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Basically, the manuscript has been accepted. However, some minor revisions are still necessary. Please address these during the proof stage.
[# PeerJ Staff Note - this decision was reviewed and approved by Gerard Lazo, a PeerJ Section Editor covering this Section #]
This is a revised and corrected manuscript dealing with the analysis of MYB transcription factors in two orchid species: D. officinale and P. aphrodite. In this version the authors attended the corrections, comments and observations made previously by the reviewers. English language used throughout is clear and professional now. Literature references were prepared according to the Instructions for Authors guide and are sufficient. This article was prepared properly with professional structure, figures, tables and suppemental material, and contained relevant results linked to the hypothesis.
Original primary research within Aims and Scope of the journal.
The research question was well defined, relevant for people in the orchids area and stated how the research fills knowledge gap.
Rigorous research performed properly with ethical standard.
Methods were described clearly and with sufficient detail to be replicated.
The findings are novel and original and contribute to the knowledge of the MYB family in orchids.
All underlying data have bee provided; they are robust, statistically sound and controlled.
Conclusions were well stated, linked to original research question.
No furtehr comments.
I had a look at the revised manuscript again and found that the authors have solved almost all my previous concerns. Thus, I’d recommend an acceptance of it provided that the authors correct the follow tiny errors:
Fig 4 legend: based->based on
Fig 5 legend: qRT-PCR validation of R2R3-MYB transcription factors-> qRT-PCR validation of R2R3-MYB transcription factor genes; The average data was-> The average data were
Figs 4&5: gene names should be spelled in italics
I’d suggest that authors check the whole text again carefully to minimize or avoid the above kinds of errors as I do not have time to point them out one-by-one.
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Comments to the Author (s):
All of my last recommendations have been well addressed. I think that this new revision can be accepted for publication now. But, there are stilll several minor problems should be addressed before it is publicated.
Hai Du
Minor comments:
1. Althought the English language of the ms has been greatly improved in the last revision by the authors, there still are some language problems in the text (such as “subfamily” in line 24 in “Abstract” should be subfamlies). So, the writing needs to be thoroughly improved by a language company.
2. To avoid confusion, the gene names in Figure 5 should be changed into “BoMYBxx”.
3. In my opinion, the letters A-T in Figure 6 and 7, and their related contents in the figure legends are not necessay, as the gene names have been marked in the figures.
Thought the authors made some improvement to the manuscript. Several important concerns from reviewers were not responded to. Please carefully read each point of the reviewer's comments and substantially revise the manuscript accordingly, especially the introduction, results and discussion sections.
I have a look at the revised manuscript, and found out that though the authors have solved most of my concerns or questions, there are still a few waiting to be solved or touched up.
For example, the title is not right, add “genes” after “factor”, since only DNA or RNA sequences were surveyed and analyzed in this manuscript. No protein work has been done at all.
Abstract, line 21, several typos or errors are identified.
Line 23: stresses->stress
Line 26: were responsible for->responded to
Still, a few grammatical errors or errors still exist in the whole text including legends.
Figs 7&8 are still obscure with too small font size, the same with the two in the Word file of responses to reviewing comments. The authors should understand that under 100% view, all figures or tables should be recognized and readable (though some figures in some other journals are also very hard to read still; I dislike those very much). At least a higher resolution figure should be provided in the supplemental data.
It is not acceptable to set up only three technical replicates for qRT-PCR assays (and many other assays, if there are). This mean that sample preparation, RNA extraction, removal of contaminated genomics DNA, RT and follow-up qPCR should be repeated three time independently, to constitute three biological replicates. Many other papers published in many journals did not abide by this simple rule either and I doubt their conclusions and relevant data.
see the above text
see the above text
see the above text
There still are a lot of problems in the full text (even some serious problems), and some of my last comments are not well addressed.
