Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL INSIGHTS INTO THE EFFECT OF DROUGHT STRESS ON THE DEVELOPMENT OF ROOT AND CARYOPSIS IN BARLEY
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the present study, barley was subjected to drought stress and the morphology and microstructural changes in root and caryopsis were observed. The pictures in the microscope seem beautiful and clear. At the same time transcriptome analysis was carried out to investigate the mechanism underlying the barley caryopsis development responding to drought stress. The results may provide valuable information for revealing the barley root and caryopsis development under drought stress. The data are enough to lead their conclusion, and should give new criteria of barley development under drought stress. Therefore, in my opinion, the paper is acceptable for publication with some revision.
Experimental design: .
Validity of the findings: .
Additional comments: 1. The abstract of the manuscript does not seem to be as informative as it should be. For example, the variety of barley used for the experiment and drought stress imposition stage should be mentioned in the abstract. Also the specific value of changes in barley traits under drought stress may be mentioned in abstract to make it more informative rather than generalized.
2. Line 91-92, the unit of soil nitrogen, phosphorus and potassium content seems incorrect. Please check again.
3. Line 107, the description of 1000-grain weight should keep consistent in the context.
4. There is a lack of the scale bar in Figure 2B.
5. There are some writing mistakes needing to be improved in the manuscript. Please check it carefully.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL INSIGHTS INTO THE EFFECT OF DROUGHT STRESS ON THE DEVELOPMENT OF ROOT AND CARYOPSIS IN BARLEY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript descripted how drought stress affects barley root growth and caryopsis development from morphological structure changes to molecular work (gene expression). The authors found drought stress increased lateral roots and decreased the vessel number and volume of root. Drought stress accelerated the maturation of caryopsis with less starch accumulation but more protein accumulation in barley endosperm. Transcriptome analysis found that differential expressions of genes in caryopsis involved in encoding storage proteins and protein synthesis pathways. These findings provide some fundamental knowledge for drought stress, which is an increasing problem in agricultural production.
However, I have some questions.
1.	How much protein contents change in caryopsis by drought stress?
2.	How to determine grain quality? More proteins is better?
3.	Lines 254-266 need to be improved.
4.	Some figures need to be improved (Fig. 1A, B, Fig. 3N,O,P, Fig. 6)
Overall, the manuscript is good. But, it still needs improving in writing (see more details in attached file).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL INSIGHTS INTO THE EFFECT OF DROUGHT STRESS ON THE DEVELOPMENT OF ROOT AND CARYOPSIS IN BARLEY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript described an experiment in which a post-anthesis water stress was applied to one barley cultivar in a single experimental replicate under glasshouse conditions. Using light microscopy, the structure of root xylem was analysed, and starch granules and protein bodies in the caryopsis endosperm were quantified by area. RNA-seq followed by a gene ontology analysis were used to detect changes in trascript abundance between the water-stressed plants and the well-irrigated plants. The introduction covered the necessary literature to understand the objectives of the study. Unfortunately the quality of the English throughout the manuscript means that some of the meaning is ambiguous, and there are a large number of grammatical errors throughout the text adding to the difficulty in interpretation. Some of these are highlighted in the General Comments section, although they are too numerous to mention every one. The findings reported are typical of water stress applied during grain filling, and the authors are advised to make it clear how their results add to this knowledge.
Experimental design: I have some serious concerns regarding the experimental design, and am not convinced that these are able to be rectified in order to make the results meaningfully interpretable.

The research question seemed to be aimed at correlating water-stressed changes in root development with development of the barley caryopses under these conditions. This is an interesting question, and had the experimental design been reported more accurately, the results could be interpreted as a meaningful correlation. However, there was no indication of any true replication in this experiment, which appeared to consist of one experiment containing 30 pots. It was not indicated that the pots were grown either spatially or temporally separated in a way that could be thought of as replication, and no indication that the caryopses and roots were taken from pots with meaningful replication in mind. Furthermore, the authors should state where the barley caryopses and roots were sampled from in order to minimise experimental bias. For example, were the caryopses always harvested from the main shoots, and at the same position within the ear ? Were the roots harvested from the main root axis, and how long from either the stem axis or the root tip ? It is therefore impossible to determine whether the correlations observed are genuine, or due to sampling bias. Why was the biomass not reported ? The root:shoot ratio is ambiguous if both were reduced by the water stress treatment.

There was a secondary emphasis on using RNA-seq to determine a genetic mechanism for these correlations. I have concerns regarding the experimental design to address these questions. The authors acknowledge that the development rate of the caryopses were affected by the water stress conditions, which means that using RNA-seq to compare caryopses at the same number of days post-anthesis would result in comparison of caryopses at two different growth stages. Therefore the reported results should be treated with caution, as the changes in transcript abundance in barley caryopses among even close growth stages will conflate these results, and would include exactly the gene types reported here (seed storage proteins for example). In addition to the lack of meaningful replication discussed above, the validity of these results are difficult to judge.
Validity of the findings: The validity of the findings is difficult to interpret as discussed above in the section for experimental design. The design does not allow for much in the way of meaningful interpretation.
Additional comments: The English quality should be improved to allow better interpretation of the meaning. There are some errors throughout the text. I suggest having the manuscript corrected for English throughout, paying particular attention to the use of plurals, and ambiguous text.
I give some examples for the abstract and the beginning of the introduction, but to address every instance would require more than a reviewer.
Abstract
Line 32 (and throughout), the plural of caryopsis is « caryopses »
Line 33 « thus ultimately led to » should be either « this ultimately led to » or « ultimately leading to ».
Lines 34 to 36, it is not made clear what these results have to do with molecular breeding, or yield and quality improvement.

Introduction
Line 42 « roots » of barley
Line 45 « Seniors pointed out », I assume Seniors is an author but this study is not referenced
Lines 46 and 47 barley does not have panicles, I suggest reading a basic cereal anatomy textbook for this
Line 52 « indices » is the plural of index, the plural of criterion is « criteria »
Line 55 « plant » and « need » should be plural
Line 59 the barley genome sources should be properly cited

Materials and methods
Lines 91 and 92 there is am odd symbol included for the soil compositions reported
Line 97 there should be some indication of how/if replication was determined. 30 pots grown at the same time is not meaningful replication.
Lines 97 to 99 please clarify what you mean by different development stage
Line 102 define what is meant by morphology
Line 104 and throughout, there should be no space between the numerals and the units for °C
Line 109 further information is needed about barley caropses harvest (see above comments on experimental design and validity). Pay particular attention to how replication was determined, which shoots, from how many pots, how was the replication biologically meaningful ?
Line 125 and throughout, I don’t know what is meant be « abdominal », perhaps « ventral ». I suggest a cereal anatomy textbook.
Line 129 Define what is a replicate
Line 132, again be very precise about how the caryopses were harvested, and define their growth stages, because otherwise these results cannot be meaningfully interpreted.
Line 152 The statistical analysis sections requires a lot more information in order to determine which tests were applied to which results. How were the results corrected for multiple comparisons ? Or were results only compared to one « control » results ? This needs to be well defined. The statistical analyses may need to be re-done according to the number of replicates, because 30 pots in one experiment does not necessarily mean 30 replicates.

Results
The p-values need to be reported throughout, because it is not always clear what is meant by « significant ».
The error bars should be clearly explained on all graphs, paying particular attendtion to stating the true number of replications. If there was no replication the errors should be standard deviation.
Line 163 « and DS » is repeated
Lines 174 to 176 Are there any analyses to support this ? If this is true, it makes it even more important to specify exactly where and how anatomically the sections were taken for microscopic analysis.
Lines 187 to 188 That water stress resulted in early caryopsis maturation is typical of water stress during grain filling, and makes it particularly important that the authors specify the growth stages (not days post anthesis) at which caryopses were harvested, otherwise readers will have to assume that the results cannot be compared between treatments as the caryopses could be at significantly different growth stages.
Lines 209 In this section, the p-values should be given alongside the correlations, as the correlation alone does not indicate significance.
Line 231 « various database(s) » could be more specific
Line 236 is « albumin » really what the authors mean ?
Line 240 to 241 is very vague
Line 252 justify why this particular pathway was chosen, what meaning does it specifically have to the research question ?
Lines 286 to 289 is very speculative, particularly regarding that the RNA-seq results were likely from caryopses at different growth stages.
Lines 298 to 300 this is very speculative and the study does not contain sufficient information to draw any causative meaning between observed correlations.
Line 316 the study present no evidence of water and nutrient absorption being affected, and this statement should be removed.
Line 320 to 321 I am not convinced the results are novel, the authors could spend furhter time addressing how their study fills gaps in the literature.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL INSIGHTS INTO THE EFFECT OF DROUGHT STRESS ON THE DEVELOPMENT OF ROOT AND CARYOPSIS IN BARLEY
Review round: 1
Reviewer: 4

Output:
Basic reporting: Li et at. al describe in their paper the morphological and structural changes in root growth and caryopsis development of barley under drought stress. Transcriptional analyses showed that drought stress induce differential expression of genes that are mainly related to protein biosynthesis in caryopsis. The authors finally suggest that the root of barley firstly senses the stress signal when subjected to drought stress which led to changes in root morphology and structure. Subsequently, the water and nutrient absorption is affected, resulting in changes in the development of caryopsis and its substance accumulation.
To my knowledge, these findings are completely new.
The quality of writing is good, the manuscript is clearly written, and the reader can easily follow the story. The manuscript provides new information concerning the relationship between root architecture and the development of caryopsis when barley is subjected to drought stress.
Experimental design: The manuscript is clearly written, the experiments are well planned, and the results are underlined by a sufficient number of replicates.
Validity of the findings: Based on this multidisciplinary approach (morphological and transcriptomic analyses), the authors proposed the model that stress signals from root to caryopsis induced by drought stress induce the differential expression of genes involved in encoding storage proteins and protein biosynthesis pathways, leading to the increase in endosperm protein accumulation in barley caryopsis. The data underlying these results are robust and sound.
Minor revisions:
Figure 3: A picture of a cross section would be helpful to the reader to orientate of which region of the endosperm the close-up was taken. Additionally, the cross section will prove an overview and underline the statistical data and correlate the close-up picture.
Additionally, can the author explain in more detail on the term “relative area” that was mention concerning statistical analyses of protein bodies and of starch granules? It is unclear what the authors finally quantified.
Line 178: The number of vessels in root under DS increased. But the next sentence describes a decrease?

