Assessing Elemental Diversity in Edible-Podded Peas: A Comparative Study of Pisum sativum L. var. macrocarpon and var. saccharatum through Principal Component Analysis, Correlation, and Cluster Analysis

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Please see my comments in the attached file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Comment: please see the comments in the attached file

Reply: All the comments marked on the attach file has been incorporated in the revised version of the manuscript. The revised version is attached herewith.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Yadav et al 3064058 Review

The authors measured pod mineral concentrations in twenty-four genotypes of edible-podded peas with the aim of assessing the extent of variation in the germplasm studied, and enabling ‘high mineral’ genotypes to be identified for future breeding. This is an important area of research, considering that more than half of the world’s population is estimated to be iron deficient and about a third are zinc deficient. By measuring minerals in pods, the authors have focused on the horticultural pea crop, rather than the field pea crop, which is novel. Most prior studies have focussed on field pea seeds. The authors identified potentially useful genotypes, such as Arka Sampoorna and Dwarf Grey Sugar, but there are deficiencies in the paper that mean the information presented is difficult to interpret.

Major deficiencies

11.      The authors need to explain exactly what pod material was taken for elemental analysis. Was it tissue from entire pods (including immature seeds), or was it isolated pod wall tissue? If the former, then the mineral measurements will pertain to two distinct tissue types, pod wall and immature seed. This would raise the question of whether, say, a high mineral concentration reflects a high concentrations in seeds, or pod walls, or do both tissues contribute evenly. This needs to be crystal clear, to allow an understanding of what the results say and how they compare with existing literature.

22.        Mineral concentration was measured, but no data on pod weight is presented. Consequently, no data on mineral content for each genotype is presented. Mineral content = average mineral concentration x average pod weight.  Throughout the paper the authors emphasise the breeding perspective on their work. Mineral content as well as mineral concentration are required to understand which pods contain more minerals. Would the genotype with the highest mineral concentration still be considered valuable for breeding if its mature pods were on average only 2 cm long? How would it compare to a genotype with the fifth-highest mineral concentration and pods on average 15 cm long?  Which pods contains more minerals? Again, from a breeding perspective, there is a problem because no yield data are presented in the Results. If a genotype with high mineral content was identified, would it be useful for breeding if it produced on average only one pod per plant? If pod weight and yield data were collected (as is usually done, especially for work aimed at breeding), then the paper would be improved by incorporating that into the manuscript. If not, then this could be part of the Discussion and the manuscript would stand as a preliminary assessment of mineral concentration variation in edible peas grown in one season and one location only.

33.        Results are not clear in Table 1. The title does not explain the figures presented. Are these mean squares, sum of squares? Normally both are given in ANOVA tables, along with the F statistics and probabilities, so the reader can better interpret the significance. Concentrations are not indicated.

44.       Results are not clear in Table 2. The means of three biological replicates are listed without errors. For example, there is a standout value of 921.42 mg/kg Fe in Arka Sampoorna. This is potentially a very interesting finding, but without any indication of the error associated with this value it is not possible to appreciate its significance. For example 921.42 mg/kg could reflect three similar readings, 921.40, 921.42, and 921.44 or, it could reflect three readings, 287.27, 276.73, and 2400.26, the last of which would be an outlier.   

55.        Results are confusing in Figure 3, the Hierarchical Cluster Analysis, which reports on PED-21-6 in Cluster I (line 259) and PED-21-8 in Cluster II (line 262), yet PED-21-6 and PED-21-8 were not in the list of genotypes tested (Table S1).

66.       Interpretation of Results is weak. For example, the first sentence of results (lines 148-150) picks out K and P being notably higher concentrations. What about Fe? Another example is the correlations in Figure 1 being described as highly significant and positive among all mineral traits (lines 220-221). This is incorrect. Cu is not significantly positively correlated with any other mineral. Neither is Fe, apart from two weak positive correlations.  These should be reported in Results and considered in the Discussion (lines 318-341). Many of the scatter plots show strong clustering of points, which could be interpreted and discussed.

77.        The Discussion contains mis-citations and irrelevant citations, as mentioned elsewhere. A paper that I would recommend as relevant reading is Ma et al (2017) BMC Plant Biology 17:43. DOI 10.1186/s12870-016-0956-4.

Minor Points

The title is unclear, and overly long. It does not convey the content of the paper concisely.

There is a lack of precision in describing the samples. These were 24 edible snow pea and snap pea genotypes. No other kinds were included.

The Discussion would be improved by considering the results more thoroughly and incisively, see all points mentioned above.  The Discussion mentions phytate binding Mg and Zn (lines 334-336). How does this explain the positive association (sic) between Zn and P, or Zn and Mg? Phosphate groups on phytic acid bind mineral cations, including Ca, Fe, Zn, Mg, Mn so it would be expected that any effects of phytate, if there were any, would pertain to all cations. Besides, the authors did not measure phytic acid, so the relevance of this discussion is not clear.   