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Comments to the Author (s):
As mentioned in my last comments, there were many problems in the manuscript, especailly some serious problems. Some of my last recommendations have been addressed by the authors, but some problems have not been well addressed, and there still are many problems in the full text. In fact, the authors focus on answering/explaining my comments point by point, instead of paying attention to the corresponding scientific issues and doing some revision in the text. Some major points listed below does not convince me that it meets the general criterion for publication in this journal.
Major comments:
1. There are a lot of format error in the whole text, even in the “Abstract” (such as line 15, 21, etc). And there are many blank spaces and lines in the text, such as line 41, 111, 178-179, etc. The authors are very careless.
2. Last time, I have suggested that “Many mistakes in English style and grammar can be found throughout the manuscript. There are many typos and some inaccuracies that should be corrected”. The writing needs to be thoroughly checked and substantially improved by English language editing company.
3. The authors defined “DoR2R3-MYB” and “PaR2R3-MYB” in “Abstract” to distinguished them from the other types of MYB super family (1R-MYB, 3R-MYB, and 4R-MYB). However, the gene names are still BoMYBxx (such as line 26 in “Abstract”). As we know, the R2R3-MYB genes are commonly named as MYB gene. If the authors want to keep this nomenclature, it is better to define it more clearly to avoid confuse, and unify the style in the whole text.
4. Last time, I have suggested that “it is inconceivable to use the MEGA5.2 software with default parameters to construct the phylogenetic tree. What is the detail information of default parameters?”. However, the authors did not understand my mean. For the phylogenetic analysis by MEGA software, the important parameters include “Tet of Phylogeny”, “Model/Method”, and “Gaps/Missing data treatment” which may impact the results directly, thus, should be clearly stated. Moreover, the NJ tree was constructed based on the full length or only the BD domains of candidate protens? It is unclear too.
5. For the “Intron-exon structure and domain analysis of R2R3-MYB genes from D. officinale and P. aphrodite” section in line 95-99, the authors should clearly state the sequences that were used in these two analyses. As we know, the MYB TFs are generally consisted of a N-terminal BD domain and a C-terminal AD domain. The BD domain is highly conserved across this gene family, thus the intron pattern is generally conserved in this region while it is not conserved in the AD region. For the MEME analysis, it is better to use the AD region, as the BD domain is actually conserved.
6. Line 132, the authors still did not state why and how they seleted the 20 genes for qRT-PCR analysis. The authors should let the potential readers know it, instead of just explain to me.
7. The method used to identify the segmental duplication was wrong, thus the corresponding results are incredible. You should note that all duplicated genes may share high sequence identity including tandem duplicaion, segmental duplicaion and so on. So, it should be identified on the basis of collinearity analysis, instead of just the sequence similarity.
8. There is even an erasure mark in Figure 1? It is bad.
9. The explain for my last comments about Figure 3 is unconvincing. Yes, the sequences between two species or even two proteins might be different, but the length of the BD domain (R2 and R3 repeats) is commonly conserved. There are a lot of published papers have proved this characteristics.
10. As you stated that you mainly focus on the MYBs in Dendrobium officinale, so the chromosomal distribution information of the MYBs in Phalaenopsis Aphrodite in line 217-229 should be deleted or moved to Supplemental data. Beacuase the chromosomal distribution of this gene family in these two species are different, it can not represent the situation in D.officinale.
11. last time, I asked the authors that “Line 219-223, what’s the main result about the 65 genes? Here, you just focus on the results of a few genes (18 genes), why?”. And the authors response to me that they just want to analyze the tissue-specific expression pattern of all 101 DoMYB genes using the transcirptome data. However, line 237-238, they wrote that “8 genes were expressed in all seven parts.” It is obvious that these genes are not tissue-specific. Accordingly, the explain for this issue is incredible. It is still hard to understand why they just anlayzed the expression profiles of the 18 MYB genes, instead of the 65 genes that have detectable transcriptome in the RNA-Seq dataset investigated.
Please carefully improve the quality of the manuscript as reviewer's comments.
[# PeerJ Staff Note: The review process has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at editorial.support@peerj.com for pricing (be sure to provide your manuscript number and title) #]
In general, this is a well written and well organized manuscript, but some corrections and observations indicated in the text of the attached file must be attended.
