Very Early Biomarkers Screening for Water Deficit Tolerance in Commercial Eucalyptus Clones

Round 1

Reviewer 1 Report

The authors present the manuscript with a serious solid research to identify and validate traits for tolerance to water deficit in eucalypt. It is a comprehensive study of mixed models, selection indexes and validation schemes estimation based on final plant height, increment in height, increment in diameter, area of mature and fully expanded leaf, nutrient contents of N, K, Ca, Mg, S, Cu, Zn, Mn and B, photosynthesis (A) and stomatal conductance (gs). The conclusions are based on clearly planned experiments and are in no doubt. The nature of the 15 selected phenotypic biomarkers support interconnected and different strategies, contributing to water stress tolerance in eucalypt. The choice for biomarkers to be used depend on the species being evaluated. These results contribute much to the fundamental analysis of genotypic parameters and early selection, and provided heritable and highly accurate biomarkers for efficient selection of water deficit tolerant Eucalyptus genotypes.

I think it is generally optimistic. Several minor items need to be revised or clarified as follows.

Line 51: “There are growth, physiological, anatomical, morphological, nutritional, gene expression and metabolism traits, among others, that contribute to water deficit tolerance” In the introduction, it is suggested to add some application of selected biomarkers in other species.

Line 78: “Initially, experiment was carried out with 22 eucalyptus genotypes, of which 3 were non-commercial and there was no information on their resistance to water deficit and, therefore, were not considered for this analysis.” If the other 3 genotypes are not used in this manuscript, they cannot be mentioned in the materials section. If necessary, they could be explained in the discussion section.

Line 88: “Six of these climatic zones exhibit annual rainfall ranging from 1,000 to 2,000 mm with two defined dry and rainy seasons that last about 6 months each.” It is suggested that the information of climate zone and genotype distribution should be corresponded by using supplemental table.

Line 151: “2 – 300 g L-1 PEG (or simply 300PEG), the standard nursery procedure (control) plus with water stress simulation with the application of 100 mL of a solution of polyethylene glycol 6000 (PEG) at a concentration of 300 gL−1 per pot, every two days during the experimental period; 3 – 100 mL L-1 water (or simply 100H2O), the standard nursery procedure (control) plus with irrigation restricted to the application of 100 mL water per pot per day.” I feel confused by the statement of “2 – 300 g L-1 PEG (or simply 300 PEG)” and “polyethylene glycol 6000 (PEG).”

Line 176: “The freezing point of the solution sample in degrees Horvet (°H) was converted into MegaPascal (MPa) according to [11]” Should be “according to the previous method [11].”

Line 476: “Likewise, it is intriguing that biomarkers such as stomatal conductance and photosynthesis were only significant when evaluated on the 30th (first evaluation), but not on the 60th days (second evaluation) in the present report.” I think this is a very reasonable phenomenon. Please refer to the difference between long-term and short-term stress for analysis.

Line 502: “Among the selected morphological biomarkers, "plant height", "increase in height", "increase in diameter" and "leaf area" showed significant heritability. Although plant height and height increase are easily measurable traits, the evaluation for early selection should be concomitant to the estimation and analysis of the other biomarkers.” In this case, why not further divide these biomarkers into early biomarkers and late biomarkers?

Line518: “Among the evaluated nutrients, N, K, Ca, Mg, S, Cu, Zn, Mn and B are emphasized in the selection of water deficit tolerant genotypes and described as appropriate biomarkers for early selection.” Here I also suggest clearly distinguishing early selection and late selection, rather than described in the order stated in the results.

Line 532: “Photosynthesis (A) decreases under water stress and is also related to the leaf area, two important biomarkers for ranking water deficit tolerant eucalypt genotypes (Tables 4 and 5).” Where is table 5? I suggest a list to show which biomarkers are strongly related or sub-strongly related, and also to distinguish for early selection and late selection.

Line 605: “Manganese (Mn) was indicated for early selection of eucalypt clones, as it has a positive association with tolerance to water deficit. Mn acts as a cofactor for many antioxidant enzymes, such as ascorbate peroxidase, SOD and CAT, which are vital for development of plant when under stress conditions as they capture free radicals [44].” However, try to explain why ascorbate peroxidase, SOD and CAT are not used as biomarkers?

