Broadening the Variability for Peanut Breeding with a Wild Species-Derived Induced Allotetraploid

Round 1

Reviewer 1 Report

This paper describes a recombinant inbred line (RIL) population developed from a cross between cultivated peanut and a reconstituted tetraploid (A. duranensis x A ipaensis).  The RILs were evaluated for numerous traits, and a considerable amount of variation was observed.  Two nice photographs are included which illustrate the variation in fruit type and canopy architecture found in this population.  A dense genetic map is presented, and several QTL for important traits were mapped.  A large amount of data has also been made available in the supplemental files.  This population and dataset will be useful resources for peanut breeders and researchers for both peanut improvement and domestication studies.  I believe this paper is acceptable for publication in its current form.

I did find a few minor errors.  Please correct these during the editing process:

Lines 234-235.  Change ‘qui-square’ to ‘chi-square’ and list the p-value as 0.335, rather than as a percent.

Line 346 (Table 3 header).  ‘WSEV, AMSEV, AWSEV’ should be ‘ISEV, AUDPC-MSEV, AUDPC-ISEV’.

Line 354 (Table footnote) and line 490.  Change ‘addictive’ to ‘additive’.

Line 490.  This should reference Table 3, not 4.

Line 370 (Table 4).  What is ‘GV MSEV’?

Figure 5.  There is a box that says ‘Plot area’ floating in the middle of the graph.

Author Response

Reviewer 1 Comments and Suggestions for Authors and Answers

This paper describes a recombinant inbred line (RIL) population developed from a cross between cultivated peanut and a reconstituted tetraploid (A. duranensis x A ipaensis).  The RILs were evaluated for numerous traits, and a considerable amount of variation was observed.  Two nice photographs are included which illustrate the variation in fruit type and canopy architecture found in this population.  A dense genetic map is presented, and several QTL for important traits were mapped.  A large amount of data has also been made available in the supplemental files.  This population and dataset will be useful resources for peanut breeders and researchers for both peanut improvement and domestication studies.  I believe this paper is acceptable for publication in its current form.

I did find a few minor errors.  Please correct these during the editing process:

General observation before reading the answers: Due to minor changes suggested by other reviewers, the number of the lines might have changed.

Lines 234-235.  Change ‘qui-square’ to ‘chi-square’ and list the p-value as 0.335, rather than as a percent.

Answer: We corrected these errors in lines 237-238.

Line 346 (Table 3 header).  ‘WSEV, AMSEV, AWSEV’ should be ‘ISEV, AUDPC-MSEV, AUDPC-ISEV’.

Answer: We corrected this error in line 346.

Line 354 (Table footnote) and line 490.  Change ‘addictive’ to ‘additive’.

Answer: We corrected these errors in lines 356 and line 493.

Line 490.  This should reference Table 3, not 4.

Answer: We corrected this error in line 494.

Line 370 (Table 4).  What is ‘GV MSEV’?

Answer: We added “*” in the last column of line 370, turning to “ * GV MSEV”, and we also added a foot note in line 373 describing “ * Genotypic value of severity means”.

Figure 5.  There is a box that says ‘Plot area’ floating in the middle of the graph.

Answer: We provided a new file without this box in Figure 5.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript under review is devoted to the issue of expanding diversity in many economically valuable traits that are important in peanut breeding. The problem of expanding variability in many traits, including economically valuable ones, is solved by the authors by introducing the germplasm of wild-growing peanut species into the cultivar Runner IAC 886, a widespread variety of peanuts in Brazil. The approach used by the authors is not new and is quite often used by breeders when creating varieties of other agricultural crops in the world. The peculiarity of using this approach for peanuts is that the cultivar of peanuts (A. hypogaea) is an allotetraploid obtained spontaneously in nature many thousands of years ago from the crossing of A. duranensis and A. ipaensis. However, plants of this species in culture have lost some traits (for example, leaf spot and rust), as well as resistance to nematode infestation. The authors applied a rather elegant approach - obtaining hybrids between A. hypogaea and an artificially obtained interspecific hybrid A. duranensis x A. ipaensis, transferred with the help of colchicine to the tetraploid level. The authors obtain F1, clone it and then get several generations (F8 and F9) from self-pollination (87 recombinant inbred lines in total). Using modern molecular genetic methods (QTL, SNP), as well as bioinformatics methods, the authors show a wide variability in many of the studied traits among these lines. The authors have established several QTLs for use in programs for marker-based breeding of new varieties, and also obtained very interesting data on various degrees of tetrasome recombination. The reviewer has no significant comments on this manuscript. The materials are of interest to breeders working to improve peanuts and the manuscript can be recommended for publication in this journal.

