Development of Polymorphic Microsatellite Markers and Identification of Applications for Wild Walnut (Juglans regia L.) in Middle Asia
Round 1
Reviewer 1 Report
The submission “diversity-2589289” mined SSR loci with 1-6 nucleotide repeats in the chromosomal sequence of walnut genome using published whole-base sequencing of walnut, the SSR data analysis results show that the conservation of wild walnut germplasm in Xinjiang urgently needs to be supplemented by walnut materials from other Central Asian countries. The results showed that the 22 SSR markers contribute to the subsequent selection of core germplasm and conservation of genetic diversity of wild walnuts in Central Asia. The manuscript has important scientific significance. According the current edit format and the effort need to be concentrated for the major result points, I suggest the deline for the current version of the manuscript. The revised version can be resubmit if it can meet the journal publication standards. The major modification points are list here, the detail version please see the revised version.
1 Abstract revision please see the attachment version;
2 The introduction revision points please see the attachment version;
3 Title “walnut” needs to be “Walnut”. In the main text section, abbreviation or the meaning of “chr” need sto be presented in the first presence location;
4 Some lines are faint in Figure 3, please set the same width for all lines in figure 3;
5 The words need to be enlargen to readable for figure 4, figure title should be put intothe below line of the picture for each figure;
6 Some lines are faint in Figure 3, please set the same width for all lines.
Considerable detail revision works need to be done based on the draft version of the submission.
Comments for author File: 
Comments.pdf
Considerable detail revision works need to be done based on the draft version of the submission.
Author Response
Review Report 1
Dear Reviewer:
We greatly appreciate your useful comments and have responded to each of them as follows:
1 Abstract revision please see the attachment version
An: We have revised the abstract based on the comments you gave. See them in Line 13-28.
2 The introduction revision points please see the attachment version
An: We have modified the introduction as per the pdf version. See them in Line 30-99, p1-p3.
3 Title "walnut" needs to be "Walnut". In the main text section, abbreviation or the meaning of “chr" needs to be presented in the first presence location
An: We've revised the article title section. See them in Line 3.
4 Some lines are faint in Figure 3, please set the same width for all lines in figure 3
An: The picture has been redrawn to show the lines, labeling clearly. See them in Line 297, p9.
5 The words need to be enlarged to readable for figure 4, figure title should be put into the below line of the picture for each figure
An: We have image formatting all changed to put the title below the image. See them in Line 300, p10.
6 Some lines are faint in Figure 3, please set the same width for all lines
An: We've bolded the lines, markers for all of the images to make them visible. See them in Line 297, p9.
Finally, thank you very much for your detailed revision of the manuscript, which has enhanced the quality of this manuscript.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments for authors of the paper: Development of Polymorphic Microsatellite Markers and identification of applications for Wild walnut (Juglans regia L.) in Middle Asia
The work seems interesting but I recommend a more accurate English revision. A map of the sampling is needed and try to use STRUCTURE genetic analysis tool for population analysis, free software package, and at least in the supplementary materials add a table of the 48 multilocus genotype obtained.
Seems there are 3 and not only one corresponding author.
Abstract Please emphasize the results obtained and on the other hand, reduce details on the analysis.
Line 24 Ili, China is written differently from in the rest of the text, anyway I would just say three country so China is enough
Introduction. Line 36 the Amercias, remove the, you mean American continent or the United States, please rephrase.
DNA extraction
Line 121-122 countries, including 23 wild walnuts (Y) from Yili, Xinjiang, China, 14 wild walnuts (D) from Tajikistan, and 9 wild walnuts (J) from Kyrgyzstan (Table 1.). Add locality and not only country for the three sampling areas
Table 1 the geographic coordinates refer to 3 different single points of sampling? A Map would be preferred. It is not clear if all the 46 samples were close in the 3 points or if you collected samples widespread in the 3 countries.
Data collection and analysis
You should also add and consider also Probability of identity (PID) and Probability of identity in siblings (PIDsib). In order to verify and validate the power of the new marker panel designed.
Figure 1. Number of SSRs of different lengths – Line 190
Please add all the length also if the number of SSR identified was 0, In the x asses 17, 19, 23, 25, 29 are missing. Or at least explain it.
Development and characterization of SSR markers: Please adjust and make the entire chapter less confusing. You use erroneous words as individual instead of alleles.
