An Evaluation of Kernel Zinc in Hybrids of Elite Quality Protein Maize (QPM) and Non-QPM Inbred Lines Adapted to the Tropics Based on a Mating Design

Round 1

Reviewer 1 Report

Overall, the topic of the study is original and it could be of high relevance for the scientific community. It contains new results that significantly advance the research on Zn biofortification in maize kernels. The methods and techniques were also appropriate. However, the manuscript should be carefully revised and some issues need to be addressed. It has not satisfied the journal manuscript format. Throughout the manuscript, different font sizes and repeated paragraphs were found. Also, the hypothesis was not clearly stated. Please consider the next comments to help you to revise the manuscript:

1. L2-4 The title seems confusing to me. It doesn’t provide clear information about the results. The expression “A Genetic Evaluation…” is too broad and makes it difficult to elucidate the topic of the work. Please replace the current title for one that resumes the most important outputs of the study.
2. L26 In the introduction, please state the hypothesis of the study. Why QPM and non-QPM inbred crosses could exhibit differences in kernel Zn concentration? It could be that the differences in Zn-binding protein content of QPM and non-QPM may impact Zn storage capacity in maize kernels. This was mentioned in the discussion but it should be part of the introduction as a starting point of the study.
3. L80 Add a period at the end of the sentence.
4. L84 Please remove “2.Materials and Methods”.
5. L85 Although inbred lines had different Zn concentrations, some of them are close relatives. This endogamy could have a negative effect mainly in inbred crosses from the same QPM or non-QPM group. On the other hand, hybrids obtained from intergroup crosses (QPM x non-QPM) may have a higher yield and Zn concentration because of heterosis effect. Please explain how endogamy and heterosis could impact on your final results.
6. L97 The inbred crosses that did not produce grain (13x16 and 15x17) should be highlighted in Table S2.
7. L115-119 This section needs revision. It has different font sizes, please revise.
8. L126 Please insert a comma after “…days to anthesis…”.
9. L126-131 The authors studied the effect of the days to anthesis and silking over Zn deposition. However, it could be interesting to study the effect of the seed filling period over that same trait because, as the authors mentioned in line 367, Zn accumulation is determined in the late stages of the crop. Please explain why traits regarding the seed filling period were not evaluated.
10. L131-137 This section has different font sizes, please revise.
11. L157 Please insert a comma after “…genotypes (σ²G)…”.
12. L257 29.69 ug/g was not the highest Zn value. Please replace it with the correct value (29.72 ug/g).
13. L265-273 This section is a duplication of the previous paragraph (L256-264). Please remove it.
14. L285 The reference to Table S4 in this paragraph seems to be a typo. The table that you may want to refer to is Table S5. Please revise.
15. L292-298 The output of these lines is one of the main conclusions of the study. Please expand this section with a more detailed explanation of the results.
16. L299 Table 6 is very confusing. Please modify it or explain it better within the results section. It seems that few headers are misaligned.
17. L334-341 These lines could also be mentioned within the introduction section to enforce a potential hypothesis of the study. The different contents of Zn-binding proteins in QPM and non-QPM inbreds could be the cause why the resulting hybrids had different Zn storage capacities within their kernels.
18. L388-391 This section needs revision. Some author’s initials are incorrect, please revise.
19. L398-602 The entire reference section needs revision. Some references are repeated (e.g. number 5) and lack of title and journal name (e.g. number 10, 43, 64, 65, and 66). Also, other references have page and URL errors (e.g. number 18, 52, and 73). Please revise this section.
20. In the supplementary files, the description under Table S2 needs to be revised. Please see that hybrid set 5 was mentioned twice and one of them should be 4.
21. The information under Table S4 also needs to be revised. Please add a comma instead of a period between the description of 0.05 and 0.01 significance levels.

Author Response

Please see the attached file, "response_reviewer 1 - 768398" for the reviewer's comments and our replies.

Author Response File: Author Response.docx

Reviewer 2 Report

General comments

 

This manuscript gives important results for the science e.g.

 

This manuscript highlights

1. the importance of inbreds' phenotype in the creation of hybrids with elevated levels of Zn in the kernel. Moreover, hybrids with a Zn content > 30 pg/g across environments were produced exclusively from inbred lines. So there is potential for the development of high- Zn hybrids
2. From nutritional part. In this study, grain yield was not correlated with Zn, which means that could be improved simultaneously, without yield penalty.
3. The largest proportion of variability for Zn among hybrids was due to GCA effects, suggesting that importance of additive gene effects in relation to non-additive gene effects for Zn.

 

 

General Instructions.

 

It would be better if the section 2. Material and Methods was separated  to subsection such as statisital analysis, Zn analysis etc.

Also, a control for grammar mistakes is necessary. I make some correction but further control is necessary.

 

Line 18. Authors could put a comma before “or”.

Line 18. Please put a comma before “which”.

Line 29. Please put a comma before “with”.

Line 30. Please change south Asia to “South Asia”

Line 32. Please put a comma before “especially”.

Line 33. Please put a comma before “which”.

Line 66. Please change have to “has”.

Line 91. Please change central to “Central”.

Line 98. Please put a comma before “and”

 

Line 65 In maize, nutritional-related research has emphasized on quality protein maize (QPM).

 

A short definition of quality protein maize maybe useful.

 

 

Line 74. “QPM and low-Zn non-QPM) and non-QPM”.

 

Could you please check this is correct?

 I think author should change it to QPM and low-Zn QPM) and non-QPM

 

Line 84. Authors should change “the Zn level in the kernel and whether classified as QPM or non QPM2. Materials and Methods”

 

to “the Zn level in the kernel and whether classified as QPM or non QPM2”.

