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The authors describe five new microsatellite loci for the Lusitanian toadfish developed from 454 sequencing.
Five variable loci seems fairly low, especially given the fact that the authors have 379 remaining loci to test. The loci exhibit few alleles, and as such, the probability of exclusion is likely relatively low. The authors should find more variable loci, especially if this marker set is going to be used for paternity analysis. I also encourage the authors to include exclusion probabilities for this marker set.
Sequences for each microsatellite locus should be deposited into Genbank.
Line 14: change “Although its widespread use” to “Despite their widespread use”
Line 17: change “close” to “closely” and delete “or when these are not applicable for the model species.”
Line 34: change “allow estimating” to “allow us to estimate”
Lines 50-51: “Finally, among 26 sequences comprising a microsatellites motif, 48 primer sets were designed.” This doesn’t make sense—how do you get 48 primer pairs from 26 sequences?
Table 2: I suggest deleting the “journal” column—readers can look that up in the literature cited.
See general comments
See general comments
See general comments
The development of microsatellite markers is laborious and expensive. So, description of new microsatellite markers, an important tool for genetic populational studies, brings a relevant contribution and deserves to be published. Especially for the biological model studied, a polygynous fish with complex male behavior. However, some considerations are important. The number of markers (five) and alleles (two to four) are extremely low. I believe that will be difficult to apply this set of markers for robust studies in population genetics, especially in parentage analysis, where is necessary to identify individuals (which require a large number of markers and/or alleles per marker). So, I have some questions and suggestions.
Regards the development of the library, some details can be better specified:
- In the Genoscreen protocols, they recommend the use of a DNA pool with samples from 8 – 12 individuals. You can specify the number of individuals used to make the pool.
- Some details are not clear in description of methodology. What was the total number of microsatellite sequences identified by 454-pyrosequencing? In table 2 you mention 427 identified loci, but is important include this information in methods.
Some aspects can be clarified about selection and validation of markers:
- What were the criteria used to select the microsatellite sequences to be tested? Did you make any consideration regarding multiplex PCR?
- Line 50. “Finally, among 26 sequences comprising a microsatellites motif, 48 primer sets were designed”. You obtained 427 microsatellite loci. In the table 2 you affirm have tested 48 loci. What means these 26 sequences in this sentence?
- Line 52. “For the validation step, a sub-group of 24 primers pairs was tested for amplification on seven DNA samples.” If you validated only 24 primers pairs, how did you tested 48 loci?
- Line 59. “Then, from 15 validated primers pairs, 12 microsatellite loci were selected for polymorphism study on seven DNA samples.” Why do not test polymorphism for the 15 validated primers pairs?
Another point is that you affirm:
- Line 94. “Nevertheless, the five polymorphic loci were sufficient to address the paternity of eggs (Amorim et al., unpublished data) and so to estimate the impact of sneaking in this population”. This affirmation is very important because is the only fact that legitimate your microsatellites set. It not seems possible to make paternity tests with five loci that are few polymorphic. So, to legitimate your markers I encourage you to present data on paternity tests in the manuscript.
Finally, you affirm that:
- Line 96. “Moreover, it seems that testing more loci does not necessarily imply a higher detection of polymorphic loci.” I disagree this affirmation. You expended money to create a good library with genomic technologies. Why not to test more markers to polymorphism? The discussion about other studies presented in the table 2 has an error that compromises your conclusions about number of loci polymorphic/ number of loci tested:
Line 102. “…the number of polymorphic loci identified in our study was higher than the mean value obtained for the considered studies (9%).” This is incorrect. According to the table 2, the mean ratio between the number of polymorphic loci and the number of loci tested (PL/TL) was 59%. Only one study obtained a ratio equal to yours (10%) and all the others were higher. Nine percent is the mean ratio between tested and identified loci.
So, my final consideration is that you need explore better the dataset obtained by next-generation sequencing and try to isolate more polymorphic markers that can be amplified in multiplex reactions and/or include the data on paternity analysis that demonstrates the applicability of your markers.
Furthermore, I have some minor observations:
- It is recommendable to deposit the sequences in GenBank
- For multiplex reactions, primers selected must be screened to hairpin and primer-dimer interactions of all primer pairs.
- Acknowledgements - Should not be used to acknowledge funders
Table 2 – It is necessary to review the values in the table. Almost one reference (Carvalho et al. 2011) is inconsistent, since they tried to amplify 23 loci and had success in 18 loci, all of them polymorphic.
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