Development and Application of a PCR-Based Molecular Marker for the Identification of High Temperature Tolerant Cabbage (Brassica oleracea var. capitata) Genotypes
Round 1
Reviewer 1 Report
The work of Song et al. provides new and interesting information related to selection of temperature stress resistant varieties of Brassica oleracea var. capitata. The manuscript is well written and the figures just has minor issues to be fixed. In order to improve the manuscript, the authors must provide the methods, data and analysis associated to RNAseq analyses, since the identification of TPPI as a possible marker for thermotolerant varieties relays completely on this approach. Otherwise, it is not clear why authors perform a whole transcriptome sequencing approach if they want to focus just on TTPI and TPPJ related genes. Indeed, figure 3 is constructed based on RNAseq data but no details how this assay was performed is provided in Materials and Methods section. Also, the introduction of the manuscript and associated references does not provide a solid basis to focus the analysis onto these two genes. Despite this important issue, the molecular identification and use of BoTPPI-2 alleles as marker for thermotolerance is described without major flaws across the document. I recommend to authors to analyze and improve figure 3, showing the transcriptional expression of all B. oleracea var. capitata TPP genes and improve the discussion connecting what is known about trehalose-6P as signaling molecule and it is role in thermotolerance. Then, complement this concept with the known function of TPPI, which is dephosphorylate trehalose-6P, and connect that to the thermotolerance phenotype observed in plants that has low or null BoTPPI-2 expression. Of course, further experiments will be requiere to test any hypothesis but are far beyond the scope of this manuscript, the idea is propose a way to continue this work on the discussion section.
As minor issues:
- Figure 1 must be a supplemental figure, since the data presented (2018) does not correspond to the data during the time of analysis (2017) as authors state in the text. Authors explain that this data (2018) is similar but that is not a scientific fact.
- Table 1 must be a supplemental figure, since it describe the sequence of the DNA oligos and this information is not fundamental to understand the approach and data presented by the authors.
-Figure 8 and 9, does not contain any indicator of molecular weight next to the MW lane.
Author Response
Author's Reply to the Review Report (Reviewer 1):
Comments and Suggestions for Authors
The work of Song et al. provides new and interesting information related to selection of temperature stress resistant varieties of Brassica oleracea var. capitata. The manuscript is well written and the figures just has minor issues to be fixed. In order to improve the manuscript, the authors must provide the methods, data and analysis associated to RNAseq analyses, since the identification of TPPI as a possible marker for thermotolerant varieties relays completely on this approach. Otherwise, it is not clear why authors perform a whole transcriptome sequencing approach if they want to focus just on TTPI and TPPJ related genes. Indeed, figure 3 is constructed based on RNAseq data but no details how this assay was performed is provided in Materials and Methods section. Also, the introduction of the manuscript and associated references does not provide a solid basis to focus the analysis onto these two genes.
>>>Response: We are really sorry about it. We would like not to mention RNA seq data simply because it made our point of this ms unclear. However, your comments will be critical in this ms. We added RNA_seq experiment, briefly mention the results, added supplementary Tables and redrew Figure 3 that included all BoTPP found in RNA_seq.
Despite this important issue, the molecular identification and use of BoTPPI-2 alleles as marker for thermotolerance is described without major flaws across the document. I recommend to authors to analyze and improve figure 3, showing the transcriptional expression of all B. oleracea var. capitata TPP genes and improve the discussion connecting what is known about trehalose-6P as signaling molecule and it is role in thermotolerance.
>>>Response: Thank you for comments. We changed Figure 3 that included transcription levels of all BoTPP genes identified by RNA_Seq. Text was also edited.
Then, complement this concept with the known function of TPPI, which is dephosphorylate trehalose-6P, and connect that to the thermotolerance phenotype observed in plants that has low or null BoTPPI-2 expression. Of course, further experiments will be requiere to test any hypothesis but are far beyond the scope of this manuscript, the idea is propose a way to continue this work on the discussion section.
>>>Response: Thank you very much for your valuable comment. We are studying mechanism(s) underlined BoTPPI-2 now. We hope we obtain desirable data soon.
As minor issues:
- Figure 1 must be a supplemental figure, since the data presented (2018) does not correspond to the data during the time of analysis (2017) as authors state in the text. Authors explain that this data (2018) is similar but that is not a scientific fact.
>>>Response: Thank you and we apologized unclear description on it. We carried out the experiment twice in 2017 and 2018, but did not record temperature in 2017 (same pattern with respect to soft rot infection was observed). The temperature indicated inside temperature of glasshouse where plants grew with high humidity, but not outside one which were available in weather website. To avoid any confusion, we changed the sentence as follow: The experiments were carried out in summer 2017 and 2018, but data shown in this paper are from summer 2018.”
In addition, we added standard deviation of mean temperature for 1 week in Figure 1.
- Table 1 must be a supplemental figure, since it describe the sequence of the DNA oligos and this information is not fundamental to understand the approach and data presented by the authors.
>>>Response: Thank you. We moved Table 1 into supplementary Table.
-Figure 8 and 9, does not contain any indicator of molecular weight next to the MW lane.
>>>Response: Thank you. We edited Figures.
Reviewer 2 Report
L242, “The experiment was carried out in summer 2017, but the data shown in this paper are from summer 2018 because results were quite similar, and summer 2018 was hotter than summer 2017.” - the weather data for 2017 should be available online (local weather website). “Similar” is not a clear word stating how significant the difference is.
L259-262, Again “similar“ is not specific enough. Is there a rating scale for plant quality to indicate the significance of the difference.
L 277, “significantly higher”, based on what statistical analysis? What software? How many replicates were used? Add error bars for Figure 2 and 3. The major issue for the manuscript is lacking of “Statistical Analysis” statements in the materials and materials.
The manuscript described a well-designed experiment and was written with clarity and depth. I recommend accepting it for publication.
Author Response
Author's Reply to the Review Report (Reviewer 2):
Comments and Suggestions for Authors
L242, “The experiment was carried out in summer 2017, but the data shown in this paper are from summer 2018 because results were quite similar, and summer 2018 was hotter than summer 2017.” - the weather data for 2017 should be available online (local weather website). “Similar” is not a clear word stating how significant the difference is.
>>>Response: Thank you and we apologized unclear description on it. We carried out the experiment twice in 2017 and 2018, but did not record temperature in 2017 (same pattern with respect to soft rot infection was observed). The temperature indicated inside temperature of glasshouse where plants grew with high humidity, but not outside one which were available in weather website. To avoid any confusion, we changed the sentence as follow: The experiments were carried out in summer 2017 and 2018, but data shown in this paper are from summer 2018.”
L259-262, Again “similar“ is not specific enough. Is there a rating scale for plant quality to indicate the significance of the difference.
>>>Response: We are really sorry about it. Appearance means leaf color. We changed it into leaf color.
L 277, “significantly higher”, based on what statistical analysis? What software? How many replicates were used? Add error bars for Figure 2 and 3. The major issue for the manuscript is lacking of “Statistical Analysis” statements in the materials and materials.
>>>Response: Thank you for your comments. We changed Figs with mean and standard deviation. In addition, we added “(11 to 12 fold)” in text.
The manuscript described a well-designed experiment and was written with clarity and depth. I recommend accepting it for publication.
Round 2
Reviewer 1 Report
Authors address most of my previous concerns.
