DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms

Selamawit Ketema; Bizuayehu Tesfaye; Gemechu Keneni; Berhanu Amsalu Fenta; Ermias Assefa; Nicolas Greliche; Eunice Machuka; Nasser Yao

doi:10.1371/journal.pone.0239122

Peer Review History

Original SubmissionFebruary 12, 2020
12 Feb 2020 Author Response Attachments Attachment Submitted filename: Declaration_ SK.pdf https://doi.org/10.1371/journal.pone.0239122.r001
7 Apr 2020 Decision Letter - Tzen-Yuh Chiang, Editor PONE-D-19-32934 DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms PLOS ONE Dear Dr. Yao, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by May 22 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Tzen-Yuh Chiang Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-016-0712-9 In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Dear Editor and Author: In the article “DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms”, the authors evaluated the genetic diversity in 357 Ethiopian cowpea germplasm, using 6498 GBS based single nucleotide polymorphism (SNPs) markers. The population structure, genetic diversity, and phylogeny were analyzed in this research. Generally, this manuscript has a large amount of data, a clear idea, and a relatively intelligent data analysis. We can see the author's research ability and efforts. Personally, I like this article very much. However, I think this manuscript is not appropriate to be published right now, until the below problems are solved: 1. This manuscript is to describe the diversity of cowpea in Ethiopian, why the author added breeding line and varieties which is not collected from Ethiopian? I agree and recommend adding a certain amount of breeding lines for diversity analysis, however, the source of the materials does not associate with the main idea of the article, it will cause confusion in the analysis. If the author implied or thought those breeding lines were all from Ethiopia, why compare breeding lines with all Ethiopian landraces as two independent groups in Table 1 and 2? 2. How did the authors pick up the 314 landraces from whole germplasms? Are these materials representative of the entire germplasm pool? Theoretically, diversity analysis must collect all local landraces, but considering the research cost. The researchers will select some highly diverse materials from all the collections to represent the whole germplasm pool. Please clarify whether these materials have such qualifications. My suggestion is that if the 314 landraces are part of germplasm, the author should provide the phenotype of all germplasm resources and prove the representativeness of the selected material; if this material is the entire germplasm of Ethiopia, it is also desirable to conduct analysis and statistics on the phenotype of this material. 3. How did the authors screen the 6498 markers from the 20276 SNPs? The main problem of GBS is missing and false data, please clarify the quality control of genotype. In my suggestion, it is also better to prove the distribution of the 6498 SNPs in each chromosome, because of the character of GBS sequencing, the average distance of markers is too much different from the real situation. 4. Please re-make Fig 2, 5 and 6 to improve the resolution, if the authors could. Except for the flaw of the scientific part, my main concerns are: 1 The germplasm in this manuscript was only from one country, which makes it of limited reference value to researchers worldwide. Please elaborate on the implications of this study for researchers worldwide. 2 Overall, although the diversity analysis of this study was relatively integrated, I did not see any innovation, depth understanding, or thinking about diversity research from the conclusions and perspectives. To sum up, I am conservative and cautious about the publication of this manuscript immediately, unless the authors can find new research ideas or impressive conclusion with a more thorough discussion and understanding from current results. My suggestion is that, since the author has mentioned the establishment of a core collection, it is better to build the “core germplasm” for Ethiopia based on the existing data combining with the phenotype of whole the germplasm. This project is more important than an only diversity study, and its results are more valuable for reference over the world. Sincerely Reviewer #2: In this manuscript the authors explored the genetic diversity and population structure of Ethiopian cowpea using DArTSeq markers. However, the paper needs improvement. The authors forgot to include line numbers in the manuscript which make it difficult to mention typographical errors. A correction of the text is recommended. In the abstract replace 357 cowpea “germplasms” by 357 cowpea accessions In the introduction the 1st sentence needs correction, also there is a lack of consensus on where in Africa cowpea was domesticated. Authors should take that in account. Methods section: a brief description of the sequencing method is recommended. Authors should provide detailed information on the SNP calling approach, SNP filtering thresholds as well as the filtering of the accessions. Population structure analysis: I would recommend a membership coefficient of ≥ 0.70. Authors should also replace the figures with high resolution figures. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0239122.r002
Revision 1