Nutritional terminology is confused. The authors mention macronutrients (lines 66, 69) and conclude that edible-podded peas serve as significant sources of macronutrients (line  398), however this manuscript did not measure macronutrients, it measured only on micronutrients. Micronutrients include vitamins and minerals. Minerals are classified into two groups, macro-elements and microelements, according to human dietary quantitative requirements in the mg/g range, or μg/g range, respectively.

Citation is needed for FAO report (line 63).

 

 

Comments on the Quality of English Language

English language is readable. Note: highest and lowest should be used instead of maximum and minimum (lines 152 and 154).

Author Response

The authors measured pod mineral concentrations in twenty-four genotypes of edible-podded peas with the aim of assessing the extent of variation in the germplasm studied, and enabling ‘high mineral’ genotypes to be identified for future breeding. This is an important area of research, considering that more than half of the world’s population is estimated to be iron deficient and about a third are zinc deficient. By measuring minerals in pods, the authors have focused on the horticultural pea crop, rather than the field pea crop, which is novel. Most prior studies have focussed on field pea seeds. The authors identified potentially useful genotypes, such as Arka Sampoorna and Dwarf Grey Sugar, but there are deficiencies in the paper that mean the information presented is difficult to interpret.

Major deficiencies

11.     The authors need to explain exactly what pod material was taken for elemental analysis. Was it tissue from entire pods (including immature seeds), or was it isolated pod wall tissue? If the former, then the mineral measurements will pertain to two distinct tissue types, pod wall and immature seed. This would raise the question of whether, say, a high mineral concentration reflects a high concentrations in seeds, or pod walls, or do both tissues contribute evenly. This needs to be crystal clear, to allow an understanding of what the results say and how they compare with existing literature.

Author Response:  Tissue from entire pods (including immature seeds) was used for elemental analysis.

22.       Mineral concentration was measured, but no data on pod weight is presented. Consequently, no data on mineral content for each genotype is presented. Mineral content = average mineral concentration x average pod weight.  Throughout the paper the authors emphasise the breeding perspective on their work. Mineral content as well as mineral concentration are required to understand which pods contain more minerals. Would the genotype with the highest mineral concentration still be considered valuable for breeding if its mature pods were on average only 2 cm long? How would it compare to a genotype with the fifth-highest mineral concentration and pods on average 15 cm long?  Which pods contains more minerals? Again, from a breeding perspective, there is a problem because no yield data are presented in the Results. If a genotype with high mineral content was identified, would it be useful for breeding if it produced on average only one pod per plant? If pod weight and yield data were collected (as is usually done, especially for work aimed at breeding), then the paper would be improved by incorporating that into the manuscript. If not, then this could be part of the Discussion and the manuscript would stand as a preliminary assessment of mineral concentration variation in edible peas grown in one season and one location only.

Author Response: Thank you for your valuable feedback. We recognize the importance of yield-related data in evaluating the practical implications of mineral concentration for breeding purposes. In this manuscript, we have intentionally focused on the nutritional diversity and mineral concentration among the pea genotypes. Our goal is to highlight genetic diversity in nutrient content, which is a critical first step towards targeted breeding interventions.

We have collected detailed yield-related data, including pod weight, pod length, number of pods per plant, pod breadth, and yield per plant. These data are presented in a separate manuscript currently under review, which aims to complement the findings reported here.

Our study identified the HPM-2 genotype as having the highest Fe concentration. Although HPM-2 may not match the yield traits of the main season variety Punjab-89, it remains a valuable genetic resource due to its enhanced Fe content. In a breeding program, HPM-2 would serve as a donor parent to enhance the Fe concentration in Punjab-89 through backcross breeding. While linkage drag, such as reduced pod number and weight, is a potential concern, this can be addressed through repeated backcrossing with Punjab-89, ensuring the retention of its superior yield traits while incorporating the enhanced Fe concentration.

We acknowledge that the absence of yield data in this manuscript might limit the immediate applicability of our findings to breeding strategies. However, this study serves as a preliminary assessment of nutritional diversity, with broader implications for future breeding programs. We appreciate your understanding of the study's focus and the direction of our ongoing research.

33. Results are not clear in Table 1. The title does not explain the figures presented. Are these mean squares, sum of squares? Normally both are given in ANOVA tables, along with the F statistics and probabilities, so the reader can better interpret the significance. Concentrations are not indicated.

Author Response: Corrected

44.      Results are not clear in Table 2. The means of three biological replicates are listed without errors. For example, there is a standout value of 921.42 mg/kg Fe in Arka Sampoorna. This is potentially a very interesting finding, but without any indication of the error associated with this value it is not possible to appreciate its significance. For example, 921.42 mg/kg could reflect three similar readings, 921.40, 921.42, and 921.44 or, it could reflect three readings, 287.27, 276.73, and 2400.26, the last of which would be an outlier.   

