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  • Yanli Yao 1,2,3,
  • Mingwei Li 1,2 and
  • Xiumei Zhang 1,2,*
  • et al.

Reviewer 1: Chat Kanchana-udomkan Reviewer 2: Anonymous

Round 1

Reviewer 1 Report

Dear authors,

 

Thank you for your manuscript in the title of “Transcriptome analysis of watercore in pineapple”. The research included the application of RNA sequencing to identify differential expression between two pineapple samples, normal and mild watercore. Transcripts were then classified and 18 were validated using qRT-PCR. The research proposed molecular pathway related to watercore in pineapple which can be useful for basic insight for this physiological disorder and can be applied to trait selection.

 

There are few typing errors, major and minor comments as detailed below.

 

Abstract:

·      More result should be reported i.e. up/ down regulated genes by how many folds.

 

Keywords:

·      “pineapple, watercore and transcriptome” are already presented in the title, please remove them from keywords.

 

Introduction:

·      Authors mentioned about watercore in pineapple and other crops and reported the disorder symptoms. The significant of this trait should be mentioned in terms of economic loss, complexity etc. Authors should also add more details in transcriptome study in pineapple, how successful? Genome size of pineapple, is there any reference genome?

 

·      Line 48: identify some genes – some should be removed from the sentence, suggested to use candidate.

 

Material and methods:

·      Plant material: Did the authors treat pineapple with any treatment to initiate watercore? If not how can you explain, why does the same cultivar differentially expressed under the same condition? If there is any treatment, please indicate in this section.

·      2.2 Determination of cell wall composition: Please specify which trait were determined? Pectin, hemicellulose, cellulose etc.

 

·      Data analysis: Please add the analysis method for sugar content and cell wall composition.

 

·      2.8 Gene expression analysis: Why only one reference genes (actin) was used in this study? Is there any related experiment to show the stability of the expression of actin across the samples? If not, please report efficiency of the primer and gene stability.

 

·      Line 94: “2.8…. real PCR” should be real time PCR

 

Result:

·      Line 111 – 113: Additionally, the total … , respectively. Please rewrite this sentence. It is difficult to understand

 

·      3.4 Isolation of genes related to pineapple watercore

o   This section needs to be clarified. From line 151, authors mentioned the expression of  AcPAL, Ac4CL etc. but these genes (or these abbreviations) were not clearly presented in any table or figure. Suggestion to change the gene_symbol in Table 4 to be related to text in the content.  Please double check consistency of abbreviated symbols throughout the manuscript.

 

o   Suggestion to rewrite this section. Authors should report the result by grouping them by 1) significantly differentially expressed between the two groups, 2) up vs down regulation, 3) gene function of key genes.

 

o   Line 149 -150: The information was not related to Figure 7 as per cited. This should still refer to Table 4.

 

·      3.5. Isolation of transcription factors (TFs) related to pineapple watercore

o   Suggest to re-write this section. The result should report 1) TFs only up (HSF, GATA, NAC, MYB) and down (MADS) regulated, 2) proportion of up/down regulated of some TFs (bZIP, ERF, bHLH, WRKY).

 

·      All genes must be italicised: For example

o   Line 149: “AcBGLU16, AcBGLU18, AcBGLU26, and AcBGLU43” are referred to genes, they should be italicised.

 

o   Line 150 – 151: AcPAL, Ac4CL, AcACT, AcCYP, 151 AcHCT, AcALDH2C4, AcADH, AcPER, AcPOD, AcBGLU43, and AcMenE

 

·      There are methods and discussions mixed within the result in most paragraphs. Please move them to relevant sections.

 

o   Line 103 – 105: Two type of ……(HPLC, Figure 2) is a method, not result.

 

o   Line 114 – 115: “Calcium……….of cell wall” should be in a discussion section.

o   Line 117 – 119: “Phenolic compounds are….and antiaging properties.” should be in a discussion section.

 

o   Line 122 – 124: “To get insight …. High-throughput platform.” is a method, not a result.

 

o   Line 129 – 132: “To identify DEGs….at p<0.05.” is a method, not a result.

 

o   Line 167: “TFs play an …to abiotic stress.” is a discussion, not a result.

 

o   Line 176 – 179: “To validate the …in Table 5” is a method, not a result.

 

 

Tables and Figures:

·      There are a lot of tables and figures in the manuscript. Some of them can be moved to supplement data (Table 1, Table 2), or some of them can be removed (Table).

 

·      There should be more detail for all titles of figures and tables. They should be able to stand alone for readers to be able to understand the content.

 

·      Figure 1. Suggestion for title: Fruit and pulp of normal fruit (CK) and mild watercore (MS) pineapple

 

·      Table 1.

o   Suggestion for title: Sequences of primers used for qRT-PCR analysis for normal and watercore pineapple

o   Please add more details in the table, genes or accession number, primer Tm (ºC), expected size (bp).