Author Response

We appreciate your time reviewing the manuscript and your comments, which were very important for its approval.

Reviewer 3 Report

The manuscript entitled with ‚Broadening the variability for peanut breeding with a wild species-derived induced allotetraploid’ describe the characterization of 87 recombinant inbred lines derived from a cross of peanut cultivar Runner IAC-886 and it’s wild ancestor A. ipaënsis. The authors identified lines with improved agronomic traits and provide interesting material for broadening the genetic diversity of peanut breeding material. The manuscript is well structured and the results are properly presented. I have just a few comments which may improve the final manuscript.

Abstract: resistance to pathogens is in my eyes an agronomic trait. Therefore, I would like to ask the authors to be more specific.

Introduction: Please introduce runner-type cultivars, since not all readers are familiar with them.

L116: The authors measured the height of the main stem at day 101. I assume (and is illustrated in Figure 2) that within the population the developmental stage differs which means that measurement at a certain developmental stage like flowering (BBCH code 61) would make the data more consistent. The same holds for the measurements of growth habit and canopy.

Fig 3: The numbers on top of the graphs and the descriptions are hardly readable. Please find a different way of presentation.

Author Response

Reviewer 3 Comments and Suggestions for Authors and Answers

The manuscript entitled with ‚Broadening the variability for peanut breeding with a wild species-derived induced allotetraploid’ describe the characterization of 87 recombinant inbred lines derived from a cross of peanut cultivar Runner IAC-886 and it’s wild ancestor A. ipaënsis. The authors identified lines with improved agronomic traits and provide interesting material for broadening the genetic diversity of peanut breeding material. The manuscript is well structured and the results are properly presented. I have just a few comments which may improve the final manuscript.

General observation before reading the answers: Due to minor changes suggested by other reviewers, the number of the lines might have changed.

Abstract: resistance to pathogens is in my eyes an agronomic trait. Therefore, I would like to ask the authors to be more specific.

Answer: We agree with you and rewrite the sentence in line 15 as “Here we report morphological and agronomic, including disease resistance, variation…”.

Introduction: Please introduce runner-type cultivars, since not all readers are familiar with them.

Answer: We agree with you, and added a sentence in line 34-36: “Runner-type peanuts are small seeded types of var. hypogaea, with two seeds per pod and tan seed coat color”. Addind this sentence would require deleting the following sentence.

L116: The authors measured the height of the main stem at day 101. I assume (and is illustrated in Figure 2) that within the population the developmental stage differs which means that measurement at a certain developmental stage like flowering (BBCH code 61) would make the data more consistent. The same holds for the measurements of growth habit and canopy.

Answer: The growth habit and canopy were evaluated at 138 days after planting, at the end of the plant cycle. At this stage, the architecture of all the plants was very well defined and, certainly, both measurements would not have changed if they were done at a certain developmental stage, as suggested by the reviewer. The difference among the plots observed in Figure 2 shows the variability among the RILs for these traits, as described in lines 226 and 227. For the height of main stem, this suggestion could probably be a better standardization than our measurements at the same day after planting. However, we do not think this has caused some significant distortion in the data because the plants germinated basically at the same time and were all harvested simultaneously, which means that the developmental stages were not so different among the 87 RILs

Fig 3: The numbers on top of the graphs and the descriptions are hardly readable. Please find a different way of presentation.

Answer: We agree with the reviewer, and we provide a file with larger numbers in the top of the graphs.