The font and the typing are inconsistent within the legends (missing space after (A), (A) is labelled in bold, ….) The legends should be corrected.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL INSIGHTS INTO THE EFFECT OF DROUGHT STRESS ON THE DEVELOPMENT OF ROOT AND CARYOPSIS IN BARLEY
Review round: 2
Reviewer: 1

Output:
Basic reporting: /
Experimental design: /
Validity of the findings: /
Additional comments: /


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL INSIGHTS INTO THE EFFECT OF DROUGHT STRESS ON THE DEVELOPMENT OF ROOT AND CARYOPSIS IN BARLEY
Review round: 2
Reviewer: 2

Output:
Basic reporting: This revised manuscript is good for publication.
Minor changes have been made in attached file.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Units in Fig. 1 should be put in parenthesis ( ), not / g.

I still think of that DS for drought stress may be not good because the manuscript has DS in many places and also has drought stress in some places. CC is not good for control.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF CHRONIC PRAZOSIN, AN ALPHA-1 ADRENERGIC ANTAGONIST, ON ANXIETY-LIKE BEHAVIOR AND CORTISOL LEVELS IN A CHRONIC UNPREDICTABLE STRESS MODEL IN ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript entitled “Effects of chronic prazosin, an alpha-1 adrenergic antagonist, on anxiety-like behavior and cortisol levels in a chronic unpredictable stress model in zebrafish (Danio rerio)” studied the effects of prazosin on behavioral and biochemical parameters in adult zebrafish submitted to unpredictable chronic stress protocol.  The manuscript presents appropriate methodological aspects, the text is clear and unambiguous, and the article presents a professional structure. The statistical analyses are correct and properly reported. Figures, table and raw data are well presented.
Experimental design: The description of the materials and methods has some limitations. Important aspects regarding animal care and randomization were not adequately described.  I suggest that the authors review the ARRIVE and PREPARE guidelines to better describe the methodology. What was the method used for random allocation in the treatment groups? Were experimenters blind to treatment? Were data analysts blind to treatment? These questions need to be addressed and clearly stated in the methods section.

I believe that beyond temperature, physical-chemical characteristic of water (for example, pH, conductivity) should be controlled during the experiments. It is not described in the methods section.

Did the authors consider the potential effect of the tank on statistical analysis?

Were experimenters blinded throughout the experimental protocols? If not, there is a possibility of bias in the interpretation of the data. This information should be described in the M&M section.

How was the sample size calculated? Was it based on a pilot experiment to determine the effects sizes? How were the calculations performed?

Did the authors check for sex effect in these tests? It is a relevant question because female and male zebrafish can respond in a different manner. In my opinion, is mandatory to check for sex effect. If there are no effects of the sex, then the data can be pooled.

How many times the prazosin solution was used?

Line 185, please change dose to concentration.

There is a clear difference between immobility time in control groups (please see figure 2 and 6; table 2 and 3). How could the authors explain this difference?

Please describe how much food was used. Was it 1% body weight per day? Please give a figure.
Validity of the findings: Although I think this approach interesting, I suggest another experimental design for the next experiments. In Marcon et al. (2019), the animals were submitted during 14 days to UCS. In the last 7 days, zebrafish were treatment and stressed concomitantly. I believe that this protocol will be more suitable to demonstrate the potential effect of the prazosin as a treatment of stress-related disorders.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF CHRONIC PRAZOSIN, AN ALPHA-1 ADRENERGIC ANTAGONIST, ON ANXIETY-LIKE BEHAVIOR AND CORTISOL LEVELS IN A CHRONIC UNPREDICTABLE STRESS MODEL IN ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is professional and intelligible, but there are some typos, some areas lack clarity, are repetitive, and/or require more work. Literature references and overall structure are good. There are some improvements needed, for example (not comprehensive list, please review the whole manuscript more carefully):

- use of word 'alter' instead of clear direction of effect (increase/decrease) throughout the manuscript, particularly problematic in the abstract and figure/table captions.
- Line 61, 'resilience of treatment' should read 'resilience to treatment'
- Line 81-84, the claim that prazosin blocks noradrenergic regulation of the HPA axis via alpha-1 receptor is highly problematic here. The authors do not cite any existing literature to support this claim and the experimental design of this study is not suited to answer whether prazosin blocks noradrenergic receptors.
- Line 98-101, this sentence implies that CUS models aspects of MDD and PTSD. Yet, the authors list two review papers (Caramillo et al., 2015; Stewart et al., 2014) that both 'suggest' that CUS (among many others) may be used to model stress/PTSD but do not show contain experimental work. In general, it is not obvious why 'chronic' mild stress would be a good model for PTSD, even when PTSD may result from a single traumatic experience. It may be more appropriate to argue that the current study is modeling general anxiety and/or stress that is shared between many mood disorders, anxiety disorders and trauma and stressor-related disorders, instead of only use the PTSD framework.
- Line 203, typo in euthanasia
- Line 313-314, and 326 please confirm that the statistics are correct here
Experimental design: The mechanisms underlying PTSD-related HPA-axis activation and behind prazosin's effectiveness are worthy topics. The research question is relevant and meaningful. It is also well-defined in the manuscript but lack key details about operationalization and procedures. Thus, it is not clear which of the measurements are relevant for PTSD modeling and why. For example, the authors measure locomotor activity but do not state whether this is to determine stress-related responses of hyperactivity/hypoactivity.

Other general and specific comments:

- Given that ample literature exists on the importance of prior housing, breeding practices and husbandry conditions for behavioural studies (some of which are mentioned in line 416-419), it is not clear why the authors used fish that were bought from a supplier and not bred in-house, a practice that would allow more control and consistency over the heterogeneity that may exist in wild-type fish. Similarly, is it possible that short acclimatisation of a week and high stocking density (5-7 fish in 1.8L tanks) may have caused stress that may have led to lack of significant differences between control and 'stressed' groups?