Author Response: Corrected

55. Results are confusing in Figure 3, the Hierarchical Cluster Analysis, which reports on PED-21-6 in Cluster I (line 259) and PED-21-8 in Cluster II (line 262), yet PED-21-6 and PED-21-8 were not in the list of genotypes tested (Table S1).

Author Response: Corrected

66.      Interpretation of Results is weak. For example, the first sentence of results (lines 148-150) picks out K and P being notably higher concentrations. What about Fe? Another example is the correlations in Figure 1 being described as highly significant and positive among all mineral traits (lines 220-221). This is incorrect. Cu is not significantly positively correlated with any other mineral. Neither is Fe, apart from two weak positive correlations.  These should be reported in Results and considered in the Discussion (lines 318-341). Many of the scatter plots show strong clustering of points, which could be interpreted and discussed.

Author Response: Corrected

77. The Discussion contains mis-citations and irrelevant citations, as mentioned elsewhere. A paper that I would recommend as relevant reading is Ma et al (2017) BMC Plant Biology 17:43. DOI 10.1186/s12870-016-0956-4.

Author Response: We have reviewed all the citations, and they are relevant to the diversity and breeding perspectives of agronomical and horticultural crops. Thank you for suggesting the paper mentioned above. However, upon examination, it does not pertain to diversity analysis.

Minor Points

The title is unclear, and overly long. It does not convey the content of the paper concisely.

There is a lack of precision in describing the samples. These were 24 edible snow pea and snap pea genotypes. No other kinds were included.

Author Response: Snow peas and snap peas are edible podded peas and we only focus on the nutritional diversity in edible podded peas

The Discussion would be improved by considering the results more thoroughly and incisively, see all points mentioned above.  The Discussion mentions phytate binding Mg and Zn (lines 334-336). How does this explain the positive association (sic) between Zn and P, or Zn and Mg? Phosphate groups on phytic acid bind mineral cations, including Ca, Fe, Zn, Mg, Mn so it would be expected that any effects of phytate, if there were any, would pertain to all cations. Besides, the authors did not measure phytic acid, so the relevance of this discussion is not clear. 

Author Response: Corrected  

Nutritional terminology is confused. The authors mention macronutrients (lines 66, 69) and conclude that edible-podded peas serve as significant sources of macronutrients (line  398), however this manuscript did not measure macronutrients, it measured only on micronutrients. Micronutrients include vitamins and minerals. Minerals are classified into two groups, macro-elements and microelements, according to human dietary quantitative requirements in the mg/g range, or μg/g range, respectively.

Author Response: Corrected

Citation is needed for FAO report (line 63).

Response: Citation is Added

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The article titled“Exploring edible podded pea genotypes through evaluation of variability parameters for mineral profile, character association studies and genetic divergence to address nutritional security” is a clear and interesting manuscript, whose goal is to identify promising parents for nutraceutical breeding. The assessment of minerals in pods of examined edible-podded genotypes offers a quantitative measure of their concentration in both macro- and micro-elements. The authors found that several genotypes exhibited superior mineral concentration and recommended their utilization in breeding programs to enhance the nutritional quality of edible-podded peas. The writing is easy to read and follow.

Author Response

Comment: The article titled“Exploring edible podded pea genotypes through evaluation of variability parameters for mineral profile, character association studies and genetic divergence to address nutritional security” is a clear and interesting manuscript, whose goal is to identify promising parents for nutraceutical breeding. The assessment of minerals in pods of examined edible-podded genotypes offers a quantitative measure of their concentration in both macro- and micro-elements. The authors found that several genotypes exhibited superior mineral concentration and recommended their utilization in breeding programs to enhance the nutritional quality of edible-podded peas. The writing is easy to read and follow.

Reply: the reviewer has give positive response for the paper and suggested no change to do in the manuscript

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Major deficiencies

11.     The authors need to explain exactly what pod material was taken for elemental analysis. Was it tissue from entire pods (including immature seeds), or was it isolated pod wall tissue? If the former, then the mineral measurements will pertain to two distinct tissue types, pod wall and immature seed. This would raise the question of whether, say, a high mineral concentration reflects a high concentrations in seeds, or pod walls, or do both tissues contribute evenly. This needs to be crystal clear, to allow an understanding of what the results say and how they compare with existing literature.

Author Response:  Tissue from entire pods (including immature seeds) was used for elemental analysis.