Author Response File: Author Response.docx

Reviewer 4 Report

Please see the attached comments

Comments for author File: Comments.pdf

Author Response

In this paper, Suassuna et al. describe a method to increase genetic diversity in peanut breeding through induced allotetraploid. As a reviewer, I appreciate the amount of input in the draft with supporting information in the supplementary files. Also, the authors were able to collect several phenotypes for genetic analysis which is impressive. Based on the draft, it seems the authors were able to achieve the objective of the research.

Overall, the draft is interesting, and appealing to the reader, however, the manuscript has failed to address the key questions for any plant breeding research.

General observation before reading the answers: Due to minor changes suggested by other reviewers, the number of the lines might have changed.

Major comments:

1) Quantitative trait loci (QTL) are highly driven by environmental factors and usually, there is variability between/or among years in the phenotypic data. This study was carried out with just one year of data without any further validation. Please provide the second-year data or any validation set to back up your results.

Answer: We totally agree with this comment. We tried to evaluate this population for leaf spot resistance traits in previous years before this successful trial. However, working with an interspecific population at this stage (first cross derived from wild x cultivated) seed increase is difficult, mainly in genotypes with wild fruit type, resulting in a highly variable seed production among the RILs. With a reduced number of plants per plot, in a previous trial, we evaluated leaf spot lesion number (LN), and we detected one QTL at chromosome 8 which is in common with a QTL described in this manuscript for severity means (MSEV) and severity index (ISEV) (see Table 3), although the variable is not the same (LN is a trait correlated to severity). We suggest mentioning these unpublished results in the text as suggested in line 499-500. We consider that the publication of this QTL data in the manuscript is important since this information could be useful and comparable to further studies. We remain working with this population, advancing the more resistant RILs with good agronomic traits and this material will be used for validation of the mapped QTLs for use in marker-assisted selection in a near future.

Growth habit and fruit type are traits with high heritability. The influence of environmental factors for height of main stem and canopy were minimized under this trial, once the management provided good conditions for plant development, as is described in Material and Methods (line 116) and can be observed in Fig 2.

2) The authors were able to collect some phenotypes in the greenhouse but others in the field. Why these phenotypes were not replicated in different conditions, please explain?

Answer: We collected data for morphological traits in the greenhouse once there was only one plant per pot (as described in line 91); these traits are not influenced by environment. The agronomic traits were evaluated in the field trial, considering all the plants in the plot (as described in line 114).

3) In the QTL analysis, only selective phenotypes were used. What is the reason behind this even though several other phenotypes were collected? What makes them more important than others?

Answer: We included in the QTL analysis the agronomic/quantitative traits. The objective was the identification of markers in wild genomic regions associated to these traits, for use in marker-assisted selection. Therefore, the four morphological traits were not included. We added a text in the Introduction (lines 73-74) to make this clear for the readers.

4) Phenotypic data for the disease severity QTL was selectively chosen for 138 days after planting even though the data were collected for multiple time points, what makes this time point so important, please explain? Why not analyze other time points individually?

Answer: In fact, the evaluations for leaf spot severity were performed at 104, 118, 138, and 146 days after planting, as described in line 128 of the manuscript.

5) QTL for leaf spot has been previously mapped in another study. The authors failed to discuss how this study is different. Please provide enough citation in the introduction as well as discuss further.

Answer: To the best of our knowledge, this is the first study identifying QTLs for leaf spots resistance using A. duranensis or A. ipaënsis as sources of resistance.

Minor comments:

Page 2, Line 81- “Lines were produced from a single F1 plant, clone by cuttings to produce enough seeds”, please explain what is enough seeds?

Answer: We included a text (lines 85-86) to make this clearer.

Page 11, Line 344- “LOD significance threshold estimated for each trait ranged from 2.5 to 3.4, and only QTLs with LOD values exceeding these thresholds were included” however, the table contains one of the LOD with 2.5 value, please explain. I think it would be nice to include a threshold for each of the traits.

Answer: The threshold for each of the traits was included in the Additional File S2. There was an error in the text, since the thresholds ranged from 2.4 to 3.1. This information was corrected in the text (line 347).

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

The comments were satisfied except few. Overall the draft looks good.