- Line 131 & 142/152. The information about housing densities does not add up. If 5-7 fish were places in 1.8L tanks (line 131), how were 50 fish were put in four tanks (line 142, implying 12 fish per tank) and 72 fish were allocated into eight tanks (line 152, implying 9 fish per tank). Similarly, in line 177, for crowding, how many fish were put in the 200 ml water?
- Line 133, please include information on any/all other parameters of housing water quality: pH, salinity, oxygenation, ammonia, nitrates/nitrite, etc.
- Line 133-134, when were fish fed on experimental days or drug exposure days? Were they fed before or after the CUS/prazosin treatment?
- Line 147 and 160, what does subsequently mean here? How long after the novel tank test were fish euthanized?
- Line 152-157, does 'handled/unhandled' mean stressed/unstressed or something else here?
- Line 153, how long was the acclimation period for experiment 2?
- Line 155-160, please stated explicitly whether the fish received stress and prazosin treatment concomitantly or in sequence. Line 158-160 seems to be the same sentence as line 145-147 and implies that fish were euthanized after the CUS treatment.
- Line 172-174 (a), while drastic temperature changes can be considered stressful, given that the fish were kept at 26 +/- 2C,  is 23C and 33C drastic enough when housing condition ranges of 24-28C? Besides existing literature (Piato et al, 2011, Marcon et al, 2016, and Rambo et al, 2017) that does the same without much explanation, why do the authors consider 23C and 33C as stressful for a fish species that can tolerate a wide temperature range in the wild and in captivity? Is this ethologically relevant?
- Line 172-174 (b), how did the experimenters maintain the temperatures at 23C and 33C for 30 min? Did the heaters/coolers used for this make noise or vibrations? Is it possible that a lack of water circulation (and re-oxygenation) for 30 minutes is perhaps more of a stressor than the temperature changes?
- Line 181-191, please provide an explanation for placement of individual fish during prazosin exposure in 50 mL of water, esp given that social isolation and little water were used as stressors (line 175 and 178).
- Line 193-204, what was temperature of the testing tank? pH? Salinity? Lighting conditions? Did the authors use system water to fill the testing tank? How often was the water changed between different trials?
- Line 194-196, please provide an explanation for why fish were acclimatized to the experiment room prior to a test of response to 'novelty'? Were the home tanks aerated during these 30 minutes? Were all fish in a home-tank tested at once or in sequence such that some fish were acclimatized for longer time?
- Line 197, please provide an explanation for using a tank of same size and dimensions as the home tank for a test of response to 'novelty'?
- Line 199, please cite previously existing literature that has validated or used this software for tracking zebrafish.
- Line 200-203, please provide more analytical information for this section. Why are distance and speed (usually quantifying hyperactivity) not considered measures of anxiety but only of general motor activity? What does 'top of the tank' means? Does the 'top' refer to top-half, top 25%, or something else? How long was the water column/height? Also, why did the authors report on 'top' and 'immobility' behavior and not include other relatively easily quantifiable measures of anxiety, bottom-dwelling, erratic movement, latency to enter 'top', and pigmentation change as measures of anxiety?
- Line 203, typo in euthanasia
- Line 207-209, please give an estimate for how long did euthanasia take typically? 1 min? 10 min? 30 min?
- Line 213, were the trunks were cleaned of digestive tract and eggs? If not, please indicate in methods the timing of last feeding prior to euthanasia and if it was the controlled for all fish.
Validity of the findings: Given the findings of experiment 1, that CUS paradigm used here had statistically significant effect on only one measure (total time spent in the top zone) out of six behavioural, cortisol, and body weights, why did the authors choose to employ the exact same CUS protocol in experiment 2?
- Line 283, given that there was an effect on time spent in the top zone, but no effect on distance traveled, and only a marginal effect of entries, it is justified to say that 'chronically stressed generally explored the top zone less'?
- Line 313-314, please confirm that the statistics for both stress (F(1,65) = 3.050, p = 0.085) and drug (F(1,65) = 3.050, p = 0.085) are exactly the same and this is not a typo or a copy/paste error.
- Line 318, please rephrase 'no strong effect' to better represent the lack of statistically significant differences. Also 'across the novel tank test' should be clarified to better represent the temporal nature (and not spatial) of these results.
- Line 326, please confirm that the F-value is correct here (F(1,65) = 956, p = 0.332).
- Line 333-335, given this statement, is it use of CUS and NTT as employed in this study appropriate for modeling persistent/long-occurring PTSD or even long-term effects of stress?
- Line 345-347, given this, is it possible that the procedure for vehicle/prazosin exposure (and/or the housing conditions) may be more stressful than the CUS itself?
- Line 374-376, this statement is misleading, as it implies that there were 'any effects of CUS' on behavior and hormone, which prazosin did not mitigate. Please rephrase this sentence to more accurately represent your data, control animals had more stress than CUS so it is not possible to determine the prazosin effects.
- Line 384-400, given previously existing literature (Song et al.,2018 Rasmussen et al., 2017, and similar studies supporting simultaneous stress and treatment), and Piato et al., 2011 (showing optimal stress after 7 days but not at 14 days) please elaborate on the choice of sequential timeline (CUS followed by prazosin) and stress responses collected on day 14 in experiment 2 (instead of day 7). This is particularly relevant for modeling of PTSD, as experiment 2 results (non-prazosin groups) do not replicate the CUS-effect findings from experiment 1, and are actually in the opposite direction.
- Line 405-406, this conclusion is problematic. The authors do not cite any existing literature to support this claim and the experimental design of this study does not substantiate that prazosin prevents further triggering of PTSD.
- Line 413, did the authors analyze the effect of sex? Especially given the relatively good sample size in experiment 1, it may be possible to see if there is an interaction between sex and CUS for cortisol?
- Line 420-423, again, this summary should be rephrased to more accurately represent the data. Same applies to the 'Conclusion' section.
- Line 430, please state the direction of the result, instead of 'alter'.
- Figures 1-8, why is minute 7 included in the figures when the testing only lasted 6 minutes?
- Figure 1 and 3. There are symbols denoting the effect of time which make these more figures complicated. Is this necessary or relevant? On the other hand, none of the significant effects are highlighted in the figures or the captions. Why?
- Figure 3 and 7, the captions are misleading. Please state the specific lack/presence of statistically significant effect of CUS and/or prazosin clearly in the caption and indicate it on the graphs. It may be of benefit to represent the data in graphs C (for both 3 and 7) as the % of total distance traveled that is in the top.
- Figure 1-8, it would help the reader if you keep the legends (colours and shapes) for the non-drug groups consistent across figures for the two experiments. In figures 1-3, red-filled squares are used for CUS that are not treated with prazosin, but the comparable groups from experiment 2, are unfilled-squares (and red-squares are CUS with prazosin).
Table 1, caption, 'throughout the light period between 9 a.m. and 4 p.m.' Please rephrase this as 9am-4pm covers only half of the 14-hrs of light.
Table 2- captions are misleading when stating 'CUS altered some behavioral measures' when the table has only one or very few statistically significant effects listed.
Additional comments: The manuscript identifies an important research question about underlying mechanism behind prazosin use for PTSD. Both effort towards animal modeling of PTSD and use of zebrafish is commendable and are strength of the paper. The weaknesses are poor construct validity, lack of key methodological details, and exaggerated implications of results.

Other specific comments:
- There is no mention of what generally causes/leads to PTSD (traumatic events) in the introduction. Prevalence rates in the US are cited from 2007. More recent and global rates may help make the need for a treatment more obvious. Similarly, while it is noted that active duty service members are prescribed more benzodiazepines for PTSD, it is not obvious that mentioned population or other people experiencing war/conflict situations have higher risk/prevalence of PTSD.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF CHRONIC PRAZOSIN, AN ALPHA-1 ADRENERGIC ANTAGONIST, ON ANXIETY-LIKE BEHAVIOR AND CORTISOL LEVELS IN A CHRONIC UNPREDICTABLE STRESS MODEL IN ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the present form the manuscript can be accept. The authors responded properly all my issues.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECTS OF CHRONIC PRAZOSIN, AN ALPHA-1 ADRENERGIC ANTAGONIST, ON ANXIETY-LIKE BEHAVIOR AND CORTISOL LEVELS IN A CHRONIC UNPREDICTABLE STRESS MODEL IN ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed my concerns and provided sufficient counter-argument or fixed the issue.
Experimental design: The authors have addressed my concerns and provided sufficient counter-argument or fixed the issue.
Validity of the findings: The authors have addressed my concerns and provided sufficient counter-argument or fixed the issue.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSESSMENT OF CRUSTING EFFECTS ON INTERRILL EROSION BY LASER SCANNING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript ID 42555 describes an interesting research focused on the dynamics of crust formation (and its effects on soil erosion) in agricultural soils during a rainfall event. This research combines rainfall simulations and measurements made by using a high accurate lasing scanner. The study was carried out in laboratory with soils from two farms with different land management in Switzerland. I understand this kind of controlled experiments shall be done in laboratory but I prefer data from real field experiment because soil transport can bias the experiment. Anyway, this latter is just a personal opinion not a critical of this research I have evaluated.

The article is quite well-written and structured, i.e. it is very easy to read and understand. Since I am not a native speaker I do not feel qualified to assess the language level of the manuscript but I think its English of high quality. I have detected a mistake in the line 28. I think you have written sol (soil in French) instead of soil. Anyway, I think the most appropriated concept to be written is land management instead of soil (please see line 28) or tillage management (please see line 106).

This research is also well-motivated. The authors remain clear in the first paragraph of the Introduction section what the research gap they are trying to cover is: “to improve the parameterization (for spatio-temporal modelling) of soil resistance dynamics as a consequence of rainfall events”. Actually, you are trying to improve the temporal (pre vs. post) and spatial (reducing the cell size of DEM) accuracy of the measurements of the variable (crust) roughness (mentioned in the last paragraph of the Introduction section).

Subsequently, they explain pretty well the existing interrelationships between crust formation and soil erosion (amount and type of sediments) in the paragraphs 2 and 3 of the Introduction section. Their affirmations are well-supported by relevant literature. The references look to be well-quoted and listed at the end of the manuscript according to the guidelines for authors of the journal. In case I am wrong I apologize because each journal requires different guidelines and sometimes it is easy to be wrong in this matter.

In the paragraph 4 they justify properly the necessity of using laser scanning in order to obtain the most precise digital elevation models. Nevertheless, I have some doubts regarding the usefulness of these precise DEMs because of up-scaling. That is very good to work at the slope scale but I guess you have to make precise DEMs in many slopes in order to prepare precise inputs for any models reliable for regional scales (personal opinion). Obviously, that is not the aim of your research because you are only testing the effectiveness of laser scanning to improve the parameterization of soil resistance dynamics (crust roughness). But, in any case, reviewing an article is the best way to discuss with researchers of your same field study.

Line 106: carbon erosion? Please check this affirmation.

Figures and tables are relevant, of high quality and well-labeled and described.

The dataset provided as raw data are 144 txt files that can be perfectly managed by MO Excel/Calc or in GIS software, amongst others.
Experimental design: This research is original and useful. This information could be used by many modelers in future tools. Obviously, this research is based on a single study case (only two farms in Switzerland) but it covers many aspects. So, it is fair to say this article fits perfectly with the scope of the journal.

