Forage Performance and Detection of Marker Trait Associations with Potential for Napier Grass (Cenchrus purpureus) Improvement

Round 1

Reviewer 1 Report

Dear authors,

Thank you for the resubmitted manuscript as well as for your reply to my review and your kind words.

However, up to my best knowledge, I have had difficulties to find out how did you follow, as you wrote, all my suggestions and corrections? Thus I would appreciate very much if you would find time, discuss in written and give a point to every of my suggestion, from the first review. I have to admit that I am used to this; as I have always received such a discussion from the authors and also have been preserving answer to the reviewers, myself.  

Only when I will receive all of your comments, I dare to make a review to your re-submitted manuscript.

Thank you very much on your co-operation and kind regards,

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript has been deeply improved after a first reviewing. Authors have followed most of my own suggestions. The use of allometry relationships of quality traits with biomass allows a better analysis of the variation of these trait among the genotypes. Authors can be deceived by the low proportion of variance explained by these allometries...But in fact taking into account these relationships should reduced substantially the variance between genotypes, and then the residual variation should remains more easy to interpret. In fact allometry are ontogénic relationships between a variable X(t) representing quality trait and crop mass Y(t)...during the crop growth process... But these relationships can be affected by different environment factors such as "Plant N nutrition status" ... "Plant N water status" etc... So they are not univocal. In consequence I find the results are very coherent: allometry with crop mass allows to explain about 30% of the total variance... so the residual variation corresponds in fact to difference of quality trait among genotypes when they are compared at similar biomass (when the effect of size of the crop has been subtracted) that allows a more accurate comparison...

I think authors should try to discuss this aspect a little bit more deeply in their discussion-conclusion. 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This paper deals with analysis of genotypic variability of Napier Grass for forage production (dry mass) and forage quality (Crude Protein and Me talolizable Energy). Authors used a very classical statistic model for comparison of several plant traits across cultivars.  These comparison of plant traits are made without any attempts for formalizing the functional relationships existing among these different plant traits. So comparisons made by ANOVA are blind as they does not integrate the basic knowledges related to the functional correlations existing among some of the traits used in this study. So authors seems to ignore the fundamental link existing between "plant size" and some plant architecture characteristics such as Leaf/stem ratio,or stem fiber content, or plant N concentration (or Crude Protein concentration)... all these trait having a high influence on forage nutritive value...  So the consequence of this ignorance.... is that authors discovered the fundamental trade-off between forage production and forage quality through the existence of a strong correlation between forage dry matter production and each of the forage quality trait. 

During preceeding decades, forage quality od different forage species has been evaluated according to plant phenology stages with a very strong normative approach, leading then to forage quality prediction in relation with plant species phenology observation. This approach has been totally contradicted by several authors working both on temperate legumes such alfalfa or temperate grasses (tall fescue or thimothy) where it has been shown that the decline in both N (or CP) concentration and in Digestibility of forage was not driven by phenology... but was the ontogenetic consequence of the increase of plant size according to a fundamental allometry between "metabolic plant tissue" linked to light interception and photosynthesis and "structural plant tissues" necessary for supporting and positionning leaves in the upper- well-illuminated layers of the plant canopy. ( see several fundamental papers and the recent review published in MDPI-Agriculture by Lemaire and Bélanger, 2019; authors should find there all the literature references and the theoretical framework describing these fundamental allometries).

So what to do with these results? I think as well indicated by authors in their manuscript, the fundamental morphogenetic parameter would be LSR Leaf/Stem ratio. So as LSR declines allometrically with crop mass (W) it would be necessary to use this allometry for calculating deviation to this allometry according to cultivars... Are differences in LSR observed among cultivars due to differences in forage mass? and Are deviations to the allometry significant? In the same way: Are the N concentration vs Crop mass allometry explain the difference among cultivars or are the deviation to this general model is significant?

In the same way... Is the difference in forage quality between season explained by difference in growth potential?

It is clear that owing to the strong and negative effect of forage mass on forage quality... any comparison between cultivars for forage quality must be made at similar biomass. Without this "biomass correction"... the comparison will be biased by the difference in biomass leading to the conclusion that the cultivar with the lowest forage mass will have the highest forage quality... that is an obvious conclusion.

So I suggest you to read carefully the literature cited in Lemaire and Bélanger (2019 in MDPI-Agriculture. Then to realize you ctatistical comparison of Napier Cultivars after having established the allometric relationship between quality traits and crop mass and calculated the deviation to model for each cultivar... It is not a too heavy task and the quality of your paper and its scientific value would be highly improved.

 

Reviewer 2 Report

The manuscript Forage Performance and Detection of Marker Trait Associations with Potential in Napier Grass (Cenchrus Purpureus) Improvement submitted by the Ermias Habte, Meki S Muktar, Asebe Abdena, Jean Hanson, Alieu Sartie, Alemayehu T Negawo, Juarez Campolina Machado, Francisco José da Silva Ledo and Chris S Jones is presenting the results about phenotyping and genotyping of forage crop Napier grass as well as correlations between the traits including environment factors and genetic markers.