Line 655: “For the confirmation of the early selection based on the phenotypic biomarkers, together with the Mulamba and Mock index, the leave-one-out validation scheme was performed. This type of validation is useful to generate confidence in the application of the proposed methodology, therefore if the early selection biomarkers are efficient, they can provide good results.” The statement here is vague. Which phenotype is used for early selection?

 

Author Response

Reviewer #1

 

Line 51: “There are growth, physiological, anatomical, morphological, nutritional, gene expression and metabolism traits, among others, that contribute to water deficit tolerance” In the introduction, it is suggested to add some application of selected biomarkers in other species.

The line numbers were changed, so, we identified the text sections highlighting it, including the performed amendments. We were in doubt if the suggestion and adjustment should deal with eucalypt species or wood tree species in general. In order to fulfill this suggestion, and avoid the introduction of more references, we chose to use the review Picoli et al (2021), reference number [8] already in use, that allow us to mention some of these uses and deduce others.

“Growth and adaptability traits are considered as markers for water stress tolerance and baseline for comparison for different eucalypt species [8]. Considering the ease access and effectiveness of traits such as petiole anatomy, allometric measures of plant height, stem and treetop diameter, and mineral content such as boron and calcium may have diverse uses such as evaluating phenotypic plasticity and GxE interactions and uncovering biomarkers associated with growth and quality traits. Since these biomarkers can be evaluated in other plant species, and bearing in mind that phenotypic plasticity is expressed in them, similar approach may also be useful for plants other than eucalypts.”

 

Line 78: “Initially, experiment was carried out with 22 eucalyptus genotypes, of which 3 were non-commercial and there was no information on their resistance to water deficit and, therefore, were not considered for this analysis.” If the other 3 genotypes are not used in this manuscript, they cannot be mentioned in the materials section. If necessary, they could be explained in the discussion section.

It was amended.

“The experiment was carried out with 19 eucalyptus genotypes, which performance data were made available by the forestry companies.”

 

Line 88: “Six of these climatic zones exhibit annual rainfall ranging from 1,000 to 2,000 mm with two defined dry and rainy seasons that last about 6 months each.” It is suggested that the information of climate zone and genotype distribution should be corresponded by using supplemental table.

We agree with the suggestion, nevertheless, we will need to ask for routine and planning information of two companies, Suzano S/A (Suzano and former Fibria) and Vallourec. This will demand a great time that is not available and still be denied as may be interpreted as strategical by the companies. Unfortunately, this information is not in our possession to disclose. For this reason we withdrawn this paragraph.

Even though, we already requested Suzano S/A the information on climate zone x genotype distribution. We also amended the item “Data Availability Statement” mentioning that climate zone and genotype information may also be requested.

 

Line 151: “2 – 300 g L-1 PEG (or simply 300PEG), the standard nursery procedure (control) plus with water stress simulation with the application of 100 mL of a solution of polyethylene glycol 6000 (PEG) at a concentration of 300 gL−1 per pot, every two days during the experimental period; 3 – 100 mL L-1 water (or simply 100H2O), the standard nursery procedure (control) plus with irrigation restricted to the application of 100 mL water per pot per day.” I feel confused by the statement of “2 – 300 g L-1 PEG (or simply 300 PEG)” and “polyethylene glycol 6000 (PEG).”

It was amended.

“Water deficit treatments was provided by the application of 100mL of a solution of polyethylene glycol 6000 (PEG), at 300 g L⁻¹ or by restricting the amount of water that was poured in the seedling recipient (plastic bag) to 100 mL L^-1 water per day.

The treatments are addressed as: 1 – Control treatment, plants growing in a greenhouse in plastic bags containing 2 L of carbonized rice, fertilizer, average temperature of 25°C, natural light and daily irrigation; 2 – PEG treatment (or simply 300PEG), the standard nursery procedure (control) plus water stress simulation with the application of 100 mL of a PEG solution 300 g L^-1 of per pot, every two days; and 3 – 100 mL L^-1 water treatment (or simply 100H₂O), the standard nursery procedure (control) with irrigation restricted to the application of 100 mL water per pot per day.”

