Evaluation of Reference Genes for Gene Expression Analysis in Eichhornia crassipes
Round 1
Reviewer 1 Report
Eichhornia crassipes is a notorious invasive aquatic plant, causing enormous ecological and economic losses worldwide. However, little is known about this plant at the molecular level. This work examined the expression of 12 candidate reference genes in samples of different tissues, samples treated with various hormones, samples supplied with different levels of phosphorus (P), and pooled samples, analyzed their expression stability using GeNorm, NormFinder, BestKeeper and RefFinder, and finally selected suitable reference genes for each experimental conditions This is the first report of selection of reference genes for qRT-PCR in E. crassipes. Therefore, this manuscript has enough novelty and scientific significance, and is worthwhile being published. In general, this manuscript is written in normative English. Besides, the experiments are set up soundly, and the results were well interpreted and discussed. Yet, there are several suggestions as following:
1. I would suggest the author add more details to the ingredients of half modified Hoagland solution mentioned in 2.1.
2. in 2.2, the author mentioned that “All RNA samples meeting the criterias, including A260/280=1.6-2.1, A260/280=1.8-2.0, and showed bright and intact bands on gels, were used for the following experiments.”. Normally, the A260/280 value of RNA used for qRT-PCR should be higher than 1.8, why did the author use 1.6 as the minimum standard?
3. there are several other publications mentioned qRT-PCR experiments on E. crassipes, for example the studies from Fu et al (2014, 2018), the author may include these work in the discussion as well.
Author Response
The authors are very grateful to the reviewer's comments. Here are the reponses:
Point 1: I would suggest the author add more details to the ingredients of half modified Hoagland solution mentioned in 2.1.
Response 1: Thank you for your suggestion. The detailed prescription of the half-strength modified Hoagland solution has been added.
Point 2: in 2.2, the author mentioned that “All RNA samples meeting the criterias, including A260/280=1.6-2.1, A260/280=1.8-2.0, and showed bright and intact bands on gels, were used for the following experiments.”. Normally, the A260/280 value of RNA used for qRT-PCR should be higher than 1.8, why did the author use 1.6 as the minimum standard?
Response 2: Thank you for your suggestion. Yes, the A260/280 value of RNA used for qRT-PCR normally should be higher than 1.8 to ensure the efficiency of following experiments. However, this value is affected by the solvents and RNA isolation procedures. In this study, we used RNA Prep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich) (TianGen, China) to isolate total RNA. The A260/280 value is possible lower than 1.8 due to the chemicals used in this kit to remove polysaccharides and polyphenolics. According to the instruction of this kit, A260/280 ≥1.0 is acceptable as long as the RNA has no degradation. Moreover, the RNA quality was verified by the gel electrophoresis, and no RNA sample showed degradation. Therefore, all RNA used for following qRT-PCR in this study was good enough.
Point 3: there are several other publications mentioned qRT-PCR experiments on E. crassipes, for example the studies from Fu et al (2014, 2018), the author may include these work in the discussion as well.
Response 3: Thank you for your suggestion. The two articles you recommended were added to the references.
Reviewer 2 Report
Comments and Suggestions
Ø The purpose of research in the present study “Evaluation of reference genes for gene expression analysis in Eichhornia crassipes” not justified properly or seems to be meaningless. Off course molecular techniques catch to the reader and researchers but research must be economic feasible, Authors should not mentioned proper hypothesis, why authors select and design this expensive research, please justified.
Ø Entire manuscript need to substantially revised with proper English and adding more suitable recent references (please see doi: 10.1111/plb.13382) for biochemical and other traits (https://doi.org/10.1016/j.sjbs.2022.01.030 ; https://doi.org/10.1016/j.envres.2022.113081; https://doi.org/10.3390/ijms23116242) etc.
Ø Line 69 to 79 need to re write and adding little account on ecological and economic importance of study.
Ø Objective of research is not clear, please re write.
Ø Line 90 to 92 not clear, add clear experimental design Plants were routinely grown in trays containing ….by adding suitable reference (Pot experiment design etc. see reference DOI: 10.5958/2320-642X.2019.00021.8; for green house https://doi.org/10.1111/plb.12173)
Ø Line 94 to 96 rewrite, please add Cleary about plant hormone.
Ø What is mean of half modified in material and method section like Line number 100, please correct and explain in entire manuscript.
Ø Write clear statistics, which used in present research.
Ø Again discussion section not write properly, many sentences are read to hard and unable to understand such as line 279 to 285 and 290 to 292.
Ø Separate conclusion section and write very clear about findings and way forwards of the research also justified your added hypothesis. .
Comments for author File: 
Comments.pdf
Author Response
The authors are very grateful to the reviewer's suggestions. Here are the response:
Point 1: The purpose of research in the present study “Evaluation of reference genes for gene expression analysis in Eichhornia crassipes” not justified properly or seems to be meaningless. Off course molecular techniques catch to the reader and researchers but research must be economic feasible, Authors should not mentioned proper hypothesis, why authors select and design this expensive research, please justified.
Response 1: Thank you for your suggestion. But, I would not agree that this research is meaningless. qRT-PCR is a prevalent techonology to study gene function, and the selection of suitable reference genes is a key step to ensure the accurance of analysis of qRT-PCR data. Moreover, different reference genes may be used in different experiment setups within one specific species. Therefore, it is indispensable to evaluate and select reference genes for specific experiment setups ahead of performing qRT-PCR to understand gene function. E. crassipes is an important invasive plant, which, however, has various utilizations. But its gene function-related research has been rarely performed, and none work regarding selection of reference genes on this plant has been published. This work could favor future research on studying gene function of E. crassipes, which could enable us better understanding on the mechanisms underlying its invasion and utilization. Besides, there are plenty of research on evaluation of reference genes on other plants, animals and microorganisms. Therefore, we believe this work exerts enough novelty and scientific significance.
