SNP in DFR1 Coding Sequence Is Tightly Associated with Anthocyanin Accumulation in Cabbage (B. oleracea var. capitata f. alba) at Low Temperature

Round 1

Reviewer 1 Report

Authors: Hayoung Song, Jong-In Park, Byung-Ho Hwang, Hankuil Yi, HyeRan Kim, Yoonkang  Hur

Title of the Paper: SNP in DFR1 coding sequence  is tightly associated with 2 anthocyanin accumulation in cabbage (B. oleracea var. capitata f. alba) at low temperature

A low anthocyanin level is an important breeding feature. The authors made an attempt to explain how and what genes regulate this trait. By examining the expression level of anthocyanin biosynthesis genes, they were able to demonstrate a relationship between BoDFR1 gene expression and anthocyanin accumulation, under LT condition. Then they cloned and compared gene and promoter sequences from cabbage genotypes characterized by high and low anthocyanin accumulation an,d the potentially crucial SNP (1118A/T) showing a strong linkage with the feature was identified. The authors developed primers and procedure for the detection of this SNP as a marker, examined activity of both BoDFR1 and BoDFR1v by expressing His-tagged recombinant proteins in E. coli. They tested the effect of temperature on expression both in the greenhouse and in the field, including inbred lines and varieties as well as related species.

In summary, although the authors could not be able to explain the direct effect of SNP on the level of expression of the BoDFR1 gene, the article presents very interesting and extensive research and describes an interesting issue. The developed marker should have a value in cabbage breeding.

My comments are:

1. Could the authors consider a change in the title? In my opinion “SNP in DFR1 coding sequence is tightly associated with 2 anthocyanin accumulation…” would be better
2. The Summary is overloaded with information and therefore difficult to understand.
3. I did not find information on which part of the BoDFR1 gene was used in qPCR and what was the length of amplified fragments.

Author Response

Author's Reply to the Review Report (Reviewer 1)

 

We appreciate reviewers' useful comments to improve our manuscript.

We have tried to edit our ms according to reviewer’s comments or suggestion.

 

1. Could the authors consider a change in the title? In my opinion “SNP in DFR1 coding sequence is tightly associated with anthocyanin accumulation…” would be better

>>> Response: Thank you very much for your valuable comment.

We changed the title according to reviewer’s suggestion as follow:

 “SNP in DFR1 coding sequence is tightly associated with anthocyanin accumulation in cabbage (B. oleracea var. capitata f. alba) at low temperature”

 

2. The Summary is overloaded with information and therefore difficult to understand.

>>> Response: Thank you for your valuable comment

We deleted some sentences to avoid confusion.

 

3. I did not find information on which part of the BoDFR1 gene was used in qPCR and what was the length of amplified fragments.

>>> Response: We are really sorry about it. That primers were those of BoDFR in Table S3. To avoid any confusion, we revised Materials and Method, and omit BoDFR from supplementary Table 3. We also mention size of amplified fragment and primer positions as exons.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,
I found your article well written.
I have no particular corrections to make, let alone detected ambiguity.
Just a few curiosities, which are the salt of our research activity and
which I hope to see clarified in a future evolutionary work on a dedicated magazine.

Author Response

I have no particular corrections to make, let alone detected ambiguity.

Just a few curiosities, which are the salt of our research activity and

which I hope to see clarified in a future evolutionary work on a dedicated magazine.

 

>>> Response:

Thank you for your comments.

We will try to extend subsequent study on the basis of your comment.

Author Response File: Author Response.docx

Reviewer 3 Report

The MS insist on the expression levels of DFR1 is linked to anthocyanin accumulation especially induced by cold temperature. Furthermore, the SNP of DFR1 CDS between BoDFR1 and BoDFR1v is linked to and used for detection of germplasm accumulation of anthocyanin by cold temperature. In addition, the authors also showed divergent sequences of promoter between BoDFR1 and BoDFR1v. Both BoDFR1 and BoDFR1v genes showed same enzyme activity. The overall conclusion is the expression levels of BoDFR1 (or BoDFR1v) linked to accumulation of anthocyanin that is caused by SNP sequence. Before publish the MS, the authors give strong evidence the SNP regulate expression levels of BoDFR1. Otherwise check the possibility that deletion of promoter sequence of BoDFR1v cause abolished expression levels of BoDFR1 (or BoDFR1v). The authors showed several germplasms used to check sequence polymorphism that classified as accumulation and non-accumulation of anthocyanin by cold temperature. However the SNP and anthocyanin accumulation is not confirmed by the genetic study, so above statements should be confirmed. In addition, It is necessary to identify the correlation between Anthocyanin biosynthesis regulator (BoMYB114, BobHLH, BoMYBL2) and BoDFR1v through analysis of expression patterns of BoDFR1 and BoDFR1v according to overexpression of Anthocyanin biosynthesis regulator (BoMYB114, BobHLH, BoMYBL2)