Regarding research questions and/or hypothesis they are not clearly mentioned, at least in the Introduction section. The authors identify perfectly the research gap and they explain properly how this gap shall be filled but there are not research questions and hypothesis to be tested addressed explicitly in the text.

The Material and methods (M&M) section is divided into 5 sub-sections that follow a logical sequence from a methodological point of view. Each methodological step is perfectly explained, i.e. this methodology could be replicable by everyone. This methodology has been made technical and ethical rigorously made (including a sufficient number of replicates). I have decided to treat each step separately in order to make easier the understanding of my comments.

Soil sampling and preparation: Experimental design and soil sampling are well-described in the previous publication (attached by the authors as supplemental material) but the line 115 should be rewritten because it is confusing. Please take into account that experimental design is the first step and soil sampling is the second one. How much time has it lasted between the finish of soil sieving and the beginning of the soil pre-wetting?

Rainfall simulations: Why are you interested in the determination of SOC eroded?

Calculation of soil surface roughness: Is it really necessary to use one sub-section for each type of measurements: 2 transects and 4 subplots?
Validity of the findings: My opinion, as reviewer, after having spent many hours in their evaluation is to “certify” the validity of these results. The process has been perfectly controlled and the results are robust. Nevertheless, I think we can discuss here about the impact and novelty of this research.

Regarding impact these findings are very useful for modelers. About novelty it depends on the personal opinion as each reader/scientist. I think it is a continuation of previous research works (kindly attached by the authors) and then it should be considered an advance in this topic.

Discussion and conclusions are consistent and they have reached my expectations as scientist. I think they are well-fitted to that the journal is grosso modo also expecting.
Additional comments: Line 122: rim or ring?

Line 195: normalized (abnormal distribution to normal distribution) or standardized (rescale the data to a known range)?

Line 253: frequent

Line 267: The fact of checking conventional farming soils flatten under low intensity rainfall events is another proof of the necessity of keeping “security” cover in our soils to keep their durability.

Line 298: crust formation (not rust formation)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSESSMENT OF CRUSTING EFFECTS ON INTERRILL EROSION BY LASER SCANNING
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the paper, the laser scanning technique (coupled with a photogrammetric survey) was used to assess the crust formation. Sorry, but I don’t understand the aim of the paper, i.e. the authors would quantify the changes of the soil microtopography due to erosive processes or test the laser scanning methodology? In the first case, I think that the paper could be interesting, but the authors have to rewrite the paper in this sense. The capability of the laser scanner technology was assessed by several authors (such as Abban et al., 2017; Zheng et al., 2014; Huang and Bradford, 1992) and the novel aspect of the paper is not clear.
The literature references are poor, I suggest to enlarge the introduction and some paper that could be interesting for the paper.

Abban, B. K. B., Papanicolaou, A. N.,  Giannopoulos, C. P., Dermisis  D.C., Wacha, K. M., Wilson, C. G., Elhakeem M., 2017. Quantifying the changes of soil surface microroughness due to rainfall-induced erosion on a smooth surface. Nonlinear Processes Geophys., 24, 569-579.
Zheng, Z. C., He, S. Q., Wu, F. Q., 2014. Changes of soil surface roughness under water erosion process, Hydrol.  Processes. 28, 3919–3929.
Huang, C., Bradford, J.M., 1992. Applications of a laser scanner to quantify soil microtopography. Soil Sci. Soc. Am. J. 56, 14-21.
Experimental design: The description of the soil sampling, the rainfall simulator, and the laser scanning survey are clear.
As commented above, from Line 183 - 219, the description is ambiguous. The authors used coefficients that there are not explained in the text (eq.1).
Furthermore, the authors treated the changes of the surface roughness as the soil surface height distribution.
In the Introduction (Line 108) "close-up photography was used..." but in the section Materials and Methods there is not a description of the photogrammetric survey.
Validity of the findings: In the paper, there are some conceptual mistakes. First of all, from Line 183, the authors describe the method used to calculate the soil surface roughness. Conceptually, the soil surface roughness is very different from the actual height of the points. In literature, numerous indices and definitions of surface roughness could be found (from Kuipers, 1957 or Allmaras et al., 1966 to more recent works where the laser scanning was used such as Abban et al., 2017). However, the surface roughness is commonly quantified as the variations (using a standard deviation for example) of the surface heights in relation to a plane (Currence and Lovely, 1970) or a surface or a residual topography (Cavalli et al., 2008). If the authors evaluated the surface roughness, probably it is necessary to clarify.
Instead, if the authors want to refer the results about the differences the term "surface roughness" has to be deleted in the paper.

Kuipers, H., 1957. A relief meter for soil cultivation studies. Netherlands Journal of Agricultural Science, 5, 255-262.
Allmaras, R. R., Burwell, R. E., Larson, W. E., Holt, R. F., 1966. Total porosity and random roughness of the interrow zone as influenced by tillage. USDA Conservation Research Report 7.
Currence, H. D.,  Lovely, W. G., 1970. The analysis of soil surface roughness. Transactions of the ASAE 13(6).710-714.
Cavalli, M., Tarolli, P., Marchi, L., Dalla Fontana, G., 2008. The effectiveness of airborne lidar data in the recognition of channel bed morphology. Catena, 73, 249–260.
Additional comments: Probably I don't understand the novel aspect of the paper in this form.
The paper could be interesting if the authors focused on the crust formation due to erosional processes and not on the laser scanning method.
Detailed suggestions are reported on the annotated PDF file.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSESSMENT OF CRUSTING EFFECTS ON INTERRILL EROSION BY LASER SCANNING
Review round: 2
Reviewer: 1

Output:
Basic reporting: My comments and those given by the other reviewer(s) have been helpful to improve considerably this manuscript. The autos have made a good job.
Experimental design: The research rationale is perfectly explained. In addition, the methodology is both consistent and useful.
Validity of the findings: The results are very interesting. In the future I would like to replicate this methodology in my study areas.
Additional comments: Dear Authors,
I am satisfied with the changes made for the current version. So, I am going to suggest the article can be accepted in the current form.
Sincerely,
Reviewer #1


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSESSMENT OF CRUSTING EFFECTS ON INTERRILL EROSION BY LASER SCANNING
Review round: 2
Reviewer: 2

Output:
Basic reporting: In this revised form the paper is clear and unambiguous.
All the critical points of the original paper were clarified.
The literature references are enlarged and the novelty and the results of the study were focused.
In this form I think that the paper could be published because the manuscript has been significantly improved.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I suggest to correct the L245 of the manuscript without revision.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANALYSIS OF THE GENETIC DIVERSITY OF THE COASTAL AND ISLAND ENDANGERED PLANT SPECIES ELAEAGNUS MACROPHYLLA VIA CONSERVED DNA-DERIVED POLYMORPHISM MARKER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Ms on Analysis of Genetic diversity in the coastal ... By CDDP markers by Wang Yi et al has been reviewed here.
The article is well written. But few technical/methodical lacunae need to be addressed to improve the quality of the study/manuscript.
1. Authors did an island-based population study, but the 'STRUCTURE' analysis and 'Loci selection' is missing. This may be included.
2. There are thousands of Conserved domain genes but authors used only few TF sequences to derive CDDP primers! Authors need to highlight the basis of selecting them.
3. Fig. 2 on DNA extraction, is not very important figure for the Ms. May be deleted.
4. Authors have provided innumerable numbers of supplementary files. May restrict to only few important ones.
Major revision is needed on the above points.
Experimental design: As mentioned earlier: STRUCTURE and Loci selection analysis needed for population study.
Validity of the findings: Mentioned in first section.
Additional comments: Article must be rechecked thoroughly and added more scientific contents to make it a relevant study for the readers.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANALYSIS OF THE GENETIC DIVERSITY OF THE COASTAL AND ISLAND ENDANGERED PLANT SPECIES ELAEAGNUS MACROPHYLLA VIA CONSERVED DNA-DERIVED POLYMORPHISM MARKER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The key point in this article is that Elaeagnus macrophylla is an endangered species and to protect the biodiversity, its genetic diversity is to be ascertained. However, the author failed to provide any appropriate suggestion, mainly because of the study was ill-conceived and several flaws in analyses of data.
Experimental design: The choice of the marker is not appropriate. Analyses should have also included STRUCTURE analyses.
Validity of the findings: The statements in lines 188-189 and 202-203 are contrary.
Again, the statements that sea current and hydrological profiles alter genetic structure is also too speculative without any valid references which explain that such things do happen
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANALYSIS OF THE GENETIC DIVERSITY OF THE COASTAL AND ISLAND ENDANGERED PLANT SPECIES ELAEAGNUS MACROPHYLLA VIA CONSERVED DNA-DERIVED POLYMORPHISM MARKER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript entitled “Analysis of genetic diversity in the coastal and island endangered plant Elaeagnus macrophylla by conserved DNA-derived polymorphism markers (#38915)” presents a molecular marker based study of this species in China, concerning its diversity and population structure.
Authors assessed conserved genes information using CDDP markers and, according to them, low levels of genetic diversity were found for the species. In addition, they reported strong population structure among sampling localities. However, no correlation between genetic distance and geographical distribution were established.
Raw data was supplied and helped to understand authors findigs.
The text presented by authors sounds grammatically ok. However, I’m concerned about the scientific writing and sequence of ideas presented. I’ve found some elements of discussion that may be well suited for a introduction. Also, there are some parts of the results that sounds more familiar for a methodological statement or for the discussion (I’ve pointed these issues below).
Figures must be reviewed. Some of them can be omitted and other must be improved due to the poor quality.
Experimental design: The research fits PeerJ scope and its questions are well defined and meaningful.  However, some concerns about methods must be checked by authors. They are listed in “Comments for the author”.
Validity of the findings: This research brought important findings concerning biological dispersion of E. macrophylla and its genetic diversity in the sampled area. Also, the lack of genetic diversity studies of island populations for this species justifies the research. However, there are important caveats that must be addressed before accepting this manuscript. So it is my opinion that the manuscript should not be accepted for publication in PeerJ at this time.
Additional comments: 1. Introduction: readers need more information concerning biological issues of E. macrophylla. Ecological and reproductive information of the species are needed to understand the results. Whats the mating system? Whats the main pollinators? How does the dispersion happen? How well this plant is fitted to the coastal/island ambient? Its important to know if a plant uses self or cross-fertilization to understand the diversity data based on molecular makers. Selfing may naturally decreases the magnitude of indexes like heterozygosity in plants and, this decrease may not necessarily represent a poor diversity for some species. So its important to give the elementary biological information of the species for readers understand your results.