14 Aug 2020 Author Response Rebuttal letter in response to the comments forwarded by the reviewers on manuscript number, PONE-D-19-32934 [DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms]. Dear Editor and Reviewers We are grateful for the reviewers who have been reviewing our manuscript during this COVID -19 pandemic time. We are also thankful for the valuable comments given to improve our manuscript. We would also like to seek apology for the delayed response for this paper, this was due to communication gap among the authors worsened by the pandemic situation and the internet restriction by the Ethiopian gouvernement. The reviewer’s comments have helped us to improve the manuscript and we have tried our best to address all the comments given by the reviewers. The changes made in the manuscript are shown in track in the manuscript. The response for each comment, along with the query of the reviewer is summarize as below. Sincerely, The Authors Revision notes Reviewer 1 Comments: Comment 1. This manuscript is to describe the diversity of cowpea in Ethiopian, why the author added breeding line and varieties which is not collected from Ethiopian? I agree and recommend adding a certain amount of breeding lines for diversity analysis, however, the source of the materials does not associate with the main idea of the article, it will cause confusion in the analysis. If the author implied or thought those breeding lines were all from Ethiopia, why compare breeding lines with all Ethiopian landraces as two independent groups in Table 1 and 2? Response 1.). One of our objectives is to see if the landraces collected from different regions of Ethiopia maintained and used by the local farmers are different from the cowpea genotypes collected from IITA or developed by breeding program in Ethiopia. The inclusion of the breeding lines is driven by the fact that the cowpea-breeding program, being dependent on the introduced cultivars imported and/or from the International Institute of Tropical Agriculture (IITA), would represent exotic/external germplasm outside Ethiopia. Our goal in comparing breeding lines/improved variety from IITA with Ethiopian landraces is to see the allelic richness of these two groups and hence develop a better germplasm management strategy and for use for breeding in Ethiopia and worldwide. In addition, to mitigate the bias that can be introduced by the difference in number of samples, we have compared the improved varieties with each of the subpopulation that constitute the whole population for landraces. Comment 2. How did the authors pick up the 314 landraces from whole germplasms? Are these materials representative of the entire germplasm pool? Theoretically, diversity analysis must collect all local landraces, but considering the research cost. The researchers will select some highly diverse materials from all the collections to represent the whole germplasm pool. Please clarify whether these materials have such qualifications. My suggestion is that if the 314 landraces are part of germplasm, the author should provide the phenotype of all germplasm resources and prove the representativeness of the selected material; if this material is the entire germplasm of Ethiopia, it is also desirable to conduct analysis and statistics on the phenotype of this material. Response 2. The 314 landraces represent the entire collection of cowpea germplasm in Ethiopia. From this study we planned to only phenotype the core collection extensively for several key and economically important traits to cut on cost usually associated with efficient broad genetic stock/germplasm characterization. Comment 3. How did the authors screen the 6498 markers from the 20276 SNPs? The main problem of GBS is missing and false data, please clarify the quality control of genotype. In my suggestion, it is also better to prove the distribution of the 6498 SNPs in each chromosome, because of the character of GBS sequencing, the average distance of markers is too much different from the real situation. Response 3. The comment is accepted, and one paragraph was inserted in material and methods under the section “SNPs calling and data filtering” regarding data filtering to respond to the request query. Data filtering conditions were included as follow: SNP markers with high proportion of missing data (>10%), individuals who have high rates of genotype missingness (>10%) and rare SNPs with <5% minor allele frequency (MAF) were discarded from further analysis using R software (version 2.8.3). The most informative SNPs were selected based on a threshold PIC value equal or higher than 0.2. Finally 357 cowpea genotypes and 6498 (32%) of SNP markers were maintained for further analysis. In addition, the distribution of SNPs per chromosomes was provided in S2 Table. The number of SNPs ranges from 463 on chromosome 2 to 849 on chromosome 3 with a mean of 591 SNPs per chromosome Comment 4. Please re-make Fig 2, 5 and 6 to improve the resolution, if the authors could Response 4. The comment is accepted, and new captures were done to improve the figure quality subsequently. The figures were also validated through the PLOS one PACE requirements. Comment 5. Except for the flaw of the scientific part, my main concerns are: The germplasm in this manuscript was only from one country, which makes it of limited reference value to researchers worldwide. Please elaborate on the implications of this study for researchers worldwide. Overall, although the diversity analysis of this study was relatively integrated, I did not see any innovation, depth understanding, or thinking about diversity research from the conclusions and perspectives. To sum up, I am conservative and cautious about the publication of this manuscript immediately, unless the authors can find new research ideas or impressive conclusion with a more thorough discussion and understanding from current results. My suggestion is that, since the author has mentioned the establishment of a core collection, it is better to build the “core germplasm” for Ethiopia based on the existing data combining with the phenotype of whole the germplasm. This project is more important than an only diversity study, and its results are more valuable for reference over the world. Response 5. The queries and suggestion of comment 5 has been addressed as follows and included in the conclusion to show the importance of the current study and its potential use worldwide � Although different reports indicated that East Africa including Ethiopia is one of the centers of origin, center of diversity or secondary center of diversity there was no cowpea collection neither any characterization to such extent in Ethiopia cowpea landraces. Though this collection was only done in Ethiopia, the result of this study has shown the existence of genetic diversity in the landraces and it expected that these germplasms might have unharnessed potential for future breeding owing different trait for cowpea improvement. Thus, these germplasms can be used globally for cowpea future breeding. Innovation, depth understanding, or thinking about diversity research from the conclusions and perspectives. � Worldwide, germplasm