 

Studying this forage crop in Ethiopia seems to be important for the dairy production and beyond. Improving of this neglected (?) crop, increasing the production and exploiting good traits could also be related to land use and climate change.

 

The manuscript contains relevant data and results. I consider that developing phenotyping and genomic tools for neglected crops is very important nowadays, this manuscript thus being great example of it and its potential for practical use.

 

However the manuscript is in need of extensive revision. The manuscript looks unfinished with many parts that are still lacking in almost every section starting from Introduction, while e.g. methodology and results are presented with mistakes. Some statements are missing to be supported either by results or references while particularly discussion should be strengthened with the inclusion of other relevant studies.

 

Introduction

It would be very comprehensive to complement Introduction with at least several more issues:

It is not very clear what kind of plant is Napier grass (elephant grass?) and why is it important to study it? It is used as a forage for dairy cattle; what is its` economic importance locally (for the region in which study is performed) and globally? What might be the importance or meaning of Napier grass in a sense of climate change i.e. greenhouse gas emission when correlated with rumen methane production? What is global importance and potentials of this grass?

Nutritional quality (or chemical composition?) is studied. Please describe its meaning and relevance in Introduction; same as phenotyping in Napier grass. Is this equivalent to “forage quality”?

Please provide overview on recent genetic analyses, existing genomic tools for Napier grass, lack of genome sequence (?) and availability and reasoning of other genomes that can serve as reference genomes.

Please provide information about recent taxonomy of Napier grass as well as most important biological characteristics of the species.

Please provide information about selection and breeding efforts.

Furthermore:

Line 31. Which Institutes?

Line 35. “Commonly improved” I am not sure what it does mean?

Line 45. Which countries? What is origin of Napier grass accessions conserved in Brazil; or Brazilian EMBRAPA, having in mind that you wrote that NG is native to Sub-Saharan Africa? Why did you choose to introduce Brazilian genotypes and not some other from other of several institutes?

Line 47. Which studies? Please provide reference(s).

Line 65. Why not all 53?

Line 70. Please describe (define) biomass yield; and metabolizable energy. From what are they comprised?

Line 74-75. Please elaborate this in detail in discussion.

MATERIALS AND METHODES

Materials and methods must be supplemented with additional information particularly in case of genetic analyses, starting from which genotypes have been used for this part of experiment; what tissues for DNA isolation? How DNA was isolated and so on… Please provide all important details as in case of description of phenotyping trial. Dividing phenotyping trial and evaluations and genetic analyses in different sub-sections under Materials and Methods and following same pattern in results might be one solution for making manuscript more comprehensive.

Table 1. Please provide more information about the genotypes used in the study. It should be given origin of the genotype; typical or specific characteristic(s) important for this particular study, place where the genotypes are conserved or available from. Also, later on in Line 430-431; I read that purple Napier grass is suddenly mentioned for the first time. Please provide this information in Table 1 for all 45 genotypes (are they all purple or how?), as well as other data relevant for the study.

Line 98. Is this data collection for biomass yield? Did you collect samples for DNA analyses at the same time?

Line 113. Which traits? Please describe!

Line 113-114. Please provide reference. Take care that you support your statements with appropriate reference throughout the text.

Line 117. Please rewrite “As described elsewhere [17]”, in order to be easier to read and more understandable. Please avoid similar phrases throughout the text?

Line 114. Please provide reference!

Line 115. Are those analyses of data from 2.3 and 2.4? Not clear from the title.

Line 124. Define biomass yield in the manuscript. Does it comprise TFW or TFW+TDW or TFW+TDW+LSR…..same for chemical composition? Clarify beforehand.

Line 132. How do you get F values? Where do we see it? How do we find out which traits are with significant F values?

Line 133. Give name of the authors.

Line 134. Plural of formula?

The careful, precise and correct naming, description and citation of the software used have to be presented in the manuscript. Outputs have to be clearly linked to the software throughout the text. I have checked 2 and find putative errors. Please check all and rewrite the manuscript accordingly:

Which version of GeneStat software has been used? 19 or 19.1. Please clarify, take care on this and similar cases and provide precise and concrete reference (give the reference for the software at the place where it is first time mentioned). Or did you use two versions of software. Please clarify. I was not able to find a version 3_1 of software DeltaGen 3_1? Could you please clarify this?

Line 142. I would rephrase description of pattern analysis in order to be easier to understand.

Another thing is that later in the text only term PCA is used. I consider this misleading as, up to my best understanding, this is not only PCA as PCA is performed as a second step in pattern analysis as e.g. as multivariate analysis of clustering. This can be misleading so please clarify this throughout the text.

Is Figure 2 an output from DeltaGen software or more precisely pattern analyses performed? Please clarify all this; rewrite it in a way that is easy to read and understand.

Accordingly, give a thorough and detailed enough title to the Figure 2. E.g. meaning of colors?...