The authors have answered the query but have not clarified the text for readers. I suggest the following wording on lines 111-112. “Entire pods, including seeds, from each genotype were harvested at the edible maturity stage before string development….etc”

22.       Mineral concentration was measured, but no data on pod weight is presented. Consequently, no data on mineral content for each genotype is presented. Mineral content = average mineral concentration x average pod weight.  Throughout the paper the authors emphasise the breeding perspective on their work. Mineral content as well as mineral concentration are required to understand which pods contain more minerals. Would the genotype with the highest mineral concentration still be considered valuable for breeding if its mature pods were on average only 2 cm long? How would it compare to a genotype with the fifth-highest mineral concentration and pods on average 15 cm long?  Which pods contains more minerals? Again, from a breeding perspective, there is a problem because no yield data are presented in the Results. If a genotype with high mineral content was identified, would it be useful for breeding if it produced on average only one pod per plant? If pod weight and yield data were collected (as is usually done, especially for work aimed at breeding), then the paper would be improved by incorporating that into the manuscript. If not, then this could be part of the Discussion and the manuscript would stand as a preliminary assessment of mineral concentration variation in edible peas grown in one season and one location only.

Author Response: Thank you for your valuable feedback. We recognize the importance of yield-related data in evaluating the practical implications of mineral concentration for breeding purposes. In this manuscript, we have intentionally focused on the nutritional diversity and mineral concentration among the pea genotypes. Our goal is to highlight genetic diversity in nutrient content, which is a critical first step towards targeted breeding interventions.

We have collected detailed yield-related data, including pod weight, pod length, number of pods per plant, pod breadth, and yield per plant. These data are presented in a separate manuscript currently under review, which aims to complement the findings reported here.

Our study identified the HPM-2 genotype as having the highest Fe concentration. Although HPM-2 may not match the yield traits of the main season variety Punjab-89, it remains a valuable genetic resource due to its enhanced Fe content. In a breeding program, HPM-2 would serve as a donor parent to enhance the Fe concentration in Punjab-89 through backcross breeding. While linkage drag, such as reduced pod number and weight, is a potential concern, this can be addressed through repeated backcrossing with Punjab-89, ensuring the retention of its superior yield traits while incorporating the enhanced Fe concentration.

We acknowledge that the absence of yield data in this manuscript might limit the immediate applicability of our findings to breeding strategies. However, this study serves as a preliminary assessment of nutritional diversity, with broader implications for future breeding programs. We appreciate your understanding of the study's focus and the direction of our ongoing research.

Although the authors have the pod weight and nutrient concentration data in hand to meet their stated goal, ie. highlighting genetic diversity in nutrient content, they have chosen not to add data on pea pod nutrient content to the manuscript

33. Results are not clear in Table 1. The title does not explain the figures presented. Are these mean squares, sum of squares? Normally both are given in ANOVA tables, along with the F statistics and probabilities, so the reader can better interpret the significance. Concentrations are not indicated.

Author Response: Corrected

The Table is still not clear. What is Mean Sum of Squares? Do the authors mean Mean Squares? Please modify the Table to give F statistics. Please indicate units of concentrations.

44.      Results are not clear in Table 2. The means of three biological replicates are listed without errors. For example, there is a standout value of 921.42 mg/kg Fe in Arka Sampoorna. This is potentially a very interesting finding, but without any indication of the error associated with this value it is not possible to appreciate its significance. For example, 921.42 mg/kg could reflect three similar readings, 921.40, 921.42, and 921.44 or, it could reflect three readings, 287.27, 276.73, and 2400.26, the last of which would be an outlier.   

Author Response: Corrected

Errors have been given, but it is not specified if they are sd or se.

55. Results are confusing in Figure 3, the Hierarchical Cluster Analysis, which reports on PED-21-6 in Cluster I (line 259) and PED-21-8 in Cluster II (line 262), yet PED-21-6 and PED-21-8 were not in the list of genotypes tested (Table S1).

Author Response: Corrected

OK

66.      Interpretation of Results is weak. For example, the first sentence of results (lines 148-150) picks out K and P being notably higher concentrations. What about Fe? Another example is the correlations in Figure 1 being described as highly significant and positive among all mineral traits (lines 220-221). This is incorrect. Cu is not significantly positively correlated with any other mineral. Neither is Fe, apart from two weak positive correlations.  These should be reported in Results and considered in the Discussion (lines 318-341). Many of the scatter plots show strong clustering of points, which could be interpreted and discussed.

Author Response: Corrected

The authors have responded to these points by adding a comment on Fe and Na concentrations, adding a correction to the statement about correlations and adding a non-interpretative comment about clustering of points.

77. The Discussion contains mis-citations and irrelevant citations, as mentioned elsewhere. A paper that I would recommend as relevant reading is Ma et al (2017) BMC Plant Biology 17:43. DOI 10.1186/s12870-016-0956-4.

Author Response: We have reviewed all the citations, and they are relevant to the diversity and breeding perspectives of agronomical and horticultural crops. Thank you for suggesting the paper mentioned above. However, upon examination, it does not pertain to diversity analysis.

Although the authors have reviewed all the citations, the errors remain unchanged. The mis-citation (previously 18, now 19) that does not attribute mineral content to genetic origin remains. The mis-citation (previously 29, now 31) remains. Citation 33 (previously 31) of a soybean paper is still described as a cowpea paper. Citation 34 (previously 32) of a chickpea paper is still described as a cowpea paper. Mis-citation 13 (previously 12) remains.

Furthermore, references on strawberry and amaranth are still present. The authors not have justified why these are relevant, apart from the fact that they happen to be horticultural crops.

Minor Points

The title is unclear, and overly long. It does not convey the content of the paper concisely.

Title has been improved

There is a lack of precision in describing the samples. These were 24 edible snow pea and snap pea genotypes. No other kinds were included.

Author Response: Snow peas and snap peas are edible podded peas and we only focus on the nutritional diversity in edible podded peas

The authors made no change to the text. I suggest the following wording on lines 90-91. “The plant material consisted of edible-podded pea genotypes, with 8 and 16 genotypes of sugar snap peas and snow peas, respectively”.

The Discussion would be improved by considering the results more thoroughly and incisively, see all points mentioned above.  The Discussion mentions phytate binding Mg and Zn (lines 334-336). How does this explain the positive association (sic) between Zn and P, or Zn and Mg? Phosphate groups on phytic acid bind mineral cations, including Ca, Fe, Zn, Mg, Mn so it would be expected that any effects of phytate, if there were any, would pertain to all cations. Besides, the authors did not measure phytic acid, so the relevance of this discussion is not clear. 

Author Response: Corrected 

The authors have replaced the paragraph about phytate with a paragraph about mycorrhiza. 

Nutritional terminology is confused. The authors mention macronutrients (lines 66, 69) and conclude that edible-podded peas serve as significant sources of macronutrients (line  398), however this manuscript did not measure macronutrients, it measured only on micronutrients. Micronutrients include vitamins and minerals. Minerals are classified into two groups, macro-elements and microelements, according to human dietary quantitative requirements in the mg/g range, or μg/g range, respectively.

Author Response: Corrected

Terminology still seems confused eg. authors replaced macronutrients with macro-nutrients (line  398). My advice would be to use macro-elements and micro-elements in the appropriate places because elemental analysis was carried out ie. minerals were measured. 

Citation is needed for FAO report (line 63).

Response: Citation is Added

A mis-citation has been provided. The link given does not lead to an FAO report on nutritional security in India. It leads to a FAO report of Food Security in Latin America and the Caribbean.

Comments on the Quality of English Language

The language is readable English. Note: maximum and minimum are used incorrectly (lines 160 and 161). Highest and lowest should be used instead.

Author Response

Major deficiencies

Comment: The authors have answered the query but have not clarified the text for readers. I suggest the following wording on lines 111-112. “Entire pods, including seeds, from each genotype were harvested at the edible maturity stage before string development….etc”

Author Response:  We have incorporated your suggested wording in the lines 111-112 of manuscript.

The revised text now reads:

“Entire pods, including seeds, from each genotype were harvested at the edible maturity stage before string development…”

Comment: The Table is still not clear. What is Mean Sum of Squares? Do the authors mean Mean Squares? Please modify the Table to give F statistics. Please indicate units of concentrations.

Author Response: 

• We have revised the table to replace "Mean Sum of Squares" with "Mean Squares" to align with standard terminology in statistical reporting. Thank you for pointing this out.
• As per the reviewer suggestion, we have added a column in the table to include the F statistics for each factor. This will provide additional clarity and detail regarding the statistical analysis performed.
• We have included the units of concentrations in the table.

Comment: Errors have been given, but it is not specified if they are sd or se.

Author Response: Actually, It is Standard Error (SE) and now added in the heading of the table.

Comment: The authors have responded to these points by adding a comment on Fe and Na concentrations, adding a correction to the statement about correlations and adding a non-interpretative comment about clustering of points.

Author Response: Comment related to Clustering of points has been added in line no. 336

Comment: Although the authors have reviewed all the citations, the errors remain unchanged. The mis-citation (previously 18, now 19) that does not attribute mineral content to genetic origin remains. The mis-citation (previously 29, now 31) remains. Citation 33 (previously 31) of a soybean paper is still described as a cowpea paper. Citation 34 (previously 32) of a chickpea paper is still described as a cowpea paper. Mis-citation 13 (previously 12) remains.

Author Response: Thanks for pointing out this major mistake which has now been corrected

Comment: Furthermore, references on strawberry and amaranth are still present. The authors not have justified why these are relevant, apart from the fact that they happen to be horticultural crops.

Author Response: The said references has been removed from the manuscript

 

Minor Points

Comment: The authors made no change to the text. I suggest the following wording on lines 90-91. “The plant material consisted of edible-podded pea genotypes, with 8 and 16 genotypes of sugar snap peas and snow peas, respectively”.