 

·      Figure 2. Please add details of CK and MS in the title, add which statistical analysis did you used to compare the variables. T-Test, ANOVA or else? The comparison, was it between the genotype variables or within the same genotype, but across sugar types? Please indicate in the title.

 

·      Table 2 and Table 3. please add more detail of which samples were used to achieve the data.

 

·      Table 3: Please add details of CK and MS in the title and more detail for statistical comparison, which statistical analysis did you used to compare the variables. T-Test, ANOVA or else? The comparison, was it between the genotype variables or within the same genotype? Please indicate in the title.

 

·      Figure 4, 5 and 6 can be grouped into one figure (a, b and c) and more detail title should be adjusted.

 

·      Table 4. Adjust gene-symbol to be the same as in the context.

·      Figure 7: This heatmap only presented up (red) and down (blue) expression without any degree of intensity. The colour chart next to the figure did not explain the figure result. Please remove the colour chart and add explanation in the title. The gene_symbol next to accession numbers should also be consistent with the gene names presented in the context and table. Please double check. In fact, this figure is the same as table 4, possibly remove it.

 

Discussion:

·      Some discussion did not relevant to the research finding. Suggested point of discussion to be

o   Pathway for watercore in pineapple and other related crop

o   Transcriptome analysis of watercore in related crop

o   Introduce key genes regulated to watercore in pineapple, and then discuss each gene.

·      Line 227 – 233: Xie et al….310 DEGs [18] these are not relevant to this research. Consider to remove.

·      Line 233 – 235: Over claim for the first time of transcriptome analysis for watercore. This research could be the first for pineapple. But there was watercore transcriptome study in pear (Liu, Xiao, et al. "Transcriptome and metabolome analyses provide insights into the watercore disorder on “akibae” pear fruit." International journal of molecular sciences 22.9 (2021): 4911.), apple (Cebulj, Anka, et al. "Alteration of the phenylpropanoid pathway by watercore disorder in apple (Malus x domestica)." Scientia Horticulturae 289 (2021): 110438.) etc.

·      Line 235 – 236: “A total of …21 KEGG pathways” is a result, not discussion.

·      Line 248 – 250: “A total of ….are differentially expressed.” is a result, not discussion.

 

Author Response

Thank you for your review for my manuscript firstly. I totally agree with you for the review changes you mentioned, and I have changed them and my reply to the article modifications as follows.

  1. Add 3Zhanjiang Experiment Station，Chinese Academy of Tropical Agricultural Sciences; yaoyanli1983@163.com of my institute.
  2. “Among them, the expressions of AcPME, AcBGLU43, Ac4CL5, AcPER1 and AcPOD genes were up-regulated by 7-21 times in watercore fruit, while the expressions of AcSUS7 were down-regulated by 16.61 times, and the expressions of other differential genes were up-regulated or down-regulated by more than 2 times.”were added to the abstract
  3. remove“pineapple, watercore and transcriptome”from keywordsand" RNA-seq" has been added
  4. The introduction was rewritten according to the opinions of the reviewers
  5. Plant material:Watercore is a physiological and metabolic disorder caused by adverse environmental conditions, and can not be directly observed from the appearance. At present, there is no treatment method to induce watercore directly.
  6. 2.2 Determination of cell wall compositionAdd the content “pectin, hemicellulose and cellulose”.
  7. Add the analysis method for sugar content and cell wall composition.
  8. Reference for the selection of reference genes [15] Li, Y.H.; Wu, Q.S.;  Xia, H.;  Liu, S.H.;  Zhang, H.N.;  Zhang, Z.;  Sun,  G.M. Molecular cloning and characterization of four genes encoding ethylene receptors associated with pineapple (Ananas  Comosus l.) flowering. Front. Plant Sci. 2016, 7, 710. https://doi.10.3389/fpls.2016.00710
  9. 3.4 Isolation of genes related to pineapple watercore and 3.5. Isolation of transcription factors (TFs) related to pineapple watercorehad rewrite these section.
  10. The mixed methods and discussions in the results have been moved to the relevant sections.
  11. Table 1and Table 2are moved to supplement data. 
  12. Supplemental changes have been made to the titles of all thefigures and tables.
  13. The discussion was rewritten as suggested by the reviewer.
  14. Other problems such as typing errorsand copy ratio were corrected.

Reviewer 2 Report

The manuscript lacks many methodological details and consequently many results cannot be followed. The authors need to complete the information (see attached file). 

 

 

Comments for author File: Comments.pdf

Author Response

Thank you for your review for my manuscript firstly. I totally agree with you for the review changes you mentioned, and I have changed them. See the revised draft for details.

Round 2

Reviewer 2 Report

The authors have addressed all previous scientific concerns.