 

Line 176: “The freezing point of the solution sample in degrees Horvet (°H) was converted into MegaPascal (MPa) according to [11]” Should be “according to the previous method [11].”

It was amended as suggested.

 

Line 476: “Likewise, it is intriguing that biomarkers such as stomatal conductance and photosynthesis were only significant when evaluated on the 30th (first evaluation), but not on the 60th days (second evaluation) in the present report.” I think this is a very reasonable phenomenon. Please refer to the difference between long-term and short-term stress for analysis.

About the results on photosynthesis, that is correct! Differences, particularly in the physiological variables, may be a reasonable phenomenon, for instance, due to acclimatization process to an imposed stress condition. We opted to correct this sentence agreeing with this observation and rewrote the sentence.

“Likewise, biomarkers such as stomatal conductance and photosynthesis were only significant when evaluated on the 30^th (first evaluation), but not on the 60t^h days (second evaluation), what was attributed to a possible acclimatization process. Nevertheless, this may comprise a disadvantage of the use of physiological biomarkers, since they are more prompt to oscillations than the others, and will need a thorough approach. In the present report our objective was to identify, validate biomarkers that will ease and help the selection process for water deficit tolerance in eucalypt. ”

…

“Further, there seems to be no consensus on the standards for water deficit tolerance tests [8]. The present results open up a glimpse for these guidelines, at least for early selection for water deficit tolerance, as were successful in the discrimination of more tolerant and more susceptible eucalypt clones considering a genetic base consisting of four species used commercially.”

 

Now, about the long-term and short-term stress for analysis suggestion. We toke the liberty to use a surrogate, but simpler, definition from Dhabhar e McEwen (1997): “Short-term stress has been defined as stress that lasts for a period of minutes to hours, and chronic stress as stress that persists for several hours per day for weeks or months.” This suit the following articles consulted, except for Neither et al (2020).

We did not evaluate “short-term”/”long-term” responses, such as could be applied to Schulze and Küppers (1979), Yang et al (2014) and Niether et al (2020), although our plant model individuals must have gone through the stress phases proposed as delineated by Lichtenthaler (1998), to date: response phase (alarm reaction, beginning of stress), restitution phase (stage of resistance, continuing stress), end phase (stage of exhaustion, long-term stress). Unfortunately, our physiological measurements occurred only at the 30^th and 60^th under stress conditions, while we did not plan further morphological and nutritional measurements during the experiments, neither performed additional steps in the experiment conduction, as Neither et al (2020), except for initial plant high. It is worth mention that these authors attributed “short” and “long-term” stress as plants submitted to a 20 and 89-day stress treatment period, which may diverge from an original concept of short and long-term analysis (Schulze and Küppers 1979; Yang et al. 2014; Sun et al 2022). The plants were maintained under water deficit stress since the PEG solution was applied every two days and the other treatment, the restriction of the amount of water poured in the plastic bags. The water stress treatment lasted 60-days. Further, the physiological measurements were performed at the same period early morning and mid-day, aiming reducing possible differences among measurements and standardization.

Therefore, we advocate that bringing elements, such as context of “short term” and “long term” stress analysis, in the present manuscript may lead to confusion. Nevertheless, if reviewer 1 understand that it is an essential contextualization, we will insert this discussion in a second round of the review process.

 

Dhabhar FS, McEwen BS. Acute stress enhances while chronic stress suppresses immune function in vivo: A potential role for leukocyte trafficking. Brain Behavior & Immunity. 1997;11:286–306.

Lichtenthaler, H.K. (1998). The stress concept in plants: an introduction. Ann. N.Y. Acad. Sci. 851, 187–198.

Niether, W., Glawe, A., Pfohl, K. et al. The effect of short-term vs. long-term soil moisture stress on the physiological response of three cocoa (Theobroma cacao L.) cultivars. Plant Growth Regul 92, 295–306 (2020). https://doi.org/10.1007/s10725-020-00638-9

Schulze ED, Küppers M. Short-term and long-term effects of plant water deficits on stomatal response to humidity in Corylus avellana L. Planta. 1979 Jan;146(3):319-26. doi: 10.1007/BF00387804. PMID: 24318185.