    2. There are some questions about CDDP markers that must be addressed. As other papers suggested, authors justifies the use of this maker due to its reproducibility when compared with other dominant markers (e.g., RAPD and ISSR). However, even the CDDP developers (Collard and Mackill, 2009) warned that reproducibility problems also exists for CDDP, “suggesting that primer length and annealing temperature per se do not ensure reproducibility”. They recommend gel scoring based on at least two independent replicates of PCRs. It is not clear if authors were careful enough considering  the reproducibility problems. Reading the text, there are no elements discussing it. I’m concerned about the confidence of data using markers like this one, mainly when authors states that “weak but distinguishable bands” were scored after electrophoresis. Whats a “weak” band? How to decide when a band is “weak”? Replicates helps to improve the information quality and must be considered during the experimental procedure.

    3. As soon as authors described the method for DNA isolation and mention that the DNA is good on quality and in with sufficient amount, I believe that Figure 2 could be omitted. I suggest that authors provide figures concerning DNA isolation as supplemental files.

    4. Shannon's information index was presented in Material and Methods section and in Table 3. However, it was not described in the results and neither was discussed. Please, properly present this result and discuss it. Or, remove it from the manuscript.

    5. Figure 1 is poor in quality and must be improved. Its resolution is not good enough. We can barely see the province names (e.g., DGD, LS and LSD). We can also barely see the “islands” for the most of sampling localities. I cannot see that Liugong is an island in Figure 1. Are Da Rushan and Laoshan mainland areas? There are too much area in this figure that is not important for the manuscript comprehension. This parts of the figure may be omitted. I don’t think that the whole China map must be present (at least in this size). Emphasizes only what is important for readers understand the main findings.

    6. Some parts of the results presentation is only based on showing maximum and minimum values of the estimates. Authors mention measures like averages, but the findings description is focused on the exposition of extreme values.  E.g., check lines 174-179. It is important to provide the descriptive statistics of the data and show the general aspects of the genetic diversity. What authors can say about the diversity of populations that didn’t show extreme values?

    7. Based on what authors use 0.94 (genetics similarity) to split groups in Figure 4? Despite of the well defined separation of some populations, it is important to mention that the figure scale starts at the 0.88 level. For a more reliable definition of groups it is important to use re-sampling methods in clustering analysis, like bootstrap. Do the authors tried to use this approach? I also suggest using other methods to find out more reliable estimates of number of groups. E. g., Bayesian inference implemented in Structure software (it goes well with dominant data). Or a Discriminant Analysis of Principal Components (implemented in Adegenet package of R). The latter is also preferred to PCoA analysis used by authors due to some particularities of the genetic data. Despite of many references found in literature use PCoA, the percentage of variation explained by the first and second Coordinates is very limited when using molecular markers. It means that with PCoA we loose too much information when we consider only the two dimension plot in analysis. However, if the authors decide to keep using PCoA because they have got high percentage of the variation explained by the two first coordinates, please, show this result.

Specific comments:
    1. Line 48: A question: authors evidenced that the species distribution include Jiangsu Province. However this province was not included in the research. Is there an specific reason for not including this province, since it was in the middle of the other sampling localities?
    2. Lines 49-51: Please, insert a reference for this statement;
    3. Line 52: Please, insert a reference for this statement;
    4. Line 68: replace “prime” by “primer”;
    5. Line 112: As authors have not used a precise method for DNA quantification, how could they guarantee that they used 30 ng/µL in PCR reactions? If no method like spectrophotometer was used, it is important to mention that this is an approximated measure;
    6. Line 128: Does Hs stand for “interspecific” genetic diversity? Isn’t it “intra”? Or better, for authors case, “local genetic diversity”? Please, consider checking it because a single species is being considered in the manuscript.
    7. Line 133: R is considered a computational language environment. Is important to mention what kind of analysis was performed using R in methods section. Also, if authors have used specific packages, it is also important to mention them giving their respective references. Include also the R reference (R core team, year).
    8. Lines 174-179: estimates of genetic distance and similarity are inversely proportional. Authors don’t have to show both of them. In my opinion it is important to choose one estimate and use it alone so there will be no redundancy.
    9. Line 200: Is “PTS” population right? Isn’t it “PTD”?
    10. Lines 208-216: Statements presented in these lines look like a discussion. So, authors have to give these information in the proper section.
    11. Lines 245-247: Unlike authors affirm, the subpopulation pattern found in Figure 4 disagree with the grouping pattern found in PcoA (Figure 6).
    12. Lines 259-264: It is very hardy to compare CDDP marker results with microsatellite (SSR) results when discussing diversity indexes. The letter is codominant and multiallelic, which means that we always expect much more information from SSR than CDDP. A dominant marker like CDDP is interpreted as a bialellic marker and the highest level of heterozygosity expected is 0,5 (or 2pq). For SSR, higher values (e.g., above 0.9) can be found due to the huge amount of alleles found.  What I mean is that for a biallelic marker, Hs = 0.5 represents high diversity. However, for SSR, 0.5 of hetherozygosity may represent low level of diversity. Also, comparisons between CDDP and ISSR should be interpreted with caution, since ISSR are potentially not affected by selection. On the other hand, CDDP is developed under the information of functional genes and, naturally, we expect lower levels of variation for these genome regions. Authors suggested in Line 294 that “results of different molecular markers are not fully comparable”, even so they compared results of different markers with no concerning about the main differences among them.
    13. Line 309-311: Values of bird propagation distance reported in discussion (480-680 m) are based on unpublished data described in Chung et al., (2002). These distances were poor discussed in Chung et al. The article only cited this values in the INTRODUCTION section and they are not part of a empiric result. Even according to these authors, these values suggests "that seeds are dispersed OVER LONG distances by birds". In addition, this paper brought information about Neolitsea sericea species. I’m not sure if these findings are fully comparable to those found in the present manuscript. I believe that the dispersion by birds should be better explored by authors even with the small gene flow found among localities.
    14. Lines 315-317: (a) Angelique (2000) is not listed in References. (b) Whats the specific dispersion information for E. macrophyta? The Angelique (2000) information is for this species? If the ocean is the main responsible for dispersion in E. macrophyta, isn't the species well fitted to this situation? As a reader I need more information to understand this issue because gene flow is a very important issue in your article that must be better explored.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANALYSIS OF THE GENETIC DIVERSITY OF THE COASTAL AND ISLAND ENDANGERED PLANT SPECIES ELAEAGNUS MACROPHYLLA VIA CONSERVED DNA-DERIVED POLYMORPHISM MARKER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Analysis of genetic diversity in the coastal and island endangered plant Elaeagnus macrophylla by conserved DNA-derived polymorphism markers (#38915)” presents a molecular marker-based study of this species in China, concerning its diversity and population structure and is being reconsidered for publication in PeerJ.
There are significant improvements in this version, mainly considering the discussion and the implementation of new statistical analysis. Authors highlighted the main biological concerns related to their findings, which is a very important point to be included in the discussion. However, some questions listed in “General comments” of this review are still present and must be addressed before considering the manuscript publication.
Experimental design: As mentioned in the last review, the research fits PeerJ scope and its questions are well defined.
Validity of the findings: The study found low genetic diversity and high genetic differences among the populations surveyed. Authors claim that the lack of studies for E. macrophylla is the main reason for this study. The research is also justified by its ecological and commercial potential.
Additional comments: a)	Authors must check the PeerJ instructions for citing references. There are some references cited (mainly in discussion) that doesn’t fit the instructions. E.g.:
•Line 236: “Weller et al., 1996”; According to the journal rules, this paper has to be cited as “Weller, Sakay & Straub, 1996”. Please, check all the “three-authors” references.
•Lines 314-316: “Leng's research also mentioned that when the seeds of Ilex integra were soaked in seawater, the germination rate was approximately zero (Leng et al., 2005).” This isn’t the most appropriate way to cite a reference. I suggest something like “Leng et al. (1995) also mentioned that when the seeds of Ilex integra were soaked in seawater, the germination rate was approximately zero.” … or … “Seeds of Ilex integra also presented germination rate close to zero when soaked in seawater (Leng et al., 2005).” There are several references cited in this way within the Discussion.

