DDIT3 Governs Milk Production Traits by Targeting IL-6 to Induce Apoptosis in Dairy Cattle

Round 1

Reviewer 1 Report (Previous Reviewer 1)

My concerns have been addressed except for the report on the SNPs identified within the DDIT3 gene. In your response to my previous comment, you identified two additional synonymous SNPs that were not reported. I did not understand the rationale behind this. Also, the statement in L200 would mean only two SNPs were identified; this is misleading. So, I recommend that all identified SNPs be included in the association and LD analysis and the results reported accordingly.

Author Response

Dear editor:

Thank you very much for providing an opportunity for us to revise our paper. We also wish to thank you and reviewers for the positive comments and constructive suggestions on the manuscript, which greatly improve the manuscript. Accordingly, we have made the essential revisions suggested by the reviewers as follows.

Reviewer 1:

My concerns have been addressed except for the report on the SNPs identified within the DDIT3 gene. In your response to my previous comment, you identified two additional synonymous SNPs that were not reported. I did not understand the rationale behind this. Also, the statement in L200 would mean only two SNPs were identified; this is misleading. So, I recommend that all identified SNPs be included in the association and LD analysis and the results reported accordingly.

Response: Thank you very much for your comments and I fully agree with you. The synonymous SNPs in the coding region means that the amino acid has not changed， so these SNPs weren’t genotyped and the promoter region were genotyped.

Reviewer 2 Report (Previous Reviewer 2)

Dear Author,

You have done interesting research work and it needs a grammar check to improve. Some of these errors are in the attached file.

Sincerely

Comments for author File: Comments.docx

Author Response

Dear editor:

Thank you very much for providing an opportunity for us to revise our paper. We also wish to thank you and reviewers for the positive comments and constructive suggestions on the manuscript, which greatly improve the manuscript. Accordingly, we have made the essential revisions suggested by the reviewers as follows.

Reviewer 2:

Dear Author, 

You have done interesting research work and it needs a grammar check to improve. Some of these errors are in the attached file.

Sincerely

Response: Thank you very much for your comments. Done as you suggested, please see the manuscript.

Line 60 - cellular functions has have

Line 65 - biological function

Line 72- by the Institutional Animal Care

Line 97- Haplotypes within these blocks will be applied to test its their

Line 129- siRNA design, synthesis, and assessment

Line 180 - shows the primers that were used in sequencing

Line 193 - by trypsinization into,

Line 202- that the SNPs of the DDIT3

Line 231 - there were no significant differences

Line 275 – proliferation, and differentiation.

Line 341 - have showed shown

Line 353 - traits remains unclear

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Generally, the research was well-designed and written concisely. The findings which provide new insight into the mechanism of milk production in cattle may be of interest to readers. However, the Material and method section requires more detail improvement for clarity. For instance, in Line 168 it is written “Mapping of reads to the reference genome was performed”, which of the reference genomes, what software and version were used?

Also, Table S1 indicated pooled DNA sequencing was performed but this was not clearly stated in the main manuscript.

I am a bit concerned about the discovery of just two SNPs in the whole coding and 5’ regulatory regions of DDIT3 and the reliance on their association with five milk traits. I am assuming the sequences were mapped against the Bos Taurus reference genome, would the result change if mapped to the Bos indicus reference genome?

The manuscript should be checked for spelling and grammatical errors.  Some of my observations include.

L39 – are influenced by

L41 – feasible for

L52 – is involved in

L121 - which correspond

L185 – The integrity

L349 - HSF1-mediated

Author Response

Generally, the research was well-designed and written concisely. The findings which provide new insight into the mechanism of milk production in cattle may be of interest to readers. However, the Material and method section requires more detail improvement for clarity. For instance, in Line 168 it is written “Mapping of reads to the reference genome was performed”, which of the reference genomes, what software and version were used?

Response: Thank you very much for your comments. As suggested, we have added the reference genomes, software and version in the manuscript.

Also, Table S1 indicated pooled DNA sequencing was performed but this was not clearly stated in the main manuscript.

Response: Thank you very much for your comments. As suggested, we have added the details of pooled DNA sequencing in the manuscript.

I am a bit concerned about the discovery of just two SNPs in the whole coding and 5’ regulatory regions of DDIT3 and the reliance on their association with five milk traits. I am assuming the sequences were mapped against the Bos Taurus reference genome, would the result change if mapped to the Bos indicus reference genome?

Response: Thank you very much for your comments and I fully agree with you. Yes, the sequences were mapped against the Bos Taurus reference genome and if mapped to the Bos indicus reference genome, the results may be very different. However, the previous results of our research group were acquired based on Bos Taurus ARS-UCD1.2. Therefore, in order to get comprehensive results, we chose Bos Taurus as the reference genome. Meanwhile, we detected other two SNPs in the whole coding, but they are synonymous mutations, so we didn’t report them here.

The manuscript should be checked for spelling and grammatical errors.  Some of my observations include.