Sun M, Yang Z, Liu L, Duan L. DNA Methylation in Plant Responses and Adaption to Abiotic Stresses. Int J Mol Sci. 2022 Jun 21;23(13):6910. doi: 10.3390/ijms23136910. PMID: 35805917; PMCID: PMC9266845.

Yang C, Liu J, Dong X, Cai Z, Tian W, Wang X. Short-term and continuing stresses differentially interplay with multiple hormones to regulate plant survival and growth. Mol Plant. 2014 May;7(5):841-55. doi: 10.1093/mp/ssu013. Epub 2014 Feb 4. PMID: 24499771.

 

Line 502: “Among the selected morphological biomarkers, "plant height", "increase in height", "increase in diameter" and "leaf area" showed significant heritability. Although plant height and height increase are easily measurable traits, the evaluation for early selection should be concomitant to the estimation and analysis of the other biomarkers.” In this case, why not further divide these biomarkers into early biomarkers and late biomarkers?

In our manuscript the “early selection” is attributed to the evaluation being performed in younger plants as a way to predict their behavior in the adult/productive period without the need for waiting a longer period of time. Accordingly, “early” refers to a precocious stage in tree lifespan. Further, this definition and use of “earl selection” is mentioned in other articles, such as approached in Pinto et al (2014) and Corrêa et al. (2017), references that were used in this manuscript.

In this a priori evaluation, the seedlings have around 6 months old. Since our goal is to foresee or estimate the clone behavior or phenotype when achieving the harvest age, further division of the biomarkers will probably cause misperception.

On the same hand, all the mentioned morphological biomarkers, "plant height", "increase in height", "increase in diameter" and "leaf area", their differences and attributed phenotypic plasticity, will be the result of exposing the seedlings to a stress stimulus in a controlled and repeatable manner for a delimited period of time. Consequently, in order to achieve the values for these traits (biomarkers), the plants need to be submitted to the stress and the differences observed consist a way to estimate the phenotypic plasticity and the expected tolerance, water deficit in the present context, will be evaluated concomitantly at the end of the experiment.

Despite of that, we acknowledge the context of “early and late selection”, probably, associating it with short-term and long-term stress analysis, as expressed by the reviewer. Therefore, we added a definition of the framework used in the present manuscript.

“Accordingly, in the present report, “early selection” is defined as the use of traits of plant individuals, at a younger stage, in the evaluation process in order to be used as predictors of features in their productive age, and allowing anticipating genetic gains [6,7].”

 

Line518: “Among the evaluated nutrients, N, K, Ca, Mg, S, Cu, Zn, Mn and B are emphasized in the selection of water deficit tolerant genotypes and described as appropriate biomarkers for early selection.” Here I also suggest clearly distinguishing early selection and late selection, rather than described in the order stated in the results.

Please, observe to the argument exhibited above dealing with morphological biomarkers.

 

Line 532: “Photosynthesis (A) decreases under water stress and is also related to the leaf area, two important biomarkers for ranking water deficit tolerant eucalypt genotypes (Tables 4 and 5).” Where is table 5? I suggest a list to show which biomarkers are strongly related or sub-strongly related, and also to distinguish for early selection and late selection.

Please, observe to the argument exhibited above dealing with morphological biomarkers.

Unfortunately, there as a mistake in calling tables 4 and 5, the later which is inexistent in the present report. The information and citation were amended.

“Photosynthesis (A) was a significant biomarker (Table 2) and decreased under water stress and is also related to the leaf area, two important biomarkers for ranking water deficit tolerant eucalypt genotypes (Figures 2 and 3). ). Similar behavior was observed for divergent eucalypt clones under water stress [9].”

 

Line 605: “Manganese (Mn) was indicated for early selection of eucalypt clones, as it has a positive association with tolerance to water deficit. Mn acts as a cofactor for many antioxidant enzymes, such as ascorbate peroxidase, SOD and CAT, which are vital for development of plant when under stress conditions as they capture free radicals [44].” However, try to explain why ascorbate peroxidase, SOD and CAT are not used as biomarkers?