Please specify how you evaluate False Allele (FA) Null Allele (NA), the number of mismatches between all the samples, and if all the samples collected have a unique multilocus genotype?
Line 194 – 195 Better clarify the detection of 316 individuals, do you mean alleles, from 48 material. In the entire chapter. With an average of 14.36 loci, again you mean alleles, per SSR marker.
Line 196-198 The number of individuals sampled (Na) Na is the number of alleles and N is the dimension of the sampled population, please utilize the correct words.
Table 3 - Line 213
The table is not easy to read, if you call each primer F and R, could be more readable WJR001F line 1 and WJR00R line 2
Table 4 – Why you do not utilize SE standard error or confidence interval for the values presented?
Line 215 Na = number of individuals sampled – ALLELES
Line 219
Add STRUCTURE analysis and bar plot as a figure as is the most widely used clustering software to detect population genetic structure.
Figure 3 A better resolution is needed. Line 261
Figure 3 Line 262 - change the number because is Fig 4
Use an ellipse in green, red and blue respectively to surround each of the 3 groups.
Line 294-295 Every time you cite other study add et al. if there are several authors.
Line 314-316 but the clustering diagram in the results sauce 8 points results into two groups, in the results of PCoA, I guess you mean Shows? Please rephrase all the sentences avoiding repetition and make it clear
Discussion and Conclusion should be implemented and a better comparison with other studies on walnut or other plant species would help to understand the novelty of the study.
Line 346 It was possible to use these 22 primer pairs to … Analizing these 22 loci it was possible to obtain individual multilocus genotype …
I recommend a more accurate English revision.
Author Response
Review Report 2
Dear Reviewer:
Abstract Please emphasize the results obtained and on the other hand, reduce details on the analysis
Line 24 lli, China is written differently from in the rest of the text, anyway I would just say three countries so China is enough
An: We have removed Ili. See them in Line 24
Introduction
Line 36 the Amercia, remove the, you mean American continent or the United States, please rephrase.
An: This was an error in the use of language words and we have corrected this grammatical issue. See them in Line 36.
DNA extraction
Line 121-122 countries, including 23 wild walnuts (Y) from Yili, Xinjiang, China, 14 wild walnuts(D) from Tajikistan, and 9 wild walnuts (J) from Kyrgyzstan (Table 1.). Add locality and not only country for the three sampling areas
An: You raised a question about the source of the material and we have added the details of the location. See them in Line 117-119.
Table 1 the geographic coordinates refer to 3 different single points of sampling? A Map would be preferred. lt is not clear if all the 46 samples were close in the 3 points or if you collected samples widespread in the 3 countries.
An: We agree with the comments you made, which helped to clarify the source of the samples, so a map of the sampling was drawn. See them in Line 148, p4.
Data collection and analysis
You should also add and consider also Probability of identity (PID) and Probability of identity in siblings (PID sib). In order to verify and validate the power of the new marker panel designed
An: The PID, PID sib, information, which helps in the examination of individual walnuts, we have added this information. See them in Line 227-231, p6.
Figure 1. Number of SSRs of different lengths - Line 190
Please add all the length also if the number of SSR identified was 0, In the x asses 17, 19, 23, 25, 29 are missing. Or at least explain it.
An: Thanks for pointing out the deficiencies in the chart. The figure is about missing values for 17,19,23,25,29, which are statistically zero. We have redrawn the picture. See them in Line 207, p5.
Development and characterization of SSR markers:
Please adjust and make the entire chapter less confusing. You use erroneous words as individual instead of alleles.
An: We have corrected the grammatical errors in this section. See them in Line 207, p5.
Please specify how you evaluate False Allele (FA) Null Allele (NA), the number of mismatches between all the samples, and if all the samples collected have a unique multilocus genotype?
An: We were looking for SSR loci that could be split out by their geographic distance, so there was no need not to judge FA and NA. Although the collection was of wild walnuts, given that there is little morphological difference between wild and cultivated walnuts nowadays, it is possible that the wild walnuts could be wild cultivated walnuts so there was no need to look for the unique multilocus genotype.
Line 194 - 195 Better clarify the detection of 316 individuals, do you mean alleles, from 48material. In the entire chapter, with an average of 14.36 loci, again you mean alleles, per SSR marker.
An: We have revised the presentation of this section. See them in Line 211, p8.