b)	Lines 77-82: This statement is too long. Breaking the sentence may provide a clearer information. Also, correct the “of and” at the end of the line 77.

c)	Lines 132-134: Structure Harvester implements the method described by Evanno et al. (2005; doi: 10.1111/j.1365-294X.2005.02553.x). Authors have to insert this reference in the text since they have used the method.

d)	Lines 162-163: This is a “discussion” statement and should not be descried within “results” section.

e)	Lines 180-195: It's still not clear which criteria the authors used to split the samples in the different groups. I can see that the “considered groups” are based in the main branches observed in the dendrogram. However, this is a little arbitrary. Are these "groups" consistent? Based on what the authors know that they really have “true groups” in this analysis? Why not presenting the bootstrap to improve the statement? If they used the "cut-line" (e.g., based on the average similarity), it is not showed in Figure 4, neither in the “Material and Methods” section. It's important to make it clear, so the reader will know the basis of the method used to split the samples in different groups. Another way to avoid this problem is bringing the focus of the statement for the fact that no cross-clustering were observed among samples of different locations. Regardless of this information is present in lines 189-190, its sounds like a “secondary” information in the text.

f)	Lines 199-214: Considering that authors have used the Evanno et al. (2005) criteria (delta K) to detect the most probable number of clusters, the results of delta K must be showed in the manuscript. Authors showed values for K=3 and K=5. But I cannot evaluate if these solutions are the best ones for the samples since I don’t have the magnitude of delta-K values. Please, provide delta-K values or the graph showing the likelyhood for each tested K value. E. g., authors said that "when K = 5, the value of delta K is also large”. But, how large is it?

g)	Line 248: “delicious” is a very relative terminology. I’m not sure if Zang (2016) have really used this word but, in general, it is not well suited in scientific language.

h)	Line 330: It’s interesting that Gst values indicates that NJD can be arranged in two subpopulations. However, this information was not presented in the Results section. It is also important to mention this approach in Methods, saying that the Gst index were calculated within localities when significant differences among specimens were found.

i)	Include the DOI in the references listed.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ANALYSIS OF THE GENETIC DIVERSITY OF THE COASTAL AND ISLAND ENDANGERED PLANT SPECIES ELAEAGNUS MACROPHYLLA VIA CONSERVED DNA-DERIVED POLYMORPHISM MARKER
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Analysis of genetic diversity in the coastal and island endangered plant Elaeagnus macrophylla by conserved DNA-derived polymorphism markers (#38915)” presents a molecular marker-based study of this species in China, concerning its diversity and population structure.
In this third review round we still have some minor questions to be addressed (see “General comments”), but, at this time, the manuscript presents most of the necessary elements to be accepted.
Experimental design: As mentioned in the first review, the research fits PeerJ scope and its questions are well defined.
Validity of the findings: The study found low genetic diversity and high genetic differences among the populations surveyed. Authors claim that the lack of studies for E. macrophylla is the main reason for this study. The research is also justified by its ecological and commercial potential.
Additional comments: Lines 124-125
Replace:
… and gene flow (Nm=1-Gst/Gst) among populations or localities when significant differences among specimens were found.
By:
… and gene flow (Nm=1-Gst/Gst) among populations. The estimates were also calculated within sampling localities when significant differences among specimens were found.

Lines 134-137
Please, revise this statement considering its grammatical improvement
Compress the result of the operation (".zip" format) and upload it to the Structure Harvester website (http://ztaylor0.bi-ology.ucla.edu/structure) to get the analysis result of the K value (Evanno, Regnaut & Goudet, 2005.)

Lines 227-230
Replace:
Frankham compared and analyzed the allelic diversity of 202 groups of land and island
populations of various species, including mammals, birds, fish, reptiles, insects and plants; in
165 cases (81.7%), the genetic diversity of island populations was lower than that of terrestrial
populations, with an average decrease of 29% (Frankham, 1997)
By:
Frankham (1997) compared and analyzed the allelic diversity of 202 groups of land and island
populations of various species, including mammals, birds, fish, reptiles, insects and plants; in
165 cases (81.7%), the genetic diversity of island populations was lower than that of terrestrial
populations, with an average decrease of 29%.

Line 288
… clustering analysis shows that there was no hybridization among individuals
Isn’t it supposed to be “there was no hybridization among individuals from different localities”?

About Delta-K values
Despite authors have answered in rebuttal letter that the Delta-K values were provided in supplemental files, I believe it could be presented within Figure 5 (e.g., Fig. 5B). This result is important to prove how good are the model chosen by authors after Bayesian analysis.
Besides, I also have tow additional comments/questions about Structure results:
a)	It’s clear that Structure pointed that E. macrophylla genetic diversity can be arranged into three main genetic groups. There was also a substructure found considering 5 genetic groups. Authors described very well the importance of the 5 subgroups. However, in discussion, the 3 main genetic groups were neglected. What are the main conclusions about these 3 main genetic groups?
b)	The occurrence of subgroups in NJD population was well described by authors. They also repeated some analysis considering only this samples (line 216; Gst and Nm of the two sub-populations of NJD). However, its clear for me that they also have another subpopulation structure in DRS samples. It can be observed in Figure 5 and, also, in the dendrogram. Part of this specific result for DRS is mentioned briefly in results, while in NJD we have an specific section to show this findings (line 216; Gst and Nm of the two sub-populations of NJD). A discussion about this result is also absent in the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POTENTIAL USE OF HUMAN HAIR SHAFT KERATIN PEPTIDE SIGNATURES TO DISTINGUISH GENDER AND ETHNICITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The background contains incorrect statements and misinterpretations of the literature.  These need correction.  See details below.
Experimental design: The 2D gel method is inferior to contemporary methods.  It dramatically limits the scope of the analysis.  Several aspects of the methods require more detail.  See details below.
Validity of the findings: If the authors want the reader to believe the conclusions, a more streamlined and quantitative approach is needed.  The alkaline SDS extraction eliminates too high a fraction of the total protein and 2D gels require too much manipulation of the samples as well as making most of the sample unusable.
Additional comments: This manuscript has a laudable goal, and the results, if believable, would be a considerable step forward.  However, the manuscript has serious shortcomings that must be addressed and is not convincing.  The main problem is the use of 2D gels.  This is an outmoded technology and is totally unjustified in this instance.  The authors are limited to comparisons of only the small number of peptides that are separated cleanly, thereby unable to use the vast majority of the protein (not quantitated but probably >>90%).  Enthusiasm and confidence are substantially eroded by incorrect statements.
[1] The Background and Discussion claim erroneously that the hair shaft is useless for forensic investigation if it does not contain nuclear DNA. The authors appear unaware of recent reports that protein profiling of the hair shaft shows promise in this direction, and the genetically variant peptides in the proteome are likely to provide definitive identification (Proteomics 17(13-14), 2017. doi: 10.1002/pmic.201600462).  Moreover, the Introduction is mistaken in that next generation sequencing of mtDNA is not limited to STRs and can be quite discriminating (Genes 9(1), 2018. pii: E49. doi: 10.3390/genes9010049).
[2] The authors cite Yamauchi et al (2008) for their assertion that the hair shaft is 80% keratin.  Actually, this reference does not do a determination but reports the work of others that the cortex is 80% of the hair shaft.
[3] The authors use their recently published (2016) method of alkaline extraction of hair, claiming to get higher yields.  No evidence is given in the present manuscript for higher yields, and the reference reports a yield of <50%.  Other methods, subjecting the entire hair shaft protein to digestion, have yields of 90%.
[4] In the Methods, the authors say they recorded phenotypic characteristics and excluded chemically-treated hair.  The reader needs to know how they determined which hair was chemically treated.  Visual inspection alone is not satisfactory.  This is critical because a high proportion of females bleach, perm or color their hair.  Not excluding some of those samples could seriously bias the results they report and could account for their reported male/female and subpopulation differences.
[5] How a supernatant was obtained by pulverizing undissolved hair shaft fractions (line 113) is not clear.
[6] How the focused strips were “equilibrated using DTT and iodoacetamide” (line 30) is not clear, nor how this did not lead to collapse of the pH gradient.  Moreover, if the purpose is to alkylate the cysteines, the subsequent reduction and alkylation of gel plugs (line 162-163) is redundant.  If it is not redundant, perhaps due to incomplete alkylation earlier (line 30), then this could be a source of variability in IEF and thus in the spot densities that are analyzed and thereby negate the results.
[7] The Discussion states that SDS and urea “are known to cause deamidation and disruption of disulfide bridges”, citing Adav et al, 2018.  Actually, Adav et al referred to deamidation from degradative/ageing processes, not denaturant treatment, and disrupting disulfide bonds is assisted by denaturants but requires a reducing agent such as DTT.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POTENTIAL USE OF HUMAN HAIR SHAFT KERATIN PEPTIDE SIGNATURES TO DISTINGUISH GENDER AND ETHNICITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: to distinguish gender and ethnicity” reports to show that there are proteins in hair that could be biomarkers associated with gender and bioancestry (based primarily on abundance). The work is preliminary and may have some value. But a bit more data are needed.
The authors have shown there is an abundance difference for some identified proteins. The abundance may be real. To support their findings they present x fold differences observed on the 2d gels. There needs to be some discussion about the robustness of the analysis. There are a lot of proteins that overlap and some areas on the gel are quite dense with protein. These areas appear that they may no longer be linear in signal. If so, then it seems suspect about rendering any abundance values. Can the authors provide support for the use such data and/or discuss the limitations.
The authors use the significant difference in abundance to infer gender and bioancestry. Yet there are probabilities, likelihoods, or confidence intervals placed on the observations. It is important to address uncertainty when offering a comparative analytical test.
Lines 53-55 – the authors cite a 2006 reference for little or no nuclear DNA. They need to search the literature better as such work was reported a decade earlier. Moreover, the statement that mtDNA cannot be used for genotyping is simply wrong. There are many papers describing the use of mtDNA. It may not be as powerful as nuclear DNA but has been used in many forensic cases as well as evolutionary studies.
Line 84 – I looked up the Wong et al paper and it is not the primary paper describing the alkaline lysis procedure. The method is cited (by the same authors) within that paper. It is important to cite the original article. However, alkaline solubilization of hair is hardly novel. It was first described by Budowle, B. and Acton, R. T. In: A technique for the detection of variable electrophoretic patterns of hair proteins. Electrophoresis 2:333-334, 1981. The authors should cite studies that have preceded their own work. Also, lysis seems an incorrect description as that refers to breaking open cells. The protein is likely being solubilized from the matrix. Perhaps the authors could correct terminology.
Line 126 – provide %C and buffers used for second dimension mass separation; provide the pH range or ampholytes used for the first dimension.
Line 138 – cite the silver stain method
Line 252 – the hair is not useless if no nuclear DNA, as mtDNA can be used. The mtDNA has higher discrimination power than the protein profiles described by the authors and it does provide bioancestry (from the maternal line).
Experimental design: Generally okay, but insufficient. Not rigorous regarding addressing statistical uncertainty.