This manuscript examine the R2R3 MYB transcription factor in two species of ornamental orchids of economic importance, and also investigate the expression in different tissues, and the effect of some abiotic stresses (salinity and drought) on the expression of them. This kind of analysis has not been explored previously in these species. The analysis of the R2R3 MYB transcription factors was well performed, and also were the expression analyses. Methods were well described and should allow to replicate them.
All underlying data have been provided. They are robust, statistically sound and controlled.
Conclusions are well stated.
Authors should prepare the corrected manuscript following very carefully the Instructions to Authors Guide.
In this study, the authors analyzed sequenced genomes of two Orchid species and identified 101 and 99 R2R3-MYB genes from Dendrobium officinale and Phalaenopsis aphrodite, respectively. After a series of bioinformatics analysis, they further classified them into 22 subfamilies based on a homologous analysis against AtR2R3-MYB genes. Using qRT-PCR, the expression patterns of six subfamily members of the DoR2R3-MYB genes in different tissues were analyzed and the results revealed that the S4 and S19 subfamily members had the highest gene expression levels in flowers. In addition, expression levels of a few DoR2R3-MYBs in response to salt, osmostic, ABA, JA etc treatments were assayed. Though these results presented may provide useful information for further studies of the R2R3-MYB gene family in the two orchid species, overall, the data were quite preliminary and very descriptive, which lack further confirmation or investigation.
obvious drawbacks exist with some experiments
limited
In this study, the authors analyzed sequenced genomes of two Orchid species and identified 101 and 99 R2R3-MYB genes from Dendrobium officinale and Phalaenopsis aphrodite, respectively. After a series of bioinformatics analysis, they further classified them into 22 subfamilies based on a homologous analysis against AtR2R3-MYB genes. Using qRT-PCR, the expression patterns of six subfamily members of the DoR2R3-MYB genes in different tissues were analyzed and the results revealed that the S4 and S19 subfamily members had the highest gene expression levels in flowers. In addition, expression levels of a few DoR2R3-MYBs in response to salt, osmostic, ABA, JA etc treatments were assayed. Though these results presented may provide useful information for further studies of the R2R3-MYB gene family in the two orchid species, overall, the data were quite preliminary and very descriptive, which lack further confirmation or investigation.
There are quite some evident grammatical errors or typos, which need to be intensively examined and corrected. To list just a few:
Lines 14-15: some words are missing in this sentence.
Line 18: delete the last “the”
Lines 59-61: several errors, need revision
Line 19: not clear what the authors wanted to say by “most of proteins were DNA binding proteins”. Are there any R2R3-MYB proteins that do not bind DNA at all (similar to members of bHLH family)?
Lines 72-88: what did the authors do to differentiate the R2R3-MYB TFs from others? The algorithm used to construct the tree should be clearly stated here.
Lines 133-139: why were the duration of treatments of salt, mannitol and ABA longer than other hormones? What is the reason or rational? Usually for transcriptional profilings, better to limit treatment duration to hours (e.g. max 2 d).
For qRT-PCR assay of tissue specificity, how were expression levels compared to? From the data presented in Fig 6, it can be seen that the expression levels in roots were arbitrarily set as 1.
For Fig 7, beside time 0 h control, how did the authors calculate the ratios? This kind of into should be stated clearly in both figure legends and materials and methods.
Line 142: What is the database acc# of reference gene β-Actin? Why was it used as a reference gene? Is there any evidence that this gene is stably expressed under various stress conditions or in different tissues and organs? Usually, at least two different and stable reference genes should be used with geometric means calculated. Leave a space between numerals and units.
In the Discussion section, result figures should be referred to at appropriate sites.
Fig. 1: the font of bootstrapping percentages is too small to be seen.
Fig 2: very obscure, font of text is too small
Fig. 4: the gene symbols are not right
Fig 6: legend is not complete or self-explanatory. The SDs are very tiny indicating no biological replicates were done. Any qRT-PCR data with only technical replicates are not acceptable. The same problem occurs to data presented in Figs 7&8.