Line 196-198 The number of individuals sampled (Na) Na is the number of alleles and N is the dimension of the sampled population, please utilize the correct words
Table 3 - Line 213
An: We have revised the presentation of this section. See them in 234, p8.
The table is not easy to read, if you call each primer F and R, could be more readable WJR001Fline 1 and WJR00R line 2
An: We have modified the format of the table to make it easier to read, based on your comments. See them in Line 232, p6-7.
Table 4 - Why you do not utilize SE standard error or confidence interval for the values presented?
An: We have written the data in the table, not written below the labeling. See them in Line 257, p8.
Line 215 Na = number of individuals sampled – ALLELES
An: This was a grammatical error, and we have corrected all of the areas that had this word error. See them in Line 257, p8.
Line 219
Add STRUCTURE analysis and bar plot as a figure as is the most widely used clustering software to detect population genetic structure.
An: Thank you very much for your suggestion to add to the content of the article, we will put the graphs for plotting the STRUCTURE analysis in the text. See them in Line 259-263, p8.
Figure 3 A better resolution is needed. Line 261
An: We changed the picture line charts to a readable size. See them in Line 287, p9.
Figure 3 Line 262 - change the number because is Fig 4
An: This was a typo and we have corrected the typo. See them in Line 291, p10.
Use an ellipse in green, red and blue respectively to surround each of the 3 groups
An: We have shown the different groups in the picture with different colored circles. See them in Line 290, p10.
Line 294-295 Every time you cite other study add et al. if there are several authors
An: This is a grammatical error and we have corrected all the places where we need to use et al. See them in Line 316-319, p10.
Line 314-316 but the clustering diagram in the results sauce 8 points results into two groups, in the results of PCoA, I guess you mean Shows? Please rephrase all the sentences avoiding repetition and make it clear
An: We fixed the grammar problem. See them in Line 334-347, p10.
Discussion and Conclusion should be implemented and a better comparison with other studies on walnut or other plant species would help to understand the novelty of the study.
Line 346 lt was possible to use these 22 primer pairs to ... Analizing these 22 loci it was possible to obtain individual multilocus genotype ...
An: We have added and modified this part. See them in Line 306, p11 and Line 370, p11.
Author Response File: Author Response.pdf
Reviewer 3 Report
In this study, 22 effective primer pairs were designed and screened to analyze the genetic diversity of 48 wild walnut samples from three countries. Ne was 5.17, PIC was 0.71, He was 0.52, while Fst was 0.09. Principal coordinate analysis that all samples were divided into three groups. Did You investigate that how the genotypes fit into the three clusters? Can some pedigree analysis be carried out using these data? For me, the PCA shows two groups, with two subgroups in one of them. Can some of the markers be used for identifying the genotypes?
The graphs are sufficiently detailed and easy to expound. The text contains occasional typos, but it is nevertheless easy to read. The literature used is enough. Figure 3 and 4 takes too much space, they should be in the additional materials.
In overall, the manuscript is complete, well written, scientifically accurate, and on a very interesting and important topic.
Author Response
Review Report 3
Dear Reviewer:
Thanks to the reviewers for their interesting questions, I will answer the following.
Did You investigate that how the genotypes fit into the three clusters?
An: The three genotypes are represented in the results in Figures 3, 4, and 5 to show how they have adapted, and the grouping in the results is a kind of demonstration of how genotypes have adapted to the group. If more details about adaptation are wanted, we have to take the approach that gives us more information, such as sanger sequencing. See them in Line 348-357, p11.
Can some pedigree analysis be carried out using these data?
An: SSR is capable of some lineage analysis, and the wild walnut collected in this study did not differ much from the cultivated walnut, so it is possible that the wild walnut is a wild species of the cultivated walnut. We'll add more about this part to the discussion.
For me, the PCA shows two groups, with two subgroups in one of them.
An: Regarding this result we need to look at Figure 4 and Figure 5 jointly together. Figure 5 shows the horizontal coordinate of the PCA plot divides it into two groups, but the vertical coordinate also divides the rest into two groups, which can only mean that these two groups are more closely related. It is better visualised in Figure 4.
Can some of the markers be used for identifying the genotypes?
An: It is possible for SSR to identify some of these specific genotypes, but this study, with the primary goal of distinguishing wild walnuts from different regions, did not need to include this. We are already adding this piece in the discussion section. See them in Line 348-357, p11.