Line 144. Please clarify which traits.

RESULTS

Please either divide results for biomass yield and feed quality; or put it all together. Tables mentioning in the text is not correct. Table 4 cannot be mentioned before 3. Please improve. Also, I would say that Table 4 belongs to section 3.2. as feed quality results are presented in greater detail there.

Line 165. Which genotypes? Present very concretely.

Line 175. What does it mean “top” in top yielding genotypes? Which genotypes concretely?

Line 176. Up to my best understanding this is not correct? Please clarify! If true, please check all the rest of your results.

Table 2. Please avoid using abbreviation in titles (in all tables!). Please add units of measurements beside traits (in all tables!) like in table 3. Please use symbol for multiplying instead of letter X. Letter X has a different meaning! Please round number to two decimals; or otherwise. (also in all tables!).

Line 182. Where is: ns = non-significant, * P < 0.05 (5%), ** P < 0.01 (1%) and *** P < 0.001 (0.1%) in table 2?

Table 3. Clarify all abbreviations.

Table 4. Would you be so kind to clarify what is the logic behind ordering the traits? What does it ASH stand for?

Lines 199-201. Wet or dry?

Line 203. Which genotypes? Same in line 204?

Line 208. This is not correct. What is in table A1?

Lines 213-218. Give the detailed result in tabular view. Like B1.

Lines 218-219. Up to my best understanding this is not correct. Please clarify!

Lines 220-246 are telling about leaf + steam. What about leaf?

Lines 248-249. Again, is it now only PCA performed? Or do you mean biplot outputs from pattern analyses? Same across the text!

Please give full names of traits in the text of the manuscript. Abbreviations should also be beside. Otherwise it is impossible to read.

Lines 253-263. Again, please associate coloring and groups given in Figure 2.

Line 274. Please replace concomitantly with some more appropriate term.

Table 6. What does ASH stand for?

Please explain section 3.4 also in methodology!

Line 290. Scatter plots – missing in methodology!

Line 312. How do you mean replicated? Explain in methodology.

Lines 319-322. Where can we find this?

Line 337. Table S1 does not exist as such? Please clarify!

Please rephrase lines 339-342. Very hard to understand.

Line 350. Up to my best understanding Supplementary Table A2 does not contain those results! Please sort this out?

Lines 350-351. Is this result from other, published study? Please clarify!

DISCUSSION

Please strengthen discussion with other relevant, comparable studies.

Line 362. Might be useful to emphasize that genotypes were introduced from Brazil.

Line 382-383. Where can we see those results?

Lines 384-385. Values?

Lines 385-387. Is this differential result for Ethiopian and Brazilian Napier grass genotypes studied in Ethiopia/Africa? Or how? Please describe this more comprehensive.

Lines 403-404. Describe in Materials and Methods!

Lines 403-407. Requires more discussion.

Lines 418-422. Please be so kind and rewrite this in more comprehensive way.

Line 432. Which findings?

Lines 445-447. To what this can be attributed? Can you tell?

Lines 455-457. Would it still be possible to present best genotypes considering coupled biomass and energy production? Is it always negatively correlated? Please discuss this more.

Line 459. In Table 2 Appendix B I can see 81 markers listed, not 82. Is this something else?

Line 463. What greater number?

Lines 469-470. Where is this described? Lines 481-482. Please provide result or reference. Please clarify what is the meaning of it for this study?

Please provide the meaning of identified genes very concretely for this study?

Line 486-487. Please rephrase the sentence.

Line 487. How cytochrome P450 was associated with plant population?

Line 492. Which ones, again?

Line 495 and 497. Why italics?

Lines 497-500. I do not understand this.

Conclusions are hard to read. I would also avoid using other references; or citation as this should be done beforehand, in discussion.

Lines 515-517. I see as a crucial (?) but not straightforward to understand.  Throughout the manuscript, the emphasize is on highest biomass producing genotypes; although this is compromised with high fiber content that is challenging digestibility quality. Therefore, is it possible to also underline those genotypes being best from the feed quality view? Or are they the same? This is confusing in the manuscript and should be cleared out and presented straightforwardly.

OVERALL COMMENTS

What is correct scientific name? Please use always the same starting from the title; and please write only name in italics…

Please use synonym name being elephant grass elsewhere in the text and not only as a key word.

Please spell the numbers 1-9.

It would be very important to synchronize use of terms throughout the text. E.g. Feed quality, chemical composition, forage quality, nutritional quality, feed nutritional quality… Are those ones all the same? Please clarify! Same example with biomass yield, biomass productivity, forage yield, forage biomass yield, agronomic performance… Are those ones all the same? Etc.

Please take care on providing result or reference for the statements given in the manuscript. This is often lacking.

Here it comes also ordering of the terms. Please use always same ordering e.g. alphabetical; or other logical ordering. Please always clarify in relevant places in manuscript.

I would improve titles of tables and figures in a way that every table and figure represents an entity understandable even if taken out of the text. Make it extensive enough and easy to read and understand.