In the present research we evaluated physiological, nutritional and morphological/growth biomarkers. Unfortunately, we did not evaluate stress enzymes, although we acknowledge their role to plants cope with water stress conditions. Unfortunately, we don’t have this data. Our goal was to link the references and information of possible associations that would grant manganese a different concentration among tolerant/susceptible eucalypt phenotypes. Further, as mentioned in the last paragraph of the manuscript introduction, “The option for this set of biomarkers depends on the ease and its common use in the routine of commercial nurseries for propagation of eucalypt”. In our view, we opted to use biomarkers that, essentially could be adapted or easily incorporated in a routine of a breeding program.

However, if reviewer 1 deem it an unsuitable relation (Nutrient – Mn èenzyme), we will withdraw the mentioned reference and discussed item.

 

Line 655: “For the confirmation of the early selection based on the phenotypic biomarkers, together with the Mulamba and Mock index, the leave-one-out validation scheme was performed. This type of validation is useful to generate confidence in the application of the proposed methodology, therefore if the early selection biomarkers are efficient, they can provide good results.” The statement here is vague. Which phenotype is used for early selection?

In the present manuscript we aim provide a routine for telling apart divergent clones according their more evidenced tolerance or susceptibility to water deficit. Hence, identifying the divergent clones, tolerant or susceptible to water deficit, a eucalypt breeder may choose a negative or positive selection according other demands and eucalypt clone characteristics.

It was further mentioned as amended in the text.

“For the confirmation of the early selection based on the phenotypic biomarkers, aiming the identification of eucalypt tolerant clones, together with the Mulamba and Mock index, the leave-one-out validation scheme was performed.”

The phenotype used as reference was the tolerant ones, based on the information we had from the commercial clone behavior in the field, and the selected biomarkers.

As in the Leave-one-out validation we had only 19 clones, the model was run 19 times, always leaving one clone out of the analysis, as in the following: 18 clones in the training group, 1 clone in the validation group. Since we have 19 clones, there were 19 rounds of the analysis. In the following, the results of the 19 rounds (predictions, one prediction for each clone) were gathered in one set, and a single estimate for the predictive capacity was performed (E.G. Pearson correlation).

The former information “The phenotype used as reference in this analysis was the phenotype of the tolerant clones, based on the information we had from the commercial clone behavior in the field, and the selected biomarkers.” was inserted in the manuscript. If necessary, we may also insert the remaining explanation for the Leave-one-out validation as well in a second round of revision.

We hope that the amendments performed, and replies to the reviewer’s comments, have fulfilled their requirements. Although we think the flaws of the manuscript were corrected and the main suggestions of the reviewers were duly addressed, we are willing to have the English language editing, if still necessary.

Reviewer 2 Report

I have listed some corrections below, but it does not mean that they are exhaustive.

 

Page 2, line 77 "…Materials and methods…"

All the subtitles in this section are disorganized, incomplete, etc. They require a thorough review.

 

Page 4, line 134 "...The seedlings were kept in a greenhouse with transparent plastic cover (Figure 1A to 1C)..."

Figures 1A and 1B do not correspond to images of a greenhouse, although Figures 1C to 1F do. This error should be corrected by re-writing both the materials and methods section and the legend of Figure 1. Nowhere in the manuscript is it indicated what Figures 1E and 1F represent. Additionally, somewhere in the materials and methods section, it should be indicated what Figures 1A and 1B represent, as they do not correspond to those of a greenhouse.

 

Page 4, line 139 "...After the acclimatization period, seedlings of similar size and development were selected to compose the experiment..."

What parameters did the authors use to say that the plants had the same size (size of what?) and the same development (development of what?)?

 

Page 4, line 161, "…(Figure 1A, 1B and 1D)…"

The same error as in line 134, page 4.

 

page 7, line 340 "...based on the average of ranks [19].."

This is redundant, it's already cited in the materials and methods.

 

page 7, linea 340 "…The lowest rank value indicates a beneficial condition for the water deficit tolerance trait. Likewise, the sum of ranks of established biomarkers will provide different numbers, the smaller the combination of traits or biomarkers the more favorable to tolerance and, the larger will indicate a more favorable combination of traits or biomarkers to susceptibility to water deficit…"

This shouldn't be here. It should be in materials and methods

 

page 8, line 348 "…(Table 3)…"

The Table 3 is good, but the authors should create a graph with the data that generated the ranking to have a more enriching view, since this result is central to the work. What about Pearson's correlation coefficients?

