Fine-Mapping of qECL7.1, a Quantitative Trait Locus Contributing to Epicotyl Length in Adzuki Bean (Vigna angularis)

Round 1

Reviewer 1 Report

General Comments

            Some previous reports are already available to improve the epicotyl length of the adzuki bean, which are mentioned below.

1)      Kaga, A., Isemura, T., Tomooka, N., & Vaughan, D. A. (2008). The genetics of domestication of the azuki bean (Vigna angularis). Genetics, 178(2), 1013-1036.

2)      Isemura, T., Kaga, A., Konishi, S., Ando, T., Tomooka, N., Han, O. K., & Vaughan, D. A. (2007). Genome dissection of traits related to domestication in azuki bean (Vigna angularis) and comparison with other warm-season legumes. Annals of Botany, 100(5), 1053-1071.

3)      Mori, M., Maki, K., Kawahata, T., Kawahara, D., Kato, Y., Yoshida, T., ... & Kato, K. (2021). Mapping of QTLs controlling epicotyl length in adzuki bean (Vigna angularis). Breeding Science, 71(2), 208-216.

            The current content of the manuscript is more closely related to Mori et al. 2021 experiment (Mentioned above; Reference number 3). More contents from the introduction to the discussion are similar to the Mori et al. 2021 article. Authors simply change the phrases. So, the authors should take more time and reconstruct the manuscript.  Apart from this, authors need to do some insilico and lab experiments. For example, authors should identify the candidate genes from the adzuki bean, common bean, or soybean genome using the nucleotide sequences of ECL trait-responsive QTL. After that, authors can analyze the expression pattern of ECL-responsive genes in the ECL tissues, which will help to improve the ECL traits. or authors said that they had identified 35 predicted genes within the qECL7.1 region. So authors can validate some genes through qRT-PCR. Why authors did not do this experiment under any abiotic stresses?

Specific comments

1)      L. No. 1111; What based authors select shorter and longer ELC of 10 lines for this experiment.

2)      Authors should write the full form of abbreviations of InDel, CAPS, dCAPS, and SNP in the abstract portion.

3)      Write the keyword in alphabetical order.

4)      Authors should check the sentences in L. Nos. 39-40. Authors confused the trait and cultivars. So, kindly clearly rewrite the sentences.

5)      In L. No. 41. Harvesting of what? Weeding or harvesting of bean?

6)      Include the appropriate reference in L. No. 43.

7)      L. No. 54, Epicotyl length should be changed into “ECL”. Authors should check and rectify this error entire manuscript.

8)      Where did the authors collect from both cultivars?

9)      L. No. 97; Potting soil does not contain micronutrients?

10)  L. No. 138, include the accession number or link for Chihayahime reference sequence.

11)  L. No. 153; 96 F2 or F7 population?

12)  Authors should include a statistical analysis section in materials and methods.

13)  What phenotypic data did the authors use to select 10 lines for this experiment?

14)  L. Nos. 246-248; authors should check the sentences and correct them.  

15)  Authors should submit the collected phenotypic data as a supplemental file.

Kindly check the manuscript again and rectify the small mistakes. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Authors report a detection of two major QTLs and fine mapping of one QTL for ECL trait in adzuki bean using different strategies of QTL mapping. Although the methodologies were generally well designed, I have some major concerns:

- The validation of 4 QTLs found by QTL-seq in a F2 population: the linkage map contains only 34 markers, which does not provide enough resolution for a reliable validation of the 4 QTLs. Maybe, for this reason, authors only could validate 2 QTLs. Indeed, other studies have found QTLs in LG 2, as here by QTL-seq method.

- Authors refer that qtls found in LG7 and LG10 are major QTLs, however, they only explain about 13 to 24 % of total variation. 

-The methodologies applied differ in the conclusions about the beneficial parental alleles for ECL.

- Since the Adzuki beans cultivars have already longer epicotyls than wild species, which was the applied/agronomic objective of this study?

 

Other comments:

Introduction: 

-Introduction organization should be improved. First, authors should mention the state of the art, followed by the gap of knowledge and then, stating how this gap could be filled ending with the objectives and novelty of the study. 

Line 46-48: provide a reference

 

Methods:

-Figure 1: How old are the plants in this picture? They seem older than 35 days after sowing, If so, please provide a picture with parental lines with 35 days after sowing (phenotypic screening age) .

-How authors define the number of 10 individuals per contrasting group of phenotypes?

Line  137: provide a reference

Line 142-144: this sentence should be included in the Results section and should include more details. How many nt did you consider short reads?

Line 179-181: provide concentration of the reagents instead of volumes

Line 216-217: Was the rotation of the trays also applied to the other phenotypic evaluations? Why in this case, the ECL survey was done 21 days after sowing? The same in line 230.

 

Results/Discussion:

-transgressive segregation observed should be mentioned and discussed together with beneficial alleles from each parental line.

-Figure 3: please provide information from all chromosomes.

 - from the 35 genes found in the qECL7.1 QTL, authors may mention the most interesting ones and discuss how they may contribute for the ECL. For example, in my opinion, the "cellulose microfibril organization" gene may have a role in the ECL trait. Please explore your results. 

-The organization of Discussion section should be improved. Please provide first your findings and then compare with previous studies to state your conclusions.

-Line 380-382 and 388-389: be careful when you state that qtls seems to be identical. About 600 000 bp is a long distance. Please, revise your sentences.

 

Minor revisions:

Line 20-21: replace "ordinary epicotyls" by  "ordinary length epicotyls"

Line 39: replace "cultivar" by "agronomic"

Line 43: be precise on the "relatively low"

Line 54: QTLs (Quantitative Trait Loci)

Line 230: replace "planting" by "sowing"

Line 448: specify "close"

English/language should be revised mostly in Introduction section to better clarify the sentences.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

No further comments. 

Some minor English check is required for this manuscript before publication. 

Author Response

We corrected the English language accordingly

Reviewer 2 Report

Figure 1: Please include in caption that picture is for the parent lines at maturity stage, to clarify the reader.

 

 

Line 256 – 259: 500 nµ and 30 ng are quantities, not concentrations, please correct.

 

 

 Minor editing of English language required

Author Response

We corrected the English language accordingly.

Author Response File: Author Response.docx