 

 

Line 81. It would be better if the section 2. Material and Methods was separated to statisital analysis, Zn analysis etc.

 

Line 92-93.  “based on their lysine and tryptophan content”

 

It would be useful if authors will make the above reference to quality protein maize in the beginning.

 

Line 106-110. Authors should reduce some parts of material and methods such as 106-110 and some others which are very explainatory.

 

Line 113. Please change “One hundred and forty-eight” to “148”.

 

Lines 120-125. Authors used very different plant densities in their experimental environment e.g. 44,444, 63,000, 93,000 plants ha-1. Please explain the reason for this, since plant density has important effect on different characteristics.

 

Line 131-132.” measured in centimeters as the distance from the base of the 131 plant to the top of the first tassel branch”.

 

Authors should change the above to proper size of font.

 

Line 137. Please put a comma after “at harvest”.

 

Line 143-147. Authors write  “six grams of kernels from each plot were ground to a fine powder” and from those they use five grams (Line 147)  for scanning. In order to for the sample to be representative, the initial sample of fine powder should be at least 4-5 times larger from the sample that used for analysis.

Why authors make only six grams of powder?

 

 

Line 167. Please correct the phrase  “To estimate combining ability of the inbred lines”,

 

Line 209. In table 2 the heritability of GY is to high e.g.  between 0.73-0.91 and always greater than heritability of Zn. Could authors please make a comment about this and also put some references in the discussion.

 

Line 218-219. Correlation takes absolute values from 0.00-1.00. So r valued 0.16-0.20 even if are statistical significantly, are not important or do not give any information, since are more close to zero than to 1.00.

 

Line 206. Please put the before “lowest”.

Line 251. Please put a comma before “which”.

 Line 251. Please put a comma before “while”

Line 272.. Please put a comma before “while”

Line 311.. Please put a comma before “which”.

Line 315.. Please put a comma before hybrids.

Line 309-311. It would be beneficial for the discussion if authors could explain why the following is happening.

“However, the Zn levels were lower for all hybrids derived from high-Zn lines compared to the respective values observed in their parental inbred lines

 

 

Line 311.. Please put a comma before “which”.

 

Line 316. Please change “have” to “has”.

 

Line 316. Please remove comma after “yield”.

 

Line 305-322  In the first two paragraphs of the discussion you give two very important results

 

1. the importance of inbreds' phenotype in the creation of hybrids with elevated levels of Zn in the kernel. Moreover, hybrids with a Zn content > 30 pg/g across environments were produced exclusively from inbred lines.
2. In this study, grain yield was not correlated with Zn, which means that could be improved simultaneously, without yield penalty.

 

Please analyze more those two paragraphs because the above results are very important for the science.

 

Lines 351-366.

 Please analyze more those two paragraphs because the results are very important

1. The GCA effects accounted for > 70% of the total variability suggesting that the accumulation of Zn in maize kernels is predominantly governed by additive gene effects.
2. The positive GCA for Zn observed for inbred lines 2, 8,11,13,16 and 20 suggest possibility of transmitting favorable alleles from these parental lines to their hybrids and could be useful for breeding to improve Zn content.

 

Lines 367-375.

 Authors found no correlation between plant height and Zn concentration, so it would be better to reduce this paragraph in the discussion.

 

Line 353. Please put a comma before “suggesting”.

 

Line 370. Please change “serves” to “serve”.

 

Line 384. Author could change “Taken together” to something else such as conclusively.

 

Comments for author File: Comments.docx

Author Response

Please see attached file, "response_reviewer 2 - 763898".

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 looks at the expression of Zn in the progeny of high and low Zn QPM inbreds, and high and low Zn non-QPM inbreds, crossed in all combinations, in two environments per season for two seasons. 

Line 205, please check this sentence as there is a mistake. 

One realizes it is not always possible but it would have been nice if the parents had been included in the trials, to compare the actual values of the inbreds and progeny in the field. 

 

 

Reviewer 2 Report

The presented manuscript A Genetic Evaluation of Kernel Zinc in Hybrids of Elite Quality Protein Maize (QPM) and Non-QPM  Inbred Lines Adapted to the Tropics submitted by Edna Mageto, Michael Lee, Thanda Dhliwayo, Natalia Palacios-Rojas, Félix San Vicente, Juan Burgueño and Arnel R. Hallauer is well written, it is easy to read and understand.

As according to the authors, a zinc deficiency in even 20% of World populations represents a serious problem and it needs prompt actions and diverse solutions. The presented research would represent an addition to the available studies about Zinc accumulation in maize kernels.

However, the presented study suffers from a serious omission in experimental design and implementation. Namely, precise phenotyping (zinc levels) of the maize inbred lines used as parents is missing for concretely this particular experiment; and this is something that has to be done. Although some of those lines showed to be e.g. high-Zn as proved in some other studies; this was supposed to be done as a part of this experiment as obligatory. As a consequence, some of the results (or even all!) could not be interpreted but only at highly speculative level (lines 360-370). Although part of the results showed to be in accordance with the expectations (or hypothesis); the rest of the results that could not be explained are putting under the questions results all in all.  

Another issue that would be necessary to have is the methodology and according results for establishing the status of homozygosity and heterozygosity in analyzed hybrids that were obtained by presented crosses (e.g. lines 348 and 349). How did authors determined that maize hybrids with the highest values of Zn were heterozygous for opaque2 (o2) locus? Was a PCR performed? This also would be requested for this kind of study; in order to be published.