Figure 3 and 4 takes too much space they should be in the additional materials.
An: We redraw the figure to be more suitable for reading in size and clarity.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Dear Editor,
Current version has been improved much. Except for minor revision in the attachment, suggest accept after minor revision.
Comments for author File: Comments.pdf
Dear Editor,
Current version has been improved much. Except for minor revision in the attachment, suggest accept after minor revision.
Author Response
Dear Reviewer:
Current version has been improved much. Except for minor revision in the attachment, suggest accept after minor revision.
An: Thank you for correcting the details in the article, we have corrected the formatting of the abstract discussion and references as per the attached article revision details.
Author Response File: Author Response.pdf
Reviewer 2 Report
Dear autors,
Despite after revisions several part of the manuscript are improved, still the paper is still not suitable for publication.
Line 198 of 14.36 ALLELES per SSR marker. (Table 4).
For STRUCTURE I recommend 100.000 iterations, 5 replicates and 30.000 burn-in You need to show Delta K plot, to demonstrate the best K value supported, once riches the plateau of the curve.
The multilocus genotypes table is still missing, and is necessary to calculate and add False Allele, Null Allele, mismatch etc.
The Genemapper plots are not needed but I see you do not use panel and bin set for automatic corrections.
Several peak are in overflow higher than 10.000, and in many case there are to many peaks or bad electropherograms … so the correction seems arbitrary and not that robust.
Some additional questions: the analysis and sequences are performed in house directly from the autors or they sent samples to an external laboratory service? In case specify. Are there any replicates to confirm results? Are utilized positive and negative controls in each step?
I recommend major revisions.
Can be improved.
Author Response
Dear Reviewer:
Thank you very much for reading our article so carefully and giving a lot of constructive comments and suggestions that will effectively improve the quality of this post! In order to ensure the accuracy of the results, in the experimental steps, we first selected the results with clear bands, then we used fluorescence capillary electrophoresis to get peaks, and finally we selected the appropriate results for analysis. Also using the advice you provided about graphing using structure software, we got similar results to dendrograms and PCA.Due to the specificity of the material selected for this paper, walnuts being an important nut food in Central Asia, and the fact that the walnut material in this experiment was not grown under purely natural conditions and was also subjected to anthropogenic interference, the boundaries of the division of taxonomic units in the results of the experiment are not clear (Figure 3, Figure 4, Figure 5).
Answer to the reviewer’s suggestion
- Line 198 of 14.36 ALLELES per SSR marker. (Table 4).
An: We have corrected the word. See them in Line 212, p5
- For STRUCTURE I recommend 100.000 iterations, 5 replicates and 30.000 burn-in You needto show Delta K plot, to demonstrate the best K value supported, once riches the plateau of the curve.
An: We have plotted line graphs and illustrated the results in the text. We would like to put the results in a supplementary chart (Supplement figure 1), do you think it is appropriate to display it as a supplementary chart? See them in Line 259-260, p8.
- The multilocus genotypes table is still missing, and is necessary to calculate and add False Allele, Null Allele,mismatch etc.
An: These values are reflected in Table 5. The calculation of the values is added to Table 4. See them in Line 232, p7.
- The Genemapper plots are not needed but I see you do not use panel and bin set for automatic corrections.
An: We have removed the relevant annexes in response to your comments (FAM1-FAM13).
- Several peak are in overflow higher than 10.000, and in many case there are to many peaks or bad electropherograms … so the correction seems arbitrary and not that robust.
An: We performed fluorescence capillary electrophoresis experiments based on previous results with clear bands done in the lab. The results of this part of the experiment are the results of all experiments of fluorescence capillary electrophoresis experiments, including unsuccessful experiments and duplicate groups. Further filtering on this basis is also required.
- Some additional questions: the analysis and sequences are performed in house directly from the autors or they sent samples to an external laboratory service? In case specify. Are there any replicates to confirm results? Are utilized positive and negative controls in each step?
An: Most of the experimental steps were done in their own experiments, except for the fluorescence capillary electrophoresis experimental step. Firstly, we first screened the suitable primers in the laboratory and chose the primers with clear results and obvious bands. Repeat the experiment using the result of the previous step, and then carry out the fluorescence capillary electrophoresis experiment, and then according to the capillary electrophoresis result then screen the primers with reliable results.
Author Response File: Author Response.pdf
