Identification of Reliable Reference Genes for the Expression of Hydrangea macrophylla ‘Bailmer’ and ‘Duro’ Sepal Color

Round 1

Reviewer 1 Report

The manuscript titled “Reliable reference genes selection for normalization genes expression of controlling Hydrangea sepals color in ‘Bailmer’ and ‘Duro’” presents interesting work on reference genes on sepal coloring in Hydrangea macrophylla. The authors lay out their way of argumentation in a structured way. Data is presented in a comprehensibly. Before publication, some aspects need to be addressed. Generally, the selection of present or past tense needs to be homogeneous within every section. Additionally, “Hydrangea” needs to be clearly referred to as name or scientifically (in italics). Furthermore, the plant material in the materials & methods part has to be described in more detail (see comment on lines 69,70). Lastly, I suggest changing the title to make it more reader-friendly by changing its structure. All in all, the manuscript needs changes to be ready for publication but contains valuable information for further studies.

 

Line 2: Please change into “genes”

Line 12 and following: Please change “hydrangea” to “Hydrangea”

Line 18: Please change into “…top in the group. All materials…“

Line 31: Please give a reference for this phenomenon. (e.g. Hayter, C.N. 1949. Growing Blue Hydrangeas and Other Hydrangea Hints. Rhodesia Agricultural Journal 46:449–451.)

Line 32: Please add the “.” at the end.

Lines 45,46: Please give more insight on why potato and hydrangea are comparable in this context.

Line 53: Please change into Hydrangea macrophylla. Did you use seedlings or cuttings?

Line 60-62: Figure 1 shows a slight blue coloring in D(Tr)-S3. Please indicate your threshold on when a flower is able to show blue coloring.

Line 69,70: 36 samples in total equal a threefold repetition. Are these taken from the same plant or different plants? Please give your reasoning on why this number of replicates is sufficient for your analyses.

Lines 73-79: I suggest to illustrate the grouping in a table to gain a clear arrangement

Line 95: Please correct to “Gene introduction”

Line 134: Please increase font size to make the axis description readable.

Lines 140-145: Please shorten the listing as it is already comprehensible from Table 2.

Line 146-167: Please use a uniform style for “vs” in your tables and text.

Line 155: Please correct to “Based on”

Lines 233-238: Please shorten the sentence to make it more reader-friendly.

Lines 247,248: Please give a scientific example for this phenomenon including reference.

Line 252: Please define your range of similarity. Additionally, make this selection clear in your Material & Methods.

Line 262: “put” instead of “putted”

Lines: 270-274: In comparison to the text, conclusions are written in a very general way. I suggest integration of the most important key-words to compilate a holistic conclusion on your findings.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript, “Reliable reference genes selection for normalization genes expression of controlling Hydrangea sepals color in ‘Bailmer’ and ‘Duro’” summarizes a series of RT-qPCR experiments using bigleaf hydrangea sepals before and after aluminum treatment. This is primarily a methods paper but the methods lack the detail necessary to repeat these experiments. In my opinion, this paper cannot be published without full details of transcriptomic analysis used by the authors and significantly more detail regarding sequence analysis for primer design. Further, the authours should at least acknowledge the fact that a reference genome exits for bigleaf hydrangea, along with a huge body of literature around aluminum pathways. Other comments are by line number below.

26: Replace “Hydrangeas….are native” with “Bigleaf hydrangea….is native”.

30: “We noticed” is not good enough, considering there is a large body of literature dealing with physiological pathways of Al-induced color change and cultivars that do not change color in the presence of Aluminum. Please review this literature thoroughly and incorporate here and throughout the manuscript.

43: Change “(EF1-a) were…” to “(EF1-a) was…”.

47: You claim that scholars are interested in hydrangea and the need for research is urgent. Please provide citations, or results from interest group surveys, economic studies, etc. that support your claim, here and throughout the manuscript.

53: More information is needed about how these plants were grown. What media was used? How often were they watered and how? Were they fertilized? What do you mean by placing containers in the “experimental field”? Was this indoors or outdoors? Were they grown under shade? You say they were cultivated for two years. Were they repotted during this time? Did you move them from the “experimental field’ during this time, or were they grown up before you placed them for this study?

53: Change “cutting seedlings” to “cuttings”.

57: Change “floral organ” to “sepal” here and throughout.

58: Remove sentence beginning, “Sepals were thinner and smaller…” since you present no data on this.

59: Change “tox” to “to”.

63: Change uppercase reference letters, (A), (B), etc. to lowercase, to match what is shown in the pictures.

65: Remove the word “type”.

66: How was Al delivered to the plants?

73: “four groups (nine subgroups)” is not clear. I counted seven groups in the list from lines 74 -79. Please make a table listing the experiments. Then refer to it here, “We divided the 12 samples into X groups of experiments (Table 1).”

84: Did you quantify, or in any other way asses for quality and purity, your RNA or cDNA samples?

85: Change “Prime” to “primer”

86: What transcriptome data are you referring to? Why did you not use the bigleaf hydrangea reference genome for these sequences?

86: More information is needed about filtering. What software did you  use?

91: How did you use BLAST? What were you searching against? It is unclear how you obtained these sequences.

95: The captions of tables and figures should stand alone. The reader should be able to  understand the table without referring to the text. Please revise all table titles accordingly.

126: What R2? Of the standard curve?

129: There are no details of this gel in your Materials and Methods. Please include all details including plant material, type of nucleic acid (DNA or RNA), running conditions, visualization conditions, etc. Did you use the same primers as for RT-PCR?

140: What does “adapt to group” mean?

203: What do you mean by “data operations?”

221 – 222: You must include the full details of this transcriptomic analysis in this paper, or cite such a paper.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Changes are acceptable, though more information on the transcriptome sequencing is warranted.