collection has been done for most crops for a long period, and it is assumed as we already had enough collection. However, this study could show, as we have not still collected the germplasm resource of the world. Thus, this study sheds light on how we could still have several germplasm in most crops that needs collection, preservation, and use for future breeding. In this study in the future, the collected germplasm will be thoroughly phenotype, and used for cowpea improvement for the target traits. A core set of diagnostic makers can be developed to serve worldwide purpose My suggestion is that, since the author has mentioned the establishment of a core collection, it is better to build the “core germplasm” for Ethiopia based on the existing data combining with the phenotype of whole the germplasm, • The suggestion for establishing core collection is well taken and included in the manuscript as shown in the introduction, material method, result, discussion and conclusion. However, we wouldn’t be able to phenotype the whole collection due to budget limitation, hence we will be focusing on phenotyping extensively the core collection for several traits Reviewer 2 Comments: Comment 1. The authors forgot to include line numbers in the manuscript, which make it difficult to mention typographical errors. A correction of the text is recommended. Response 1 Comment accepted and line numbers included Comment 2. In the abstract replace 357 cowpea “germplasms” by 357 cowpea accessions Response 2. Comment accepted and correction made according to the suggestion Comment 3. In the introduction, the first sentence needs correction; also, there is a lack of consensus on where in Africa cowpea was domesticated. Authors should take that in account. Response 3. Comment accepted and the sentence was rephrased as “Cowpea [Vigna unguiculata (L.) Walp., 2n = 2x = 22] originated and was domesticated in Africa though the exact location of origin of domestication is still a matter of speculation and different authors suggest different areas in Africa, Northeastern Africa including Ethiopia (1–4), Central Africa (5), South Africa (6), and West Africa (5,7,8). (Line 45 to line 48) Comment 4. Methods section: a brief description of the sequencing method is recommended. Response 4. A brief description of the GBS protocol as developed and implemented by DArT was added to the Material and Method under the section “DNA extraction and sequencing” Comment 5. Authors should provide detailed information on the SNP calling approach, SNP filtering thresholds as well as the filtering of the accessions. Response 5. One paragraph was inserted in Material and Methods section with respect to data filtering under a new title “SNP calling and data filtering” Comment 6. Population structure analysis: I would recommend a membership coefficient of ≥ 0.70. Response 6. The comment accepted and membership coefficient of ≥ 0.70 were used and the results were corrected accordingly. Comment 7. Authors should also replace the figures with high resolution figures. Response 7. The comment is accepted, and the figures have been replaced with figures with higher resolution quality. Response to reviewer comments, August 05 The reviewer comments are shown below in black and the response by the authors is provided as response to reviewers in italics Dear Dr Yao, Thank you for submitting your manuscript entitled "DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms" to PLOS ONE. Your manuscript files have been checked in-house but before we can proceed we need you to address the following issues: 1) Thank you for updating your data availability statement. You note that your data are available within the Supporting Information files, but no such files have been included with your submission. At this time we ask that you please upload your minimal data set as a Supporting Information file, or to a public repository such as Figshare or Dryad. Please also ensure that when you upload your file you include separate captions for your supplementary files at the end of your manuscript. As soon as you confirm the location of the data underlying your findings, we will be able to proceed with the review of your submission. Response 1 to reviewers: The genotyping data set was included as supporting information and a caption was added at the end of the manuscript under the title: S3: Genotyping supporting data obtained after filtering used for the study 2) Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. We note that you have included affiliation numbers 1-6 and , ¶ however only affiliations 1-6 and have authors linked to them. Please amend affiliation ¶ to link an author to it or remove if added in error. Response 2 to reviewers: The affiliation “¶” was added by mistake and was removed from the author list 3) Please include a copy of Table 4 which you refer to in your text on page 10. Response 3 to reviewers: Table 4 and Table 6 should read the same. The numbering as Table 6 came from the fact that S1 and S2 tables were previously included in the manuscript body as Table 4 and Table 5 before been moved as supplementary files. Then the actual Table 6 was amended as Table 4 in the table caption and in the text body. Attachments Attachment Submitted filename: Combined response to Reviewers_August 13.docx https://doi.org/10.1371/journal.pone.0239122.r003
1 Sep 2020 Decision Letter - Tzen-Yuh Chiang, Editor DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms PONE-D-19-32934R1 Dear Dr. Yao, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Tzen-Yuh Chiang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: Authors have addressed the comments. I recommend to italicize the scientific name of the species in the references section. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No https://doi.org/10.1371/journal.pone.0239122.r004
Formally Accepted
28 Sep 2020 Acceptance Letter - Tzen-Yuh Chiang, Editor PONE-D-19-32934R1 DArTSeq SNP-based markers revealed high genetic diversity and structured population in Ethiopian cowpea [Vigna unguiculata (L.) Walp] germplasms Dear Dr. Yao: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Tzen-Yuh Chiang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0239122.r005

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .