Development of SSRs Based on the Whole Genome and Screening of Bolting-Resistant SSR Marker in Brassica oleracea L.

Round 1

Reviewer 1 Report

The manuscript provides information about development of SSR markers based on Brassica oleracea L. genome and their application for BSA (Bulked Segregant Analysis) among other things. The core idea and the results seems to be alright but the current manuscript suffers from some technical, formatting and language issues diminishing whole work. The issues will be pointed out in the following sections of the review and I believe that solving them will substantially improve overall quality of the manuscript.
1) Part of the second paragraph [line 49-55] in the Introduction section should be modified because it does not make sense. There are mentioned methods for development of SSR markers including in-silico analyses [line 49-50]. Then followed by sentence about how inefficient is it [line 52-53] and how accessibility of whole genome sequences in public databases made it more easier [53-55]. How is it different from in-silico analysis? It is not, only more data are available now.
2) Provided link [line 65] is not functional.
3) What is the meaning of following sentence: “. . . including 52,469,534 bp. . . ” [line 100-102]? I suppose it could be chromosome sizes, but purpose of the sentence is unclear.
4) Structure of the Material and methods (MM) and related parts of the Results (Res) section is necessary to improve. Generally, in its current status it is difficult to navigate through and connect it with related results. Moreover, some information more suitable for the MM can be found in the Res section. For example, the part 2.5 of the MM informs the reader about 54 tested SSR markers for transferability between the species [line 123-125], but which species were tested is mentioned in the part 3.3 of the Res section [line 249-251]. Similarly, first paragraph of part 3.4 Res section [line 271-276] should be in the MM section. Further, parts 2.6, 2.8 and 2.9 [line 136-143, 165-175 and 176-181] are closely related. Therefore, it would be better to make only one more (dense) part.
Both PCRs are almost the same (parts 2.8 and 2.10) [line 125-133 and 185-192]. Maybe only one part describing both PCR versions looks like better alternative. By the way, there are typos in the name of thermocycler “BioRad S1000TM” [line 132] (BioRad S1000^TM ).
Description of PCR products sequencing is poorly written [line 194-200].
Experimental design of BSA is unclear. In the part 2.6 is mentioned crossing between P1Y9805 and P2 Y5-3-41, then only one F₁ plant and bulked DNA of 15 plants F₂ generation per bolting type. On the other way, in the part 3.4 [line 271-276] are parents Y9805 and Y5-3-14 (probably two typos?), F₁ population (?) and 224 “individual population” [line 275-276]. What does it mean? It was really tested 224 populations? If so, how it is connected with the description in the part 2.6?
My conclusion is that the authors should carefully reconsider how to change the structure of the MM and Res sections in order to make it more readable.
5) Was it necessary to wrote whole paragraph about that all traits followed normal distribution?
6) Text with explanation what is recombination [line 328-333] is pointless because every geneticist already known it.
7) The manuscript should be better formatted. The authors did not remove some MDPI template notes like “Type of the Paper. . . ” [line 1], “Background:” [line 17], “;Methods” [line 20], “;Results:” [line 22-23], “Interventionary studies. . . ” [line 200-202],“6. Patents. . . ” [line 470-472] and “All authors have read . . . ” [line 192-495], respectively.
There are other small formatting issues or typos like “DNA(RAPD), . . . ” [line 39] (space between DNA and RAPD), “bulked segregate analysis (BSA) method” [line 73] (bulked segregant analysis), “Raphanus sativus” [line 119] (Raphanus sativus), “ddH2O” [line 128] (ddH₂O), “Brasica oleracea” [line 167] (Brassica oleracea), “E. coli” [line 199] (E. coli), “. . . 82,984 SSR primers. . . ” [line 237] (. . . 82,984 SSR loci. . . ), “TM value” [line 238] (T_m value), “Morgen” [line 329] (Morgan).
8) Why are the Figures S2 and S3 uncompressed? It is better to use lzw compression for the tif format in order to minimize picture size without sacrificing resolution quality.