L39-are influenced by; L41-feasible for; L52-is involved in; L121-which correspond; L185-The integrity; L349-HSF1-mediated

Response: Thank you very much for your comments. As suggested, we have revised the spelling and grammatical errors. Furthermore, we have polished the language to fix the grammatical errors and improve the overall readability of the paper.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

To improve this manuscript, it is recommended to consider the points in the attached file.

Sincerely

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

Line 27- correct: apoptosis and increase the number

Response: Thank you very much for your comments. As suggested, we have revised the word.

Line 28-9: correct: inflammatory markers, such as IL-6, IL6R, IL1B, IL7R, IL1RL2, IL1A, STAT1-5, MYC, IGFBP4, and IGFBP5, were differentially

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 30: correct: increased lipid accumulation

Response: Thank you very much for your comments. As suggested, we have deleted “the”.

Line 38- correct: fat percentage (FP), and protein percentage (PP),

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 39- correct: which are influenced by numerous genes

Response: Thank you very much for your comments. As suggested, we have revised “were” to “are”.

Line 40- reference no. 2 is not a suitable for this sentence.

Response: Thank you very much for your comments. As suggested, we have revised the cited reference.

Line 41- correct: it feasible for gene discovery

Response: Thank you very much for your comments. As suggested, we have revised “to” to “for”.

Line 44- correct: DNA damage-inducible transcript 3

Response: Thank you very much for your comments. As suggested, we have added “-”.

Line 46- correct: that DDIT3 has located only

Response: Thank you very much for your comments. As suggested, we have revised the word.

Line 48-9: fat percentage and protein percentage-

Response: Thank you very much for your comments. As suggested, we have deleted the word.

Line 49- correct: These findings indicated that the DDIT3 gene could

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 64: correct: potential causal genetic variants

Response: Thank you very much for your comments. As suggested, we have revised “causal”.

Line 68-9: correct: to better understand the molecular mechanisms

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 70- correct: by which DDIT3 regulates milk production ability

Response: Thank you very much for your comments. As suggested, we have deleted “involved in”.

Line 83- correct: from the whole blood of the cows

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 84- correct: from the semen of the sires

Response: Thank you very much for your comments. As suggested, we have added “the”.

Lines 86-8: the ID number of gene is not written. It also seems that the region amplified by these primers is less than 1500 bases. In addition, it is better to write the sequence and information of the primers in the article.

Response: Thank you very much for your comments. As suggested, we have added ID and revised the sentence in the manuscript.

Line 90- correct the sentence: The SNPs were genotyped for all individuals using

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 94- correct: The measure of pairwise LD for all three

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 106- the definition of Z matrix is not appeared in the next paragraph.

Response: Thank you very much for your comments. As suggested, we have added more details in the manuscript.

Line 107- correct: the five milk production traits, and μ is the overall mean

Also, change the font of “five milk production traits“

Response: Thank you very much for your comments. As suggested, we have added “and”.

Line 109- x is the fixed effect vector(?)

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 111- correct: The additive (a), dominance (d), and allele substitution

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 112- correct: a = (AA-BB)/2, d = AB-(AA + BB)/2, and α= a + d (q-p)

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 114- A and B in the corresponding loci, respectively (Falconer and Mackay, 1996).

Response: Thank you very much for your comments. As suggested, we have added “respectively”.

Line 115- analyses above, the Bonferroni method

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 118- SNP loci or haplotype blocks tested

Response: Thank you very much for your comments. As suggested, we have revised “blocks”.

Line 121- correct: which correspond to

Response: Thank you very much for your comments. As suggested, we have revised “corresponding”.

Line 131- Three siRNAs’ sequences targeting DDIT3 mRNA

Response: Thank you very much for your comments. As suggested, we have revised the word.

Line 134- Grand Island

Response: Thank you very much for your comments. As suggested, we have revised the word to capital.

Lines 137-9: It has a different color font!

Response: Thank you very much for your comments. As suggested, we have revised the color font.

Line 137- Next, the total RNA

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 138- (Invitrogen), and qRT-PCR analysis

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 139- Each of the siRNAs was

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 141- 2.7. Construction of

Response: Thank you very much for your comments. As suggested, we have added “.”.

Line 153- (Sigma), and 450μl

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 157 - of the experimental group

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 158- The integrity of obtained

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 159- Agilent Technologies

Response: Thank you very much for your comments. As suggested, we have revised the word to capital.

Line 161-2- a sequencing library was prepared

Response: Thank you very much for your comments. As suggested, we have added “a”.

Line 173- . A q-values of 0.05 was considered significant.

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 175- pathways were evaluated

Response: Thank you very much for your comments. As suggested, we have revised “was” to “were”.

Line 179- Table S1 shows the primers that used in sequencing 

Response: Thank you very much for your comments. As suggested, we have added the sentence to the manuscript.

Line 185- fixed with 4% paraformaldehyde

Response: Thank you very much for your comments. As suggested, we have revised “by” to “with”.