Methods need more citation or details.
Validity of the findings: May not be statistically sufficient - need to address uncertainty and support for abundance assessments
Additional comments: The authors have shown there is an abundance difference for some identified proteins. The abundance may be real. To support their findings they present x fold differences observed on the 2d gels. There needs to be some discussion about the robustness of the analysis. There are a lot of proteins that overlap and some areas on the gel are quite dense with protein. These areas appear that they may no longer be linear in signal. If so, then it seems suspect about rendering any abundance values. Can the authors provide support for the use such data and/or discuss the limitations.
The authors use the significant difference in abundance to infer gender and bioancestry. Yet there are probabilities, likelihoods, or confidence intervals placed on the observations. It is important to address uncertainty when offering a comparative analytical test.
Lines 53-55 – the authors cite a 2006 reference for little or no nuclear DNA. They need to search the literature better as such work was reported a decade earlier. Moreover, the statement that mtDNA cannot be used for genotyping is simply wrong. There are many papers describing the use of mtDNA. It may not be as powerful as nuclear DNA but has been used in many forensic cases as well as evolutionary studies.
Line 84 – I looked up the Wong et al paper and it is not the primary paper describing the alkaline lysis procedure. The method is cited (by the same authors) within that paper. It is important to cite the original article. However, alkaline solubilization of hair is hardly novel. It was first described by Budowle, B. and Acton, R. T. In: A technique for the detection of variable electrophoretic patterns of hair proteins. Electrophoresis 2:333-334, 1981. The authors should cite studies that have preceded their own work. Also, lysis seems an incorrect description as that refers to breaking open cells. The protein is likely being solubilized from the matrix. Perhaps the authors could correct terminology.
Line 126 – provide %C and buffers used for second dimension mass separation; provide the pH range or ampholytes used for the first dimension.
Line 138 – cite the silver stain method
Line 252 – the hair is not useless if no nuclear DNA, as mtDNA can be used. The mtDNA has higher discrimination power than the protein profiles described by the authors and it does provide bioancestry (from the maternal line).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POTENTIAL USE OF HUMAN HAIR SHAFT KERATIN PEPTIDE SIGNATURES TO DISTINGUISH GENDER AND ETHNICITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Literature reference inaccurately interpreted.
Experimental design: The authors use an approach (2D gel) that prevents analysis of most of the protein.  What remains for comparison is only a tiny fraction of the total.
The comparison (small differences between large numbers) depends on accurate normalization.  Linearity of the "volume"measurements is not addressed.  It is hard to know whether the measurement is saturated in regions of the gel with the highest protein concentration.
Validity of the findings: The original goal and the results, if believable, are interesting.  However, the shortcomings undermine the conclusion.
Additional comments: This manuscript is considerably improved, especially with respect to correction of some misinformation in the previous version.  Several points need further attention, however.
1] An important advantage of the present approach is the claim of higher protein yield using the alkaline extraction method.  Lines 70-71 refer to extraction of a “substantially higher percentage of hair shaft proteins within only 2 hrs”.  Lines 207-208 refer to yields “generally higher compared to the methods of Lee et al (2006)….”  Line 267 refers to their “newly developed protocol that enhanced the extraction yield of proteins from the human hair shaft….”  Line 210 cites “an average extraction yield of 63.56 +/- 6.59”, presumably % of the starting amount, but does not explain how this figure was obtained.  How much protein the method of Lee et al (2006) would extract after 2 hr was not reported, but close reading of the methods section of that paper reveals that “the final insoluble material comprised 13.3 +/- 3.9% of the total protein”, which means about 86% was soluble.  Thus the presumed higher yield of the alkaline SDS method is not substantiated by the cited literature.  The speed of the method could be an advantage but is canceled by coupling it with an unnecessary 2D gel separation.
2]  The authors still refer to the 2D gels as giving “highly resolved profiles” (line 36).  The resolution may well be higher than before, but the protein available to be analyzed is still only a tiny fraction of the total.  “A larger scale analysis … using antibodies” could indeed “increase the robustness of the results” (line 323-325), but this ignores the likelihood that a direct mass spectrometric analysis (no 2D gels) is going to be more accurate than western blotting.
3]  Figure 1 has two problems.  First, it strains credulity that spot 7 can be analyzed with the accuracy claimed.  Poorly resolved (smeary) 2D gel spots often contain more than one protein.  Second, the source of the protein in panel A is not given.
4]  Although the methods are greatly improved, with considerable detail now included, verification of linearity of the volume measurements is not addressed.  This is critical for the normalization of spot amounts for comparison of the different samples and would be inaccurate if, for example, the measurement saturates in regions of the highest protein content.
5.  The rationale for limiting the crime scenes to “old” ones (line 25) is unclear.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMMUNOSTIMULATORY ACTIVITY OF POLYSACCHARIDES FROM GLYCYRRHIZA URALENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study from Aipire et al. investigates the immunostimulatory effect of Glycyrrhiza uralensis in vitro and in murine vivo studies and is definitely worthy of publication. The experiments carried out, fit into the logic of argumentation and are all in all well performed. Minor changes have to be done to enhance its quality.

Some general English language changes have to be made.

- The authors should think about changing DC into DCs if they are using the plural form of multiple cells to increase reading fluency.

Specific changes:
29: add an article to "mouse"
28: add an article to "immune" and a comma before "and" (same error is repeated several times)
49: "therefore DCs are pharmacological targets of immunostimulatory agents..."
108: article to "supernatant"
139: strange sentence --> rearrange or add an article to "photo"
200: add article
add several articles to nouns in the whole text! there are several errors!
Experimental design: Materials and methods:

- please mention how you isolated the cells where the BM-DCs were derived from with GM-CSF and also add information about the culture conditions.
- where did side-effects observe in mice injected with CTX?
- be accurate: you show +SEM and SD and not +/- SEM and SD as described under statistics.
- what LPS concentration was used for the in-vivo studies?
- please add the number of replicates and what is presented to the figure legends, also mention if data are representatives (fig. 4?)
- what t-test did you utilize, because 5 animals per group may not fulfill test requirements

Results:

- please explain the different purpose for the use of BM-DCs and Mo-DCs
- specify the difference between control and untreated
- you only used 20ng LPS but startet from 1 µg GUPS --> discuss the differences in the dose dependent effects
- fig. 3: % negtive is a rare cell analysis and maybe the MFI of CFSE would also be helpful. Alternative: show unstained T-cells and start the gating from these population so that no junk is included (as seen at 10^0?!)
- fig. 6: highlight key results and important changes!
- fig. 8 and table 1: you have to discuss the smaller thymusses in the LPS group. Why are they smaller than the control thymuses?
- fig. 9a: color-coding would be helpfull (rethink that for some other figures too)

Figure legends:

- put more attention to the figure legends (see above)
Validity of the findings: Discussion and conclusion:

- you mention signaling pathways but only investigated some factors. Explain how they interfere with each other or make an overview.