Point 2: Entire manuscript need to substantially revised with proper English and adding more suitable recent references (please see doi: 10.1111/plb.13382 ) for biochemical and other traits (https://doi.org/10.1016/j.sjbs.2022.01.030 ; https://doi.org/10.1016/j.envres.2022.113081; https://doi.org/10.3390/ijms23116242) etc.
Response 2: Thank you for your suggestion. However, I checked these publications you recommoned, and they are irrelevant to the topic of or the methods used in this study, except for this one (doi: 10.1111/plb.13382), which evaluated the reference genes in Arabidopsis in response to temepture changes. Since this work aims to select reference genes in different tissues of E. crassipes , and in E. crassipes in response to hormones or P levels stimuli, we cited publications on the subjects of qRT-PCR, commonly used reference genes in plants, softwares that used to analyzing our data, the biology and significance E. crassipe, and other studies involves with similar treatments. We did included more relevant references in the latest version of manuscript.
Point 3: Line 69 to 79 need to re write and adding little account on ecological and economic importance of study.
Response 3: Thank you for your suggestion. This part has been re-written, and the ecological and economic importance of study was highlighted, please see the changes in the latest manuscript.
Point 4: Objective of research is not clear, please re write.
Response 4: Thank you for your suggestion. This part has been re-written to make the objective more clear, please see the changes in the latest manuscript.
Point 5: Line 90 to 92 not clear, add clear experimental design Plants were routinely grown in trays containing ….by adding suitable reference (Pot experiment design etc. see reference DOI: 10.5958/2320-642X.2019.00021.8; for green house https://doi.org/10.1111/plb.12173)
Response 5: Thank you for your suggestion. This part has been re-written to make the experimental design more clear, please see the changes in the latest manuscript. However, we looked through the two articles you recomonded very carefully and we do not think it’s suitable to cite them in this work for the following reasons: 1) for the reference you recommonded for pot experiment design (DOI: 10.5958/2320-642X.2019.00021.8), that work cultured plants with field soil in pots, whilst our work employed hydroponic system to grow E. crassipes. The two work used very different system to grow plants. 2) as to the reference you recommonded for greenhouse, the authors simply mentioned “A growth chamber pot experiment” without describing any growth conditions.
Point 6: Line 94 to 96 rewrite, please add Cleary about plant hormone.
Response 6: Thank you for your suggestion. This part has been re-written, please see the changes in the latest manuscript.
Point 7: What is mean of half modified in material and method section like Line number 100, please correct and explain in entire manuscript.
Response 6: Thank you for your suggestion. Modified Hogland Solution is a widely used nutrition solution in plant culture. Since E. crassipes grows too fast in modified Hogland Solution, this research used half strength of modified Hogland Solution. The detailed recipe has been added in section 2.1, please see the changes in the latest manuscript.
Point 8: Write clear statistics, which used in present research.
Response 8: Thank you for your suggestion. The data presented in this research was generated by the four softwares mentioned in the manuscript. The output data of these softwares contains no statistics. Similar data presentation can be found in other studies on reference genes selection, which have no statistics as well. But we added some sentences to explain the algorithms of the four softwares, please see the changes in the latest manuscript.
Point 9: Again discussion section not write properly, many sentences are read to hard and unable to understand such as line 279 to 285 and 290 to 292.
Response 9: Thank you for your suggestion. This part has been re-write, please see the changes in the latest manuscript.
Point 10: Separate conclusion section and write very clear about findings and way forwards of the research also justified your added hypothesis.
Response 10: Thank you for your suggestion. This part has been re-write, please see the changes in the latest manuscript. According the authors’ guildlines of Sustainablity, if the conclusions are concise,there is no need to separate the conclusion section. Therefore, we placed the conclusions in the last paragraph of Disscussion.
Round 2
Reviewer 2 Report
Authors not mention proper hypothesis again, methodology are not very clear and methods adopted by authors are not properly cited and supported by relevant citations, again statistics portion in not improve only software story highlighted by authors, few statements in discussion part look like a part of conclusion and author not separated conclusion properly in revised manuscript. Ecological importance, novelty and significant of study is not clear again in revised manuscript. After critical examination of revised manuscript, I find that authors addressed very casually taken to comments and suggestion. Therefore, I recommended to rejection of this revised manuscript.
Dear author, I examined very carefully your responses and find that vary casual approach was adopted during revision, Authors stated in response section “This work could favor future research on studying gene function of E. crassipes, which could enable us better understanding on the mechanisms underlying its invasion and utilization. Besides, there are plenty of research on evaluation of reference genes on other plants, animals and microorganisms. Therefore, we believe this work exerts enough novelty and scientific significance”.
Science and research not based on your belief, this must require sufficient explanation with data, even I found that authors not addressed properly about hypothesis of the present research.
Dear author, the cited manuscript does not highlight about less report of your chosen study and unnecessarily you write your study is less reported to gain attention towards novelty of your presented work, this way you miss guide to the researchers. Authors try to demonstrate the novelty of research on the basis of a few reports such as Fu et al., 2014, 2018 and Zhang et al., 2021, (line 79-81).
Author Response
Please see the attachment。
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
No need