Statistical analysis is missing in either Fig 7 or 8. Without it, it is not scientific to say if a change is significant from mock treatment or not.
The article must be written in better English. any mistakes in English style and grammar can be found throughout the manuscript. There are many typos and some inaccuracies that should be corrected.
The “introduction” did not provide sufficient background, thus it needs more literature highlighting this topic, and more bibliography should be included. The ms should include more sufficient intronduction and background as well.
The “material and method” section is not clearly nor concisely written, and at the same time lacks of many essential details.
The results and conclusions should be more well stated. Most of the results are not explained and presented well. Many parts are descriptive and lacks of scientific soundness, and should be extended to unravel the story in detail.
MYB proteins have interested biologists for a long time and their roles have been deciphered in a number of organisms. The manuscript by Fan et al. reports a genome-wide anlaysis of the R2R3-MYB family in two Orchid species, Dendrobium officinale and Phalaenopsis aphrodite. First, the authors identified R2R3-MYB coding-genes in these two species. The work continues with a phylogenetic and genomic analysis (chromosomal distribution, gene and protein structure analysis), followed by a global expression profile analysis for different tissues at different developmental stages, as well as in response to stress treatments. Furthermore, the authors studied by qRT-PCR the steady-state levels of transcripts of 20 representative genes in response to salt stresses. This work is basically bioinformatics, which is fundamental for a genome-wide analysis of gene families. Although the experimental strategy in this manuscript is appropriate, the study is very descriptive and lacks of scientific soundness. The authors have drawn hasty conclusions. Many mistakes in English style and grammar can be found throughout the manuscript. There are many typos and some inaccuracies that should be corrected. Turning it very difficult for the reviewer to evaluate their scientific significance.
Additional comments follows.
1. Line 12, replace “plant growth and development” with “plant growth, development and stress response”.
2. line 14-15, it is a bad sentence. You must re-write it.
3. Line 17-18, bad sentence. It is hard to under your mean of “There were different numbers of introns, exons and conserved sequences in all of the identified genes”. The statement is unclear and very confusion.
4. Line 18, “They” indicate what? Intron, exons and conserved sequences? If yes, how intron “evolved at the protein level”? And the statement of “conserved sequences” is very confusing.
5. Line 20, you just performed the chromosomal distribution analysis of PaMYBs? How about the BoMYBs, as the title and main contents of this ms are about the R2R3-MYB genes in these two species. Similar problems are existed in the whole text. Thus this paper is disordered in many parts, and the authors should state your idea and results more clearly to avoid confusion. The authors should check logic in the whole text.
6. Abbreviation, such as PaMYBs and BoMYBs in line 20 and 23, should be defined at the first time in Abstract, Figure legend, and main text respectively. Moreover, PaMYBs and BoMYBs may be replaced with Pa2R-MYBs and Bo2R-MYBs to distinguished from thr other types of MYB super family (1R-MYB, 3R-MYB, and 4R-MYB).
7. Line 22, “further quantitative analysis to determine their function” is not rigorous, because it is hard to determine gene function merely by quantitative analysis.
8. Line 23, delete “As a result”.
9. Line 24, “Parts of” is obscure. And, as mentioned above, why do you choose to analyze the expressions of BoMYBs in stead of PaMYBs?
10. Line 27, replace “Bioinformatics” with other key word(s) related to this study.
11. The “introduction” did not provide sufficient background, thus it needs more literature highlighting this topic, and more bibliography should be included. For example, first paragraph in this section, the major functions of R2R3-MYB genes are involved in metabolism, growth/development, and stress.
12. Line 44, the size of “Rabinowicz” is different from the main text?
13. For the logic issue, the second paragraph in this section should be the first; the first and the third paragraphs are somewhat overlaped.