Author Response: The updated sentence now reads:

“The plant material consisted of edible-podded pea genotypes, with 8 and 16 genotypes of sugar snap peas and snow peas, respectively.

Comment: Terminology still seems confused eg. authors replaced macronutrients with macro-nutrients (line  398). My advice would be to use macro-elements and micro-elements in the appropriate places because elemental analysis was carried out ie. minerals were measured. 

Author Response: The suggested changes have been incorporated in the manuscript.

Comment:A mis-citation has been provided. The link given does not lead to an FAO report on nutritional security in India. It leads to a FAO report of Food Security in Latin America and the Caribbean.

Author Response: The link has been corrected as per suggestion.

Round 3

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have modified and improved the manuscript.

Interpretation of Results remains rather weak. On line 332-333 the authors have again stated incorrectly that “a significant positive relationship was observed between the concentrations of all macro and micro elements (Figure 1)”. As discussed in a previous review, this statement is simply not true (the correlations of Cu and Na and Cu and Ca are 0.14 and 0.15 respectively) therefore it must be corrected or removed. Furthermore, this newly inserted statement disagrees with the authors modifed statement in Results section (lines 229-230) where it is stated “This study also identified a significant positive correlation among all elements, except for certain pairs such as Cu with Na and Cu with Ca which exhibited weak correlations”.  The authors have pointed out the exceptions, however, it would be more accurate to say This study also identified a significant positive correlation among elements, except for certain pairs such as Cu with Na and Cu with Ca which were not significantly correlated”.

The authors seem to have an urge to focus only on positive correlations, yet those that are not positively correlated are also interesting and deserve discussion, since these might require special attention in terms of breeding. Negative correlations and non-correlations are not “bad” results. There is no such thing as a bad result. Indeed it’s the opposite. Results that buck the trend or go against expectation are the very ones that tend to advance the scientific research endeavour. For Cu, the trend lines seem to change from positive to negative about half way along, suggesting there is a positive correlation when the elements are at lower concentrations, but this correlation does not hold at higher concentrations. The same sort of effect can be seen in some of the Fe graphs. What might explain this? 

In a previous review I noted that many of the scatter plots show strong clustering of points, which could be interpreted and discussed. The authors responded by adding a comment “The strong clustering of points in the scatter plots between K vs. Mg and K vs. P within the correlation matrix suggests various underlying relationships and patterns among the elements analyzed”. This is a vague statement that does not interpret the data. I would suggest that clustering of points indicates low variation in measured element concentrations. Looking at the graphs, one can see that Mg, P and S are highly clustered vs all other elements, suggesting there is low variation for Mg, P and S in this germplasm. But since the clustering is located in the upper right quadrant of the graphs, most of the germplasm contains similarly high concentrations of these elements.

Author Response

Comment: Interpretation of Results remains rather weak. On line 332-333 the authors have again stated incorrectly that “a significant positive relationship was observed between the concentrations of all macro and micro elements (Figure 1)”. As discussed in a previous review, this statement is simply not true (the correlations of Cu and Na and Cu and Ca are 0.14 and 0.15 respectively) therefore it must be corrected or removed.

Response: Corrected

Comment: Furthermore, this newly inserted statement disagrees with the authors modifed statement in Results section (lines 229-230) where it is stated “This study also identified a significant positive correlation among all elements, except for certain pairs such as Cu with Na and Cu with Ca which exhibited weak correlations”.  The authors have pointed out the exceptions, however, it would be more accurate to say This study also identified a significant positive correlation among elements, except for certain pairs such as Cu with Na and Cu with Ca which were not significantly correlated”.

Response: Corrected

The authors seem to have an urge to focus only on positive correlations, yet those that are not positively correlated are also interesting and deserve discussion, since these might require special attention in terms of breeding. Negative correlations and non-correlations are not “bad” results. There is no such thing as a bad result. Indeed it’s the opposite. Results that buck the trend or go against expectation are the very ones that tend to advance the scientific research endeavour. For Cu, the trend lines seem to change from positive to negative about half way along, suggesting there is a positive correlation when the elements are at lower concentrations, but this correlation does not hold at higher concentrations. The same sort of effect can be seen in some of the Fe graphs. What might explain this?

Response: Thank you for your insightful feedback. We appreciate your emphasis on the importance of analyzing negative and non-correlations, as well as your observation regarding the trend shifts in copper (Cu) and iron (Fe) correlations. In response to your comments, we have expanded our discussion to include a detailed analysis of these aspects. We agree that these findings are important and deserve careful consideration in the context of breeding programs and scientific research.