Line 188-9 – Which one did you use? (APPLYGEN, Beijing, China) (Sangon Biotech Co., Ltd., Shanghai, China).

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

line204- five milk production traits

Response: Thank you very much for your comments. As suggested, we have deleted “related”.

Table 1: Considering that the comparisons are inside the column, it is not necessary that the symptoms are different from column to column.

Response: Thank you very much for your comments. As suggested, we have revised the note.

Line 225- significance level of 0.05

Response: Thank you very much for your comments. As suggested, we have added “of”.

Line 231- there were no significant changes

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Lines 232-3: correct: the allele of T showed 68% higher luciferase activity compared with the allele of T

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 234: can be considered a functional SNP

Response: Thank you very much for your comments. As suggested, we have revised “as” to “a”.

Lines 238-9: This result is not consistent with Figure 2A.

Response: Thank you very much for your comments. As suggested, we have revised Figure 2A.

Line 238- of the DDIT3 gene

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 240- have an obvious effect

Response: Thank you very much for your comments. As suggested, we have added “an”.

Line 243- fluorescence signals

Response: Thank you very much for your comments. As suggested, we have added “s”.

Line 244: This is not seen in Figure 2A.

Response: Thank you very much for your comments. As suggested, we have revised “Figure 2A” to “Figure 2B”.

Line 246- were found to decrease

Response: Thank you very much for your comments. As suggested, we have revised “decreased” to “to decrease”.

Figure 2A: For what purpose did you draw a red box on D394 in Figure 2A?

Response: Thank you very much for your comments. Actually, we want to indicate D287 instead of D394 and we have revised it now.

Figure 2A: MARVELD1?

Response: Thank you very much for your comments. Actually, MARVELD1was an internal reference gene and a combined utilization of two internal reference genes is also recommended now.

Line 274-5: Of these, there are 1940 genes down-regulated and 1898 genes up-regulated.

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 275: The volcano

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 276: Explain how you chose these top 10 genes?

Response: Thank you very much for your comments. Actually, we chose the top 10 genes based on Log₂fold change.

Line 277- with the regulation

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 279- pattern significantly

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 282- only the regulation

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 283- cellular senescence, and

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Table 3: The font color of the contents of the table is not same.

Response: Thank you very much for your comments. As suggested, we have revised to same font color.

Line 291- group were significantly

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 292- Figure  2B

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 293- determined the role of

Response: Thank you very much for your comments. As suggested, we have deleted “the”.

Line 295- and increase the number of

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 296- Figure  2C

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 309- MYC, IGFBP4, and IGFBP5,

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 313- MYC, IGFBP4, and IGFBP5

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 315- shown in Figures 3A, 3C, and 3D

Response: Thank you very much for your comments. As suggested, we have added “s”.

Line 317- , IL7R, MYC, and IGFBP4

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 318- IL1RL2, IL1A, and

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 319 - in the control group

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 320- Figure 3A, 3B, and 3E

Response: Thank you very much for your comments. As suggested, we have revised full-width to half-width.

Line 320- All the expressions of these

Response: Thank you very much for your comments. As suggested, we have added “s”.

Table 3: The location of Figure 3 comes before it is referenced. It should come after the results 3.6.

Response: Thank you very much for your comments. As suggested, we have moved Figure 3 to after 3.6.

Line 331- protein and fat percentages

Response: Thank you very much for your comments. As suggested, we have added “s”.

Line 332- cause significant potential

Response: Thank you very much for your comments. As suggested, we have deleted “ly”.

Lines 333-4- in the promoter region were significantly associated with MY, FY, and PY.

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Lines 334-5: Individuals with genotype CC had higher MY, FY, or PY than

line 336- . By using phenotypic values

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

line 337: This reference (Li et al. (2014)) does not exist in the list of references.

Response: Thank you very much for your comments. Sorry for my spelling errors, the year is 2019.

line 339- milk MY and PYThese results

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

line-341 association analyses basically

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

line 342: was considered a major gene

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 344: increased promoter activity

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 346: may up-regulate the expression

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 349: roles in regulating HSF1 mediated stress response

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 352: milk fat and protein production traits,

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 355: with MY, FY, or PY may

Response: Thank you very much for your comments. As suggested, we have added “，”.

Lines 362-3: correct the sentence: cows with the TT genotype had lower MY, FY, and PY than those with the TT genotype 

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 367: inhibited a more increased number of

Response: Thank you very much for your comments. As suggested, we have revised the sentence.

Line 369: in the bovine MAC-T cell line

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 374: of stimulation

Response: Thank you very much for your comments. As suggested, we have deleted “a”.

Line 376: through the activation of

Response: Thank you very much for your comments. As suggested, we have added “the”.

Line 379: the JAK/STAT pathway was

Response: Thank you very much for your comments. As suggested, we have revised “were” to“was”.

To improve this manuscript, it is recommended to consider the points in the attached file.
Response: Thank you very much for your comments. As suggested, we have made the essential revisions suggested by the reviewers.

Author Response File: Author Response.pdf