Below are minor comment:

Line 101-102: The authors target the BoDFR1 in this study, However I could not find background why the authors target the BoDFR1 for control anthocyanin biosynthesis in cabbage.

Lime 125-128: Give more detail information (temperature) of inside and outside greenhouse, during cabbage cultivation.

Line 147: T&A àTA

Line 195 and Line 256: adjust format, same as line 190.

Line 271-272: BoDFR1 is increased in LT??? Furthermore, the sentence is not logical. Give clues why BoDFR1 was selected as a tareget.

In figure 3 and 4: which stage of plants were shown in Fig, 3B. and Figure 4. In addition, show both picture of Ck and LT culture condition, in Fig 3 or 4.

Line 354 –362: Please describe all the SNPs in BoDFR1 between 5 LAAs and 5 HAAs. Is there any conserved SNP between 5 LAAs and 5 HAAs. In the text, related description is not clear.

Line 420-421: It doesn’t make sense

Line 459-462: There is no evidence for support this sentence. The authors misunderstand the results of Fig. 9.

Line 494-505: Need more evidences for the TA conversion is related to anthocyanin accumulation. The authors showed the relationship between genotype and levels of anthocyanin using only two germplasm for each genotypes.

Lime 530-531: Is there evidence to support this sentence? How SNP is related to expression of the gene???

Line 570-571 and line 578-581: if the authors excluded the sequence polymorphism of the promoter sequence, how explain the different expression levels the genes??

Author Response

 

Author's Reply to the Review Report (Reviewer 3)

Comments and Suggestions for Authors

The MS insist on the expression levels of DFR1 is linked to anthocyanin accumulation especially induced by cold temperature. Furthermore, the SNP of DFR1 CDS between BoDFR1 and BoDFR1v is linked to and used for detection of germplasm accumulation of anthocyanin by cold temperature. In addition, the authors also showed divergent sequences of promoter between BoDFR1 and BoDFR1v. Both BoDFR1 and BoDFR1v genes showed same enzyme activity. The overall conclusion is the expression levels of BoDFR1 (or BoDFR1v) linked to accumulation of anthocyanin that is caused by SNP sequence. Before publish the MS, the authors give strong evidence the SNP regulate expression levels of BoDFR1. Otherwise check the possibility that deletion of promoter sequence of BoDFR1v cause abolished expression levels of BoDFR1 (or BoDFR1v). The authors showed several germplasms used to check sequence polymorphism that classified as accumulation and non-accumulation of anthocyanin by cold temperature. However the SNP and anthocyanin accumulation is not confirmed by the genetic study, so above statements should be confirmed. In addition, It is necessary to identify the correlation between Anthocyanin biosynthesis regulator (BoMYB114, BobHLH, BoMYBL2) and BoDFR1v through analysis of expression patterns of BoDFR1 and BoDFR1v according to overexpression of Anthocyanin biosynthesis regulator (BoMYB114, BobHLH, BoMYBL2)

>>>Response: We really appreciate for your valuable comments. First of all, we have tried to figure out the association of SNP in BoDFR1 and its expression levels (and/or anthocyanin accumulation), even the association of the BoDFR1 SNP with differential expression patterns of the upstream regulatory genes. However, we did not identify the critical gene(s) so far. Regarding to promoter sequence, difference between HAAs and LAAs was MYBST1 element (Lines 362-370), but it was not related to anthocyanin accumulation. In addition, we performed Arabidopsis transformation of promoter::GUS constructs, but no difference in GUS expression was found between two promoters (therefore, we did not mention this experimental result because above sentence will be enough for that). Currently we can declare that SNP in BoDFR1 is associated with its expression level as well as anthocyanin content (our conclusion). We are still trying to find out genes (or factors) which regulate these DFR1 genes expression. We hope its results are coming soon in another paper. As you know, cabbage breeding is required for many years. We are trying to generate genetic population using HAA and LAA cabbages, but it will take several years. Transformation of cabbage also takes long period time as well as not easy. We hope we can perform these experiments you mentioned and we can present data soon as possible. Again we really express our sincere thanks.