Comment: In a previous review I noted that many of the scatter plots show strong clustering of points, which could be interpreted and discussed. The authors responded by adding a comment “The strong clustering of points in the scatter plots between K vs. Mg and K vs. P within the correlation matrix suggests various underlying relationships and patterns among the elements analyzed”. This is a vague statement that does not interpret the data. I would suggest that clustering of points indicates low variation in measured element concentrations. Looking at the graphs, one can see that Mg, P and S are highly clustered vs all other elements, suggesting there is low variation for Mg, P and S in this germplasm. But since the clustering is located in the upper right quadrant of the graphs, most of the germplasm contains similarly high concentrations of these elements.

Response: In response to your feedback, we have revised our manuscript to include a more detailed discussion on the observed clustering, particularly focusing on the low variation in element concentrations for Mg, P, and S, as well as the implications of these findings.

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

Comments and Suggestions for Authors

Manuscript entitled »Diversity of mineral profiles of edible-podded pea (Pisum sativum L. var. macrocarpon and Pisum sativum var. saccharatum) to address nutritional security« by Yadav et al. reports on the element profiles of 24 genotypes of two subspecies of Pisum sativum. The study is very interesting and timely, but it will benefit significantly by native English speaker edits (this should be done because it will increase the quality of the paper significantly). In addition, there is some information lacking in the MM section, such as details to the experiment itself, for example, were pods harvested on the same day (I suspect not all mature at the same time), it is not clear how many replicates were really analysed and information on biomass (or at least yield) is essential to be able to conclude on the appropriateness of certain genotype for further breeding. In addition, conclusions are too ambitious, too pompous based on one experiment with limited repeatability.

 

Some minor comments:

L79: This analysis was crucial for characterizing genetic diversity > This is phenotypic diversity and not genotypic.

… with K and P content being notably higher than other minerals across all pea genotypes >this is expected, as these are macronutrients…

Captions need more data: for example, Table 2: what do the letters next to numbers mean? What is being compared (specify), what is the number of repetitions for each genotype? Genotype HPM-2 has significantly smaller concentration of all elements. Could this be due to measurement error (this is why repetitions are important).

Keep the sequence of elements constant.

Do not use the same number of decimal places in all instances. When a number exceeding three digits, no decimal places are needed.

Use concentration instead of content, consistently.

…all traits studied – one trait was studied: element profile, thus amend to specify what is meant by traits.

L240: the reference is not in the right format.

L267: Notably, high heritability estimates (>60%) were evident for all traits (Table 3), suggesting their lesser susceptibility to environmental influence [26]. > as these plants were grown under the same environmental conditions AND there was significant variability in concentration of elements father discussion is needed for this argument.

L280: The traits exhibit favorable additive gene action, making them suitable for inclusion in a breeding program > I am not confident that this conclusion is supported by the data. In addition, what kind of breeding program? Are there any existent (or planned) programs for element superiority in Pisum sativum in India?

L292: This correlation may be attributed to the similar chemical properties of these elements, resulting in competition for absorption sites, transport, and function within plant tissues > which exactly (be specific, it is not clear)? In addition, if there was a competition between elements, there would be a negative correlation between their concentrations.

L296: Zn also demonstrated a significantly positive correlation with all other mineral nutrients, consistent with findings in peas > language issues: Zn does not demonstrate a correlation, but Zn concentrations correlate positively with xxx (specify).

Consistently use past tense when writing. L334: The mean of cluster I surpasses the overall mean of the population (Supplemental file 3).

Amend: L335: This finding suggests cluster I consistently and significantly demonstrated superior performance in virtually all studied traits, owing to its elevated genetic diversity > how consistently? Experiment was not repeated thus consistency of element concentrations was not determined. Replace “virtually” with exact values. What is elevated genetic diversity?

Amend: L333: All 24 genotypes were divided into two clusters (Figure 3). > This implies they were divided on purpose, better: Genotypes clustered into two contrasting clusters.

Comments on the Quality of English Language

The study is very interesting and timely, but it will benefit significantly by native English speaker edits (this should be done because it will increase the quality of the paper significantly).

Author Response

Query: Were pods harvested on the same day (I suspect not all mature at the same time), it is not clear how many replicates were really analysed and information on biomass (or at least yield) is essential to be able to conclude on the appropriateness of certain genotype for further breeding.?

Response: All the genotypes were sown in three replications and the nutrient analysis was also done for each replication individually when the pods reach edible maturity for each genotype. All the pods don’t get mature on the same day, we take multiple picking. However concerning the yield data, it has been taken but it is not included in this paper since this paper focused only on nutrient analyses.

Query: Conclusions are too ambitious, too pompous based on one experiment with limited repeatability.

Response: The conclusion has been improved.

Some minor comments:

L79: This analysis was crucial for characterizing genetic diversity > This is phenotypic diversity and not genotypic.

Response: Suggestion has been incorporated.

… with K and P content being notably higher than other minerals across all pea genotypes >this is expected, as these are macronutrients…

Response: We have analyzed five macronutrients and out of these five, the concentration of K and P was high.

