. The number of microsatellites of each type was classified and counted respectively for each species”. Line 109-113 in the manucript.
Point 5: Lines 137-138: see the comment above
Response 5: We have modified the line 137-138 to “The 46 published SSR sequences of S. chinensis were derived from previous studies (Chen and Yang, 2008; Dai et al., 2021). These microsatellites have been identified via PCR amplification in S. chinensis. We aligned the microsatellites sequences to the S. chinensis genome sequence with an e-value < 1e-5 by BLAT (v. 36) (-t = dna, -q = dna, -out = blast8) (Kent, 2002). The location and motifs of the published markers were compared with our results, and the consistent markers were considered to be verified. Other markers were shorter SSRs, which were verified by searching SSRs with parameters: flank length of SSRs = 20 bp, length of SSRs ≥ 8 bp”. Line 130-137 in the manuscript.
Point 6: In addition, section 4 of the manuscript (Conclusions) would benefit from a rephrasing. For instance, what does mean the staements between lines 342-347? Authors should also consider that mining microsatellites does not mean they could also useful for population genetic studies. They need to be further tested.
Response 6: We agree with your suggestion. We have rephrased the conclusion section as follows: “We explored and identified useful microsatellite markers in S. chinensis from a genome-wide scale and designed primers for these markers. Comparison with the published marker sequences revealed that the markers developed in this study had high reliability. Microsatellites in S. chinensis genome were mainly distributed in the non-coding regions, and only approximately 0.5% were distributed in coding regions. Their associated genes presented potential methylation impacts in dolphins. Dinucleotide repeat motif type microsatellites were the most abundant, and this motif type was dominated by the AC-rich type. Cetacean species shared the same microsatellites composition characteristics, which possibly revealed the conserved nature of the cetacean genomes. The study also yielded polymorphic microsatellites data for male and female S. chinensis, which provided a valuable database for the investigation and demography of dolphin populations. Moreover, the male-specific marker mining provided useful candidate markers for sex identification of S. chinensis, as well as other cetacean species. Given that many marine mammals are species at risk of extinction, there is an urgent need to develop diverse conservation tools. The extensive and reliable genome-wide microsatellites information we identified in this study is expected to expand to other cetacean species and will play an important role in their population genetics studies and conservation”. Line 321-337 in the manuscript.