 

Below are minor comment:

1. Line 101-102: The authors target the BoDFR1 in this study, However I could not find background why the authors target the BoDFR1 for control anthocyanin biosynthesis in cabbage.

>>>Response: Thank you for your comment. We have thought that the explanation of importance of DFR in anthocyanin biosynthesis (Lines 62-94) in “Introduction” will be enough to choice the gene as a target. In addition, we have experimentally proved why we selected BoDFR1 as the target gene in Figs. 1 and 2 (Lines 264-304 in text). To make concise introduction, we did not put the explanation

2. Lime 125-128: Give more detail information (temperature) of inside and outside greenhouse, during cabbage cultivation.

>>>Response: Thank you for the comment. We added parenthesis as follows:

3. Line 147: T&A àTA

>>>Response: We are sorry we do not understand it. According to manufacturer’s protocol, they express it as T&A cloning vector or T&A^TM cloning vector.

4. Line 195 and Line 256: adjust format, same as line 190.

>>>Response: Thank you for your comment, we changed it.

5. Line 271-272: BoDFR1 is increased in LT??? Furthermore, the sentence is not logical. Give clues why BoDFR1 was selected as a target.

 >>>Response: We are sorry about it. We changed the sentence.

6. In figure 3 and 4: which stage of plants were shown in Fig, 3B. and Figure 4. In addition, show both picture of Ck and LT culture condition, in Fig 3 or 4.

 >>>Response:  Thank you for your comment. We changed figure legend of Fig. 3 to avoid confusion. We did not put photo for CK because there was no color variation between HAAs and LAAs (all green color).

7. Line 354 –362: Please describe all the SNPs in BoDFR1 between 5 LAAs and 5 HAAs. Is there any conserved SNP between 5 LAAs and 5 HAAs. In the text, related description is not clear.

>>>Response: We are sorry to make confusion. Nucleotide sequences (enrolled in NCBI) were identical except SNPs mentioned in Text. To avoid any confusion, we edited sentence as follow:

 

8. Line 420-421: It doesn’t make sense

 >>>Response: We are sorry about it. We speculate that the lower activity of DFR1 might produce lower substrate for LDOX, thereby lowering its expression at this point. Therefore, we changed the sentence.

 

9. Line 459-462: There is no evidence for support this sentence. The authors misunderstand the results of Fig. 9.

>>>Response: Thank you for the comment.  You are absolutely right and we did not have direct evidence for cold tolerance. As mention in Lines 451-454, however, anthocyanin content (or DFR expression) is related to cold tolerance, which subsequent associated with CBF expression. Therefore, two contrasting genotypes of cabbage exposed to LT appear not to be related cold tolerance (Both genotypes showed similar expression pattern). As mentioned in “Introduction”, one of breeding target in cabbage is selection of genotype having no or less anthocyanin accumulation under low temperature.

 

10. Line 494-505: Need more evidences for the TA conversion is related to anthocyanin accumulation. The authors showed the relationship between genotype and levels of anthocyanin using only two germplasm for each genotypes.

>>>Response: Thank you for your comments. As mentioned before, we have tried to collect more genotypes of cabbages and production of genetic population to prove the SNP involvement for anthocyanin biosynthesis. However, it will require more time from now.

.

11. Lime 530-531: Is there evidence to support this sentence? How SNP is related to expression of the gene???

>>>Response:  Thank you for the comment. Again we did not find out scientific evidence (except several TF expression pattern in Fig. 8) on the SNP of BoDFR1 and anthocyanin accumulation. Your comment will be subsequent topic for our labs.

12. Line 570-571 and line 578-581: if the authors excluded the sequence polymorphism of the promoter sequence, how explain the different expression levels the genes??

>>>Response: Thank you for the comment. We explained that promoter sequences did not provide or explain different expression between BoDFR1 and BoDFR1v. We have speculated that upstream components of MBW complex or types of MBW complex formation would be different in two genotypes (HAAs and LAAs). However, we did not obtain direct evidence yet, but we can declare only that SNP in BoDFR1 is associated with its expression as well as anthocyanin content.