 

page 8, line 355 "…Of the 15 variables evaluated, only those selected by the model are represented in Figure 3 and appear in the boxes…"

Correct this sentence with something similar to the following: "..Figure 3 only includes and displays the variables that were selected by the model out of the 15 that were evaluated, and these selected variables are the ones that appear in the boxes.."

 

page 8, line 357 "… When the bar is blue, the contribution of the variable is positive in the tolerance probability, on the other hand, when the bar is red, the contribution is negative in estimating the probability…"

This is not a result! It should be in the figure's legend!

 

In general terms, the work is well developed, as are the experiments and techniques employed.

 

However, the manuscript lacks a well-structured discussion in its entirety. Therefore, the discussion section requires a thorough revision. It contains paragraphs that do not provide a contrasting discussion between the results of this work and the previously published works. Examples of such paragraphs are found on page 13, lines 457-466, and page 14X, lines 485-494.

 

Overall, particularly in the materials and methods and discussion sections, it appears that the manuscript was written without a comprehensive review by all authors. Therefore, the manuscript requires an extensive revision to correct many formalism errors, which are necessary for the work to be of scientific quality.

Author Response

Reviewer #2

 

I have listed some corrections below, but it does not mean that they are exhaustive.

 

Page 2, line 77 "…Materials and methods…"

All the subtitles in this section are disorganized, incomplete, etc. They require a thorough review.

We understand the reviewer 2 point of view and tried to ameliorate the organization of the section. Nevertheless, we argue in our favor that, rather than incomplete, we went down to several details (such as mineral nutrition, light conditions, and seedling management). Some details that were omitted depended on each forestry company and would turn the manuscript rather wordy. This was reasoned to describe as far as we could the procedures that were conducted.

If reviewer 2 understand that the needed through review we will work on the summarize of the information, what we foresee, indeed, will turn the report incomplete.

 

Page 4, line 134 "...The seedlings were kept in a greenhouse with transparent plastic cover (Figure 1A to 1C)..."

Figures 1A and 1B do not correspond to images of a greenhouse, although Figures 1C to 1F do. This error should be corrected by re-writing both the materials and methods section and the legend of Figure 1. Nowhere in the manuscript is it indicated what Figures 1E and 1F represent. Additionally, somewhere in the materials and methods section, it should be indicated what Figures 1A and 1B represent, as they do not correspond to those of a greenhouse.

We are terribly sorry for this mistake. It went unperceived as figure 1 was further handled to focus on our research and avoid the mention of activities that were not performed as activities of this project. For instance, the deleted images approached the photographic documentation of adult plants in the field by the occasion of one of the evaluations performed by Suzano. This was withdrawn thought that we were not responsible for this field evaluation, hence, it was not supposed to be exhibited in this manuscript.

Despite of that, if reviewer 2 deem it is proceeding we may update the material and methods section with this further information in a second-round review, clearly expressing that phenotype evaluation in field were performed by each company.

The updated figure 1 was inserted.

 

Page 4, line 139 "...After the acclimatization period, seedlings of similar size and development were selected to compose the experiment..."

What parameters did the authors use to say that the plants had the same size (size of what?) and the same development (development of what?)?

It was amended. Since we are dealing with different eucalypt clones, and that they may have little differences in plant height according each genotype and time they were delivered at the nursery, we may provide a further Supplemental table with the average initial height of each clone if necessary, although we think it may be excessive. In our view, this information will be superfluous considering that, if anyone, us included, will repeat the experiment or selection procedures, we will face similar variation according genotype, routine and provision of the seedlings, nursery management differences, etc.

 

“Considering that seedlings were approximately the same age, after the acclimatization period, seedlings of similar size were selected to compose the experiment. Plant height was used as size parameter and standard for the plant chosen for the experiment.

 

Page 4, line 161, "…(Figure 1A, 1B and 1D)…"

The same error as in line 134, page 4.

Please, observe the argument mentioned above.

 

page 7, line 340 "...based on the average of ranks [19].."

This is redundant, it's already cited in the materials and methods.

This sentence is exhibited for the first time at the “Materials and Methods” section.

It will only be further mentioned at “Results” section.