The experiments you carried out are not enough to conclude the therpy as "without side-effects" and as "safe and effective".
- please specify that you only have hints regarding safety levels
- specify the use for cancer treatment

To better clarify the use of the study the authors
should discuss the possible clinical application of Glycyrrhiza uralensis:
- possible side-effects or hyper-inflammation (septic shock, application routes?)
- possible other TLR agonists as LPS or imiquimod, that is currently used in the therapy of cancerogenious lesions.
Additional comments: For further investigations, it would be helpful to investigate the M1/M2- polarization of macrophages, as a very relevant setting for immunity and tumor defense.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMMUNOSTIMULATORY ACTIVITY OF POLYSACCHARIDES FROM GLYCYRRHIZA URALENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There is sometimes a lack of information to understand the reasons for the following experiments.
Experimental design: In general, the initial observations of this paper are very interesting, however, not to be despised part of the data is interpreted incorrectly or the experimental planning is partly not well designed.
The other major drawbacks of the paper are the superficialities or inconsistencies.
Validity of the findings: The validity of the data and statements can be improved. Based on the data too much is speculated.
Additional comments: Please, work on the experimental design and care of your interpretation and presentation of the data.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CANDIDAL CARRIAGE IN SALIVA AND SUBGINGIVAL PLAQUE AMONG SMOKERS AND NON-SMOKERS WITH CHRONIC PERIODONTITIS—A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see attached PDF page with all comments to the authors.
Experimental design: The experimental work was well conducted. Though, some explanations are needed to justify the use of morphological characters only to identify Candidal isolates? and why the authors only chose to test very few antifungal agents? Other comments are highlighted in the enclosed PDF report.
Validity of the findings: Please see attached comments.
Additional comments: Please make sure to check spelling and language.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CANDIDAL CARRIAGE IN SALIVA AND SUBGINGIVAL PLAQUE AMONG SMOKERS AND NON-SMOKERS WITH CHRONIC PERIODONTITIS—A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.The rules in scientific naming of organisms should be adhered to
2. Rephrase the statement in line 287-291 and clearly describe a point at a time. The presence of diversity of Candida spp. should be discussed separately from the point of C. albicans dominating with clear reference of each.
3. Should only discuss the results presented. Since female were not included in the study line 291-296 should be deleted.
4. It is important to explain how did the stated limitations affect the study findings for reproducible of the study
5. With citation clear description of the relationship between smoking and C. krusei and C. tropicals is warranted
6. Conclusion should built from discussion that based on the study findings. Line 384-387 should be rephrased
Experimental design: 1. More clarification on the pooled subgingival pockets samples. How were they pooled, how many subgingngival pockets were pooled together, what happened in the culture positive pooled samples
2. Any scientific explanation of excluding female participants even when the population of smokers is pronounced to be low they still had the right to participate. Wont there be any interesting finding in this small
3. More description of the inclusion criteria in line 143-145 are warranted. Did the investigator considered all the mentioned criteria for each patients? Were there any superior criteria in recruitment?
4. Methodology need more detail. Clear description of Fagerstom test  in line 166-167 is required
5. With reference clear description on how were the measurement taken in line 169-177 used in the study. What was concluded if some measurement were abnormal while others are normal in a single patients? Which measurement weight more than others
6. More details on methodology line 221-224 regarding any discrepancies between the 24 hrs readings and 48 hrs reading. Which reading was considered, How many times were the test repeated in case of discrepancies
7. What was the considered level of confidence interval on statistical analysis
8. What was the considered normal range of the mean plaque index
Validity of the findings: 1. For easy understandings of the reader all the reports on the abstract should be reported with frequency and percentage
2. All statistical significant data should be presented with their statistical test and strength
3. Quantitative part which is highly pronounced in the title is neglected in the abstract section
4.More details on the meaning of fair correlation and marginally significant correlations  as used in line 278-280. Statistical numbers representing the terms should be used
Additional comments: 1. Authors should be carefully with scientific naming of bacteria
2. In abstract the repeated world "as" should be deleted
3. It is not clear what was the sample size. Was it 90 or 30 more clarifications are needed
4. Sentence should not start with number
5. Several grammatical errors need corrections before publications example use of capital letter in the middle of sentence and small letters at the beginning of the sentences
6. some words are joined and lose the meaning example in abstract, conclusion section "....althoughC albicans ...."


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CANDIDAL CARRIAGE IN SALIVA AND SUBGINGIVAL PLAQUE AMONG SMOKERS AND NON-SMOKERS WITH CHRONIC PERIODONTITIS—A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is clearly written with good background and context being provided.
Experimental design: The methodology was well defined with sufficient information. Some questions are however still need to be answered as shown in the attached comments document.
Validity of the findings: The conclusions need to be revised. The authors concluded that  smoking significantly contributed to the colonization effect by Candida among their study group.  However, the same trend was evident among the non-smokers. Only when you compare these two groups with the healthy group, then there seem to be some differences and it is mainly in the saliva samples.
Additional comments: Please see the attached document.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CANDIDAL CARRIAGE IN SALIVA AND SUBGINGIVAL PLAQUE AMONG SMOKERS AND NON-SMOKERS WITH CHRONIC PERIODONTITIS—A CROSS-SECTIONAL STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: The article is well written, clear and conform with professional standards.
Experimental design: The experiments were well designed and executed.
Validity of the findings: The data  presented was very sound and controlled. Conclusions were linked directly to the questions raised and were supported by the findings of the results.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A SIMPLE AND EFFICIENT CLONING SYSTEM FOR CRISPR/CAS9-MEDIATED GENOME EDITING IN RICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: -	the paper was written correctly, although minor language errors are present, such as "second round of PCR to producing" should be "to produce".
-	the introduction should focus more on the topic of research and not on CRISPR technique and achievements in general. There is a lack of information about what other methods are used to obtain plasmids
-	the results contain a lot of information that is repeated from the methods
-	there is no comparison as to how this method compares with others
-	there is no information in the literature regarding
Arite et al. 2007;
 Arite et al. 2009
Khan et al. 2017
-	In turn, Symington LS &Gautier J (2011) does not appear in the manuscript
-	in the manuscript table s2 is before table s1
-	no restriction sites are marked in table s2 (Line 111)
-	Fig 3 i 4:
colony PCR rather than clone PCR
incubation and plasmid extraction rather than palsmid
Transformation into….
-	Fig 5 a and b
more clearly mark the editing places and PAM
-	Fig 5d sign the size marker bands and add the product from the control plant
Experimental design: There is a lack of details in the methodology so that the presented technique can be easily used in one's own research. The purpose of this research is to propose an alternative method to others, so the more it should be very clear and leave no doubt.
Validity of the findings: no comment
Additional comments: no comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A SIMPLE AND EFFICIENT CLONING SYSTEM FOR CRISPR/CAS9-MEDIATED GENOME EDITING IN RICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants. Genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA via Agrobacterium tumefaciens-mediated transformation or particle bombardment. Although CRISPR/Cas9 integration increases the chance of off-target modifications and foreign DNA sequences cause legislative concerns about genetically modified organisms, transformation of plants with Cas9-guide RNA (gRNA) expression cassette DNA is most popular and widely used approach for making genome-edited plants.
	In the manuscript (peerj-42378), the authors established simple and efficient system for constructing CRISPR/Cas9-gRNA expression vectors to produce genome-edited rice plants. In this system, overlapping PCR and Gateway/Golden Gate system were successfully used for reducing the step and time for vector construction. The reviewer considers that present CRISPR/Cas9-gRNA expression vector construction system is highly useful to reduce molecular lab works such as PCR, subcloning, ligation. However, the reviewer thinks that the manuscript is too preliminary to be published in the The PeerJ.

The reviewer thinks that readers cannot understand what the authors conducted for producing new donor and destination vectors even if they carefully read Materials and Methods section. To avoid this, detailed flowcharts for the procedures of making donor and destination vectors should be presented as Figures (or Supplementary Figures), and please refer these figures in Materials and Methods section. Although showing several figures in Materials and Methods section is not so usual in scientific journal, it is not problematic but will be recommended for the present manuscript because contents of this study are mostly protocol-like for efficient producing of the CRISPR/Cas9-gRNA expression vector. Although present Figures 1 and 2 are flowchart-like, however, these figures are too rough to understand precise procedure. Figures 3 and 4 are also just outline, and important technical information are mostly omitted.

Lines 271-273: Please add descriptions for the estimated size (bp size) of long deletion of inter-region between targets 1 and 2 in OsRCD gene in the text, and discuss whether the estimated size fits with the experimentally detected deletion size in Figure 5D, or not.

Figure 5A and B: Sanger sequencing chromatograms for control and mutants should presented as top-bottom set (aligned) to clearly show the mutated position. In addition, please add information for the exact insertion/deletion regions like as “+1, A”, “-2 GG)” and etc.

Figure 5D: Information of base pair length for maker DNA is essential to estimate the size of deletion.

Supplementary Table 1: Please show number of plants that possessed both editions/mutations in targets 1 and 2 of OsRCD gene.