Phenotypic Characterisation for Growth and Nut Characteristics Revealed the Extent of Genetic Diversity in Wild Macadamia Germplasm

Round 1

Reviewer 1 Report

Dear authors,

I have read your manuscript with great interest. In general, I like the manuscript. Overall, the manuscript is basically written clearly, and materials and methods used for data analysis are acceptable. The results are clearly presented, and the discussion is reasonable and sound. However, list of references should be updated. All corrections to minor errors, as well comments and suggestions are marked in the attached document.

Best of luck with the future work!

Comments for author File: Comments.pdf

Author Response

Thank you very much for your comments. We addressed all your comments and made the necessary modifications. Please see the attachment for the details.

Author Response File: Author Response.docx

Reviewer 2 Report

The paper is well written, the results and their context described clearly. Further references to molecular data among wild species may be useful in highlighting elements of future research in introgressing yield traits. 

Author Response

Thank you very much for your kind review. The genetic structure of macadamia wild species using DArTseq-based markers was reported by Thuy, M et al (2020). Molecular and phenotypic data of wild species will be used in future research to find out markers associated with yield traits, which we have suggested in the discussion part of this document.

Reviewer 3 Report

Title:

Phenotypic characterisation for growth and nut characteristics revealed the extent of genetic diversity in wild macadamia germplasm

Authors:

Thuy T.P. Mai 1, 2 *, Craig M. Hardner 1, Mobashwer M. Alam 1, Robert J. Henry 1, and Bruce L. Topp 1

Reviewer’s Summary:

The authors present a study of macadamia accessions aimed at identifying promising source material for crop improvement breeding based on observed trait expression that they associated with source genetics and geographic distribution. The nearly 250 accessions they examined were derived from four species and inter-specific hybrids propagated in a common garden from cuttings collected in the wild or planted field.

General comments:

The authors report on the results of a straight-forward phenotypic characterization of an economically important tree crop. While the general study design itself is not novel, they report careful implementation of the same adapted to a long lived tree species are to be applauded for this effort. The general organization and flow of the presentation is clear. However, there is critical lack of clarity and specificity in the sampling methodology underlying the measurements and reported results. While the interpretation of the results follow, the methodological specificity need to be improved in order to be improved for the reader to understand the quality/validity of the results.

My recommendation is to satisfactorily addressing the observations – in particular those regarding lack of clarity of sampling methods, species representation, and meaning of associated terminology described below.

Specific comments:

94-95: The photo in Figure 1 does not add to the interpretation of the results; interested readers could likely find this in reference 17. Since the interpretation of results includes latitudinal variation among accessions, it would be meaningful for Figure 1 to be a line diagram map showing the accession collection locations in relation to the location of the planting site. Such a map would also be useful in clarifying the field sampling and terminology issues describe immediately below in reference to lines 99-112.

99-112: Beyond citing details published elsewhere, please clarify the sampling scheme both in terms of how accessions were selected in the field (wild or planted) and how plantings in the two blocks were selected for inclusion in the study sample. As written, this is entirely opaque to the reader and thus prevents meaningful assessment of the reported measurements and statistical analyses.

In Table S1, each row is a unique Accession – does this refer to a single tree (genet) that was sampled from the wild or a planted field? Or does Accession mean a specific “site” within a “Locality”? Line 102 states “number of accessions sampled/site ranged from one to eight” which seemingly corresponds to the “No. of trees” column in Table S1. The authors indicate in Line 108 that a random sample of 247 accessions were used and there are 247 rows/accessions in Table S1. Were 247 individual trees randomly selected from the garden (two blocks) containing 448 (2 x 8 x 28; lines 101-102) planted cuttings? How does this relate to the “one to eight” trees per “site” indicated in line 102 (which sums to 397 in the last column of Table S1)? Is an accession a site and the number of trees (one to eight) cuttings from one to eight different trees at the site? Or, is it one to eight cuttings from the same tree where “accession” refers to a single source tree? In either case, please clarify why are there multiple “no. of trees” per accession planted in the blocks and why the number differs for each accession. Lastly, please specify what is meant by “selected randomly” – does this refer to selection of individuals in the wild or planted field or to selection of trees from within the blocks to include in the analysis?

There is also inadequate distinction of what precisely is meant among “locality”, “region”, and the geo coordinates as indicated in Table S1. “Locality” would normally be more specific than “Region”, yet there are multiple Regions tied to the same Locality – some with the same coordinates and some with differing coordinates. Looking at Table S1, Locality Gundiah, Qld, is represented in multiple rows with distinct accession numbers tied to the same set of geo coordinates. Again, the reader is left confused about whether accessions are individual trees? Individual “sites” within a locality? 

Clarifying all of the above is necessary to evaluate the reported results and the corresponding interpretation of the analyses.

115-140 (Data collection): In general, the illustration of measuring techniques in Figures 2 and 3 is superfluous. It is not necessary, for example, to illustrate taking a stem diameter measurement with a tape, using a caliper to measure nut parameters, or a scale to measure weight (mass). A single illustration – either line diagram or annotated photo(s) showing the various measured spans of the nuts, locations of hull thickness measurements, and an intact nut and separated kernel would be sufficient.

Line 130-131: Please specify how ten nuts were selected from all the nuts harvested from a given tree. Since nut traits are among the measured subjects of analysis, the reader needs to know how investigator bias was avoided – presumably with some kind of randomization or consistently applied heuristic applied in selecting the 10 nuts from the trees. This is much more important than showing a photo of ten nuts labeled 1 - 10 (Figure 3a) – your readers can count to ten.

141 – 174 (Data analysis): Specifying the explicit variance models used in the analysis is to be applauded. In reporting estimated genetic variances and heritability, the authors presumably do so as a function of assumed genetic differences determining measured trait values among accessions. That is fine but points back to the critical need to clarify the sampling scheme and use of the terms accession vs. number of trees vs. locality detailed above. Also please clarify the replication term in Models 1 and 2 – were there multiple replicate measurements for each accession? If so, does this correspond to the number of trees per accession (from one to eight)? Table 1 indicates N values for each species that sum to (nearly) the 247 sample size. Is N both the number of accessions/species with one measurement per accession or are there multiple replicates measured for some accessions (up to eight?) and single (one) measurement for others? If the latter, how does this difference in # of replicates not weight/influence the results?

149 and 158: Both of these lines refer to analyses involving multiple comparisons within the same experimental design. Please specify and use an appropriate experiment-wise correction for significance testing (e.g. Tukey HSD for means comparisons, Bonferroni correction for multiple correlations) to avoid Type 1 errors.

255 (Figure 4): The PCA figure is quite noisy. The background should be white, the grid lines should be removed or, if retained should be thin black or light grey, and the trait vectors should be black because color is used to differentiate species among the accession dots. The trait vector labeling is unreadable; it needs to be simplified with numeral coding and/or otherwise offset so individual vector trait labels are distinct and legible. Please specify the analytical basis for positioning the ellipses – if arbitrary, then remove since the overlap of the three species is evident from the placement of the dots as is the separation for M. ternifolia.

Typographical and/or word usage errors in need of correction are indicated by highlighting in the attached marked-up PDF.

Author Response

We would like to express our heartiest thanks to the reviewer for your valuable suggestions to improve the manuscript. We agreed that all the comments are valid, which we addressed in the revised version of the manuscript. Please see the attachment for the details of the responses.

Author Response File: Author Response.docx