 

page 7, linea 340 "…The lowest rank value indicates a beneficial condition for the water deficit tolerance trait. Likewise, the sum of ranks of established biomarkers will provide different numbers, the smaller the combination of traits or biomarkers the more favorable to tolerance and, the larger will indicate a more favorable combination of traits or biomarkers to susceptibility to water deficit…"

 

This shouldn't be here. It should be in materials and methods

It was amended and the paragraph transferred to the proper section.

 

page 8, line 348 "…(Table 3)…"

The Table 3 is good, but the authors should create a graph with the data that generated the ranking to have a more enriching view, since this result is central to the work. What about Pearson's correlation coefficients?

We invite reviewer 2 to visit our previous reports, Corrêa et al (2017); Dias et al. (2017); and Condé et al. (2020), DOI:10.5039/agraria.v15i3a7515; and Andrade Bueno et al. (2020), https://doi.org/10. 1016/ j. cpb. 2020. 100136.

We agree that a graphical exhibition suit a friendlier data observation, particularly when multivariate analysis is performed. Nevertheless, in the present report we used only the ranking of the clones (according Mulamba and Mock index) focusing on the unfolding and use of this proposal and observations. Considering a greater number of genotypes, a graphical approach will allow the superficial view of the genotype assortment but, the table display, allow us to observe and identify precisely as needed the rank of the genotype. It will also ease the cross-reference access of other clone/genotype trait, such as production, yield, among others.

Again, if reviewer 2 think otherwise important to work on a graphical exposition of data, will work it on a second round-revision.

Further, Pearson correlation would allow us identify the traits or biomarkers that were associated, which was not our goal. We managed to identify and validate several traits (biomarkers) that could be associated with water deficit tolerance in eucalypt as proposed and, in the same hand, Suzano is has stablished plans to screen some of their clones aiming water deficit tolerance. The biomarkers that are evidenced here may be assembled to their strategy and test these clones, observed that the similar species genetic base be used.

 

page 8, line 355 "…Of the 15 variables evaluated, only those selected by the model are represented in Figure 3 and appear in the boxes…"

Correct this sentence with something similar to the following: "..Figure 3 only includes and displays the variables that were selected by the model out of the 15 that were evaluated, and these selected variables are the ones that appear in the boxes.."

It was amended. The information was sustained in the “Results” section, although not mentioned by reviewer 2.

“Figure 3 only includes and displays the variables that were selected by the model, out of the 15 that were evaluated. The selected variables are the ones that appear in the boxes.”

 

page 8, line 357 "… When the bar is blue, the contribution of the variable is positive in the tolerance probability, on the other hand, when the bar is red, the contribution is negative in estimating the probability…"

This is not a result! It should be in the figure's legend!

 

It was amended.

 

In general terms, the work is well developed, as are the experiments and techniques employed.

However, the manuscript lacks a well-structured discussion in its entirety. Therefore, the discussion section requires a thorough revision. It contains paragraphs that do not provide a contrasting discussion between the results of this work and the previously published works. Examples of such paragraphs are found on page 13, lines 457-466, and page 14X, lines 485-494.

The original lines and paragraphs were out of order. Further, the amendments also helped with this. We kindly ask reviewer 2 that, inform us the paragraphs that will need further attention and revision if still needed after the re-structuring the original manuscript. This will allow us a deeper and well-structured discussion in the examples mentioned (but that lost track in the correction process. The modifications are highlighted or set with the word correction tool.

 

Overall, particularly in the materials and methods and discussion sections, it appears that the manuscript was written without a comprehensive review by all authors. Therefore, the manuscript requires an extensive revision to correct many formalism errors, which are necessary for the work to be of scientific quality.

We apologize for this lack of order and flaws. We hope that, in this revised version of the manuscript, these have been properly corrected.

 

We hope that the amendments performed, and replies to the reviewer’s comments, have fulfilled their requirements. Although we think the flaws of the manuscript were corrected and the main suggestions of the reviewers were duly addressed, we are willing to have the English language editing, if still necessary.

 

Round 2

Reviewer 2 Report

La nueva version del manuscrito ha mejorado respecto de la anterior.

One more detail. The figure 1 still has issues, with repeated photos and pictures of eucalyptus trees that have no relation to figure according to the legend,