14. Line 50-51, rewrite this sentence.
15. Line 56, “Arabidopsis thaliana” should be “A. thaliana”. Similar problems should be corrected in the whole text.
16. Line 64, repace “in all aspects” with “in many/diverse aspects”.
17. The “material and method” section is not clearly nor concisely written, and at the same time lacks of many essential details. For example, line 82, it is dangerous to delete the redundant MYB sequences based on sequence alignment, as some recently duplicated genes may have high sequence identity (even the same). Line 85, it is inconceivable to use the MEGA5.2 software with default parameters to construct the phylogenetic tree. What is the detail information of “default parameters”?
18. Line 87, replace “Arabidopsis thaliana” with “A. thaliana”.
19. Line 104, “MeGA 5.0” should be “MEGA 5.0”?
20. Line 111, “PaR2R3-MYB” should be unified in the whole text.
21. Line 117, should state why just analyze the expression profiles of MYB genes in D. officinale.
22. Line 122, the raw data was normalized or log2-transformed before making heatmap?
23. Line 123, why do you choose the 20 DoR2R3-MYB genes to do co-expression analysis? And the co-expression analyses should be performed at the genome-wide level, in stead of just the 20 genes.
24. Line 130, why use “a tissue culture seedling”?
25. Line 142, supply the GenBank ID of β-Actin gene.
26. Line 144-145, re-write this sentence.
27. “Results” section, most of the results are not explained and presented well. Many parts are descriptive and lacks of scientific soundness, which include a lot of contents about the corresponding methods. The authors should focus on the scientific issues. Many sections should be extended to unravel the story in detail. Figures 1-5 should be explained further to unravel their significant findings.
28. Line 156, why S6, S12, and S15 did not have members in these two species investigated? The information about the results in this part is too simple. The distribution situation of the candidate genes in the same subfamily between the two species is the same?
29. Line 166-168, in fact, there are many literatures have proved that the intron patterns in the MYB domains of 2R-MYB genes were generally conserved in many subfamilies. The analyses and results in this part are too obscure.
30. Line 202, “99 Pa R2R3-MYB genes” should be “99 PaR2R3-MYB genes”? “85 genes were mapped”, how about the remainings? Moreover, how about the situation of BoR2R3-MYB genes?
31. Line 207, how did you identified the “33 pairs” genes?
32. Line 209, what’s the method that was used to identify the segmental duplication?
33. Line 213, the statement of “almost identical (> 80%)” is not rigorous.
34. Line 214, why did you just focus on DoMYB? How about the PaMYB genes?
35. Line 219-223, what’s the main result about the 65 genes? Here, you just focus on the results of a few genes (18 genes), why?
36. Line 224-234, How about the results between the RNA-Seq and qRT-PCR analyses.
37. Line 237, replace “under stresses” with “under drought stresses”.
38. Line 249-258, given the homologs may have similar expression patterns, thus the co-expression analysis of them is meaningless. It is better to perform this assay at a genome-wide level to speculate the co-expression network of them.
39. In the qRT-PCR expression analyses, statistical analysis should be clarified and the softwares used should be mentioned in details. The statistical analyses performed to find significant differences are not shown in Figure 6, 7 and 8.
40. Figure 1: Increase the size of the letters. It is difficult to see the names of the genes and the bootstrap value on the phylogenetic tree. Moreover, the legend of the picture should be more clarified, and the “Dendrobium officinale”, “Phalaenopsis aphrodite” and “Arabidopsis thaliana” should be in italic.
41. Figure 2: Increase the size of the letters. It is difficult to see the names of the genes the illustration, and the lables in the picture. Figure 2C is hard to read, and should be great imprvoed.
42. Figure 3: Why the number of amino acid in A & C, and B & D are different? Increase the size of the letters.
43. Figure 4: Increase the size of the letters. It is difficult to see the names of the genes the illustration, and the lables in the picture.
44. Figure 5. The size of the letters should be increased. The gene names are different from these in the text? The figure represent log2 expression values (FPKM>1)?.
45. Figure 6, 7 and 8 should be replaced with high resolution figures.
46. The discussion still needs improvement and explanation in detail. The discussion should show how these new findings in this study support the research question and hypothesis.
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