Amplification of Cherimoya (Annona cherimola Mill.) with Chloroplast-Specific Markers: Geographical Implications on Diversity and Dispersion Studies
, Gustavo Fontecha 3, Federico J. Albertazzi 4, Margarita Palmieri 5 and Jose I. Hormaza 1,*Round 1
Reviewer 1 Report
1）The raw sequencing figure to determine the nucleotide (Figure 2) is not clear.
2）It is better to show the two different haplotypes you found in a separate figure, which will clearly to display the genetic structure of Annona cherimola.
3）The evolutionary relationship of the samples collected from different locations also needed to be presented. This result will help to identify the core germplasms of this fruit tree for resource utilization and conservation in the future.
4）Using only chloroplast data, we cannot to infer the possible explanation of hybridization. The result presented in manuscript must been supported by data.
5）The language throughout the manuscript needs further smooth.
6）I think the title of this manuscript is not clear.
Author Response
We thank the reviewer for his/her thorough review that has helped to significantly improve the manuscript over our original submission. Here we detail the changes made in the new version in response to the reviewer's comments:
1）The raw sequencing figure to determine the nucleotide (Figure 2) is not clear.
This figure was included to show the mixed peak pattern in the trnLF gene of some samples, but we are deleting it in the new version due to this comment and the one made by the other reviewer. A new figure, providing phylogenetic relationships based on matK sequences, is included instead.
2）It is better to show the two different haplotypes you found in a separate figure, which will clearly to display the genetic structure of Annona cherimola.
We appreciate this comment but, since one of the haplotypes is only present in Central America, one of the maps would be mostly empty, so we think that it will be better to keep the map showing the two different haplotypes.
3）The evolutionary relationship of the samples collected from different locations also needed to be presented. This result will help to identify the core germplasms of this fruit tree for resource utilization and conservation in the future.
The cherimoya matK sequences obtained (20 + 3 of the previous article) have been used to construct a NJ tree (new Figure 3), but we did not sequence all samples used in this study. Nevertheless, a core collection for the germplasm bank of Malaga has already been stablished (Escribano et al 2007) and the Central American accessions characterized using SSR markers (Larranaga et al 2017). Effectively, this information, plus the one provided in the present work, could be used in the future to efficiently establish ex situ collections in Central American countries.
Escribano et al (2007) Establishment of a core collection to optimize the conservation of cherimoya (Annona cherimola Mill.) genetic resources using SSR information. Acta Horticulturae 814.
Larranaga, N.; Albertazzi, F.J.; Fontecha, G.; Palmeri, M.; Rainer, H.; van Zonneveld, M.; Hormaza, J.I. A Mesoamerican origin of cherimoya (Annona cherimola Mill.). Implications for the conservation of plant genetic resources. Mol. Ecol., 2017, 26, 4116–4130.
4）Using only chloroplast data, we cannot to infer the possible explanation of hybridization. The result presented in manuscript must been supported by data.
We have modified the sentences related to this topic following the recommendation of the reviewer and we have also eliminated the reference to hybridization in the abstract.
5）The language throughout the manuscript needs further smooth.
The English has been further revised along the manuscript
6）I think the title of this manuscript is not clear.
The tittle has been re-written as follows: “Amplification of cherimoya (Annona cherimola Mill.) with chloroplast-specific markers: geographical implications on diversity and dispersion studies”
Reviewer 2 Report
Dear Authors
I have carefully reviewed your manuscript, which shows good originality.
However, I have found several critical points that need to be resolved.
Please take note of my comments/suggestions in the attached file and respect them in order to improve the quality of your manuscript.
I wish you good work.
Comments for author File: 
Comments.pdf
Author Response
We would like to thank the reviewer for his/her thorough review that has helped to significantly improve the manuscript over our original submission. We detail below the changes made in response to the revierwer's comments:
118: Please specify whether sequencing was performed in both directions (forward and reverse). Please also specify whether the same primers used in nucleotide amplification were used for sequencing.
Sequencing was performed in the forward direction and the same primers were used for amplification and sequencing. This has been added in the text in the new version of the manuscript.
Figure 1. This is not a very good image to represent good DNA amplification. The bands go beyond the levels indicative of the Ladder used in the electrophoretic run. Please delete or replace it.
The bands beyond the ladder are, very likely, due to primer dimer (size and presence in the water sample). The band we are interested in is the one that appears next to the shorter ladder band (200pb) in the first column, showing that only Annonna cherimola DNA is amplified with this marker.
Figure 1. Please specify in the caption the length of the nucleotide amplifications obtained.
Done.
Figure 2. This is not a valid method for representing the existence of genetic polymorphisms in a species.
This figure was included to show the mixed peak pattern in the trnLF gene of some samples, but we are deleting it in the new version due to this comment and the one made by the other reviewer. A new figure, providing phylogenetic relationships based on matK sequences, is included instead.
Figure 3. This figure should be moved to the section 'Plant material' to indicate the geographical locations where the samples were collected. It makes no sense to indicate on a map the amplified samples from the non-amplified ones.
Since the figure does not include all the samples used in the study (only 411 out of 516 due to missing coordinates) we would rather prefer to maintain it in the results section to show the distribution of the samples that do amplify/no amplify with the matK based specific cherimoya marker.
- How many sequences were produced? Have they been deposited in the database? If not, deposit the sequences and report their accession numbers in the results. I suggest that the authors perform a phylogenetic analysis of the sequences produced, and highlight any genetic polymorphisms by constructing a phylogenetic tree.
In total 20 new sequences were produced. They were not included in a repository before, but, following the recommendation of the reviewer, they have been submitted to GenBank and the codes included in the new version. In addition, a NJ tree based on p-distance has been constructed to show the topology of the phylogenetic inference of this method.
- Did authors exclude the presence of pseudogenes (i.e. frame-shift mutations or stop-codons) through the translation in amino-acid of obtained sequences? I would suggest to check with the used MEGA software or other available tools;
Using the tool Open Reading Frame Finder of the NCBI (in the URL https://www.ncbi.nlm.nih.gov/orffinder/) to record the features of the sequences uploaded to GenBank, no pseudogenes were encountered.
- The discussion needs to be revised after substantial changes have been made to the results. I ask the authors to add a short Conclusion paragraph.
The discussion has been revised after the changes made in the other sections of the manuscript and a short conclusion paragraph added at the end of the manuscript.
Round 2
Reviewer 1 Report
The authors have addressed my previous comments and the revised manuscript is much improved. I appreciate the detailed responses of the authors and I have no further comments.
Reviewer 2 Report
Congratulations to the authors! I am pleased with the improvements to the manuscript.