Captions need more data: for example, Table 2: what do the letters next to numbers mean? What is being compared (specify), what is the number of repetitions for each genotype? Genotype HPM-2 has significantly smaller concentration of all elements. Could this be due to measurement error (this is why repetitions are important).

Response: Suggestions have been incorporated. Additionally, for the case of HPM-2, the quantity of samples taken for all the genotypes was the same, and analysis was done in 3 replicates. So, there were very few chances of measurement error. Conclusively it can be said that HPM- 2 may have an inherently low concentration of studied nutrients.

Keep the sequence of elements constant.

Response: the suggested change has been made

Do not use the same number of decimal places in all instances. When a number exceeding three digits, no decimal places are needed.

Response:

Use concentration instead of content, consistently.

Response: Done

…all traits studied – one trait was studied: element profile, thus amend to specify what is meant by traits.

Response: Ammended

L240: the reference is not in the right format.

Response: Corrected

L267: Notably, high heritability estimates (>60%) were evident for all traits (Table 3), suggesting their lesser susceptibility to environmental influence [26]. > as these plants were grown under the same environmental conditions AND there was significant variability in concentration of elements father discussion is needed for this argument.

Response: Further discussion has been added.

L280: The traits exhibit favorable additive gene action, making them suitable for inclusion in a breeding program > I am not confident that this conclusion is supported by the data. In addition, what kind of breeding program? Are there any existent (or planned) programs for element superiority in Pisum sativum in India?

Response: It is generally agreed that high heritability combined with high genetic advance indicates a trait is controlled by additive genes (Ogunniyan et al., 2014). Our study also found similar results. Regarding breeding programs for element superiority in Pisum sativum in India, to the best of our knowledge, very few programs have been conducted to improve edible podded peas.

Ogunniyan, D. J., & Olakojo, S. A. (2014). Genetic variation, heritability, genetic advance and agronomic character association of yellow elite inbred lines of maize (Zea mays L.). Nigerian Journal of Genetics, 28(2), 24-28.

L292: This correlation may be attributed to the similar chemical properties of these elements, resulting in competition for absorption sites, transport, and function within plant tissues > which exactly (be specific, it is not clear)? In addition, if there was a competition between elements, there would be a negative correlation between their concentrations.

Response: Respective changes have been made in the manuscript.

L296: Zn also demonstrated a significantly positive correlation with all other mineral nutrients, consistent with findings in peas > language issues: Zn does not demonstrate a correlation, but Zn concentrations correlate positively with xxx (specify).

Response: The line has been improved

Consistently use past tense when writing. L334: The mean of cluster I surpasses the overall mean of the population (Supplemental file 3).

Response: Respective Changes have been made.

Amend: L335: This finding suggests cluster I consistently and significantly demonstrated superior performance in virtually all studied traits, owing to its elevated genetic diversity > how consistently? Experiment was not repeated thus consistency of element concentrations was not determined. Replace “virtually” with exact values. What is elevated genetic diversity?

Response: All the respective changes have been done.

Amend: L333: All 24 genotypes were divided into two clusters (Figure 3). > This implies they were divided on purpose, better: Genotypes clustered into two contrasting clusters.

Response: Amended

Reviewer 2 Report

Comments and Suggestions for Authors

The article addresses a crop that is not widely consumed but is important for feeding the population, taking into account that this will continue to increase. The issue of nutritional security is not addressed much in the article and is part of the title, in addition to being of great importance to further explain the results referring to these current terms.

The article has some errors and omissions noted in the text.

Author Response

The article has been improved and we tried to omit errors in the manuscript

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

Thank you very much for the request to make this review.

 

In my opinion the Article entitled „Diversity of mineral profiles of edible-podded pea (Pisum sativum var. macrocarpon and Pisum sativum var. saccharatum) to address nutritional security” can not be published in the Horticulturae.

 

In my opinion the biggest problem which I see in materials and methods chapter is connected with numbers of experiment years. The Authors carried out the experiment only one years/one season..... it's not enough.  In this kind of experiment,  to be able to prepare conclusions, we need more years of experiment. Moreover, increasing the number of research years would have provided more solid conclusions. Besides after reading of this chapter I didn’t know: it was field experiment? Greenhaus? Pots? What size of plots? Weather conditions? – this factor is very important for chemical compositions…

 

Out of concern for the high scientific level of the journal and the published content in journal, in my opinion the research results from this experiment should not be published as a scientific article. Perhaps a way out of this situation will be publish these results as an announcement or preliminary research results?

Kind Regards,

  Reviewer

Comments for author File: Comments.pdf

Author Response

The experiment was conducted for one year and the crop was sown in plots in the field and the pods were harvested at edible maturity stage and afterwards the mineral analysis was done. The plot size is 1 x 2 m which is mentioned in the section 1.2 cultural practices. The increasing the number of years data is not possible at this stage because the experiment was finished.