The authors should use some language editing service specialized in biology and/or genetics field. Even I’m able to recognize, as a non-native English speaker, many incorrect sentences, grammar issues etc. Sometimes was really difficult to understand what the authors meant by given sentence(s) and I would be lost without the knowledge of problematics they tried to address. Here I provide only a few examples as I do not have any intention to supply specialized language service.
“We analyzed the PCR product that observed the difference of SSR marker between resistant-bolting gene bulk and easy-bloting gene bulk. The result shows that maybe linked to the target character (bolting). To confirm the screening SSR markers in F2 generation plant and 10 plants each of the two
parents.” [line 172-175]
“Using the recombination values of marker loci for linkage analysis and were converted into genetic map distances (cM) using the Kosambi mapping function.” [line 177-178]
“The arrow points to the difference band, and the bands amplified in bolting-resistant plants have slightly larger than those in bolting-easy plants.” [line 299-300]
“Based on the result of screening the resistant-bolting gene, BolSSR040196, and we need to validate whether the SSR marker has the same preceding difference in the F2 generation offspring and their parents.” [line 313-315]

Author Response

Response letter to reviewer 1

Dear Editor-in-Chief,

The response letter to reviewers is as follows:

Reviewer 1:

The manuscript provides information about development of SSR markers based on Brassica oleracea L. genome and their application for BSA (Bulked Segregant Analysis) among other things. The core idea and the results seems to be alright but the current manuscript suffers from some technical, formatting and language issues diminishing whole work. The issues will be pointed out in the following sections of the review and I believe that solving them will substantially improve overall quality of the manuscript.
1) Part of the second paragraph [line 49-55] in the Introduction section should be modified because it does not make sense. There are mentioned methods for development of SSR markers including in-silico analyses [line 49-50]. Then followed by sentence about how inefficient is it [line 52-53] and how accessibility of whole genome sequences in public databases made it more easier [53-55]. How is it different from in-silico analysis? It is not, only more data are available now.

Response: Thanks for your valuable comment. We have revised the description herein and omitted the unjustifiable sentences. Modify it to: “With the publication of more and more high-quality genome data, the development of SSRs becomes faster and more accurate.”

2) Provided link [line 65] is not functional.

Response: Thanks for your valuable comment. The invalid connection has been substituted with: http://brassicadb.cn/#/

3) What is the meaning of following sentence: “. . . including 52,469,534 bp. . . ” [line 100-102]? I suppose it could be chromosome sizes, but purpose of the sentence is unclear.

Response: Thanks for your valuable comment. The numerical value presented here pertains to the magnitude of each chromosome. The preceding inappropriate description has been rectified to: “The size of each chromosome is: 52,469,534 bp, 66,012,635 bp, 74,510,869 bp, 65,432,303 bp, 57,884,551 bp, 47,844,321 bp, 55,965,641 bp, 52,084,256 bp, and 67,469,775 bp, respectively.”

4) Structure of the Material and methods (MM) and related parts of the Results (Res) section is necessary to improve. Generally, in its current status it is difficult to navigate through and connect it with related results. Moreover, some information more suitable for the MM can be found in the Res section. For example, the part 2.5 of the MM informs the reader about 54 tested SSR markers for transferability between the species [line 123-125], but which species were tested is mentioned in the part 3.3 of the Res section [line 249-251]. Similarly, first paragraph of part 3.4 Res section [line 271-276] should be in the MM section. Further, parts 2.6, 2.8 and 2.9 [line 136-143, 165-175 and 176-181] are closely related. Therefore, it would be better to make only one more (dense) part.

 Both PCRs are almost the same (parts 2.8 and 2.10) [line 125-133 and 185-192]. Maybe only one part describing both PCR versions looks like better alternative. By the way, there are typos in the name of thermocycler “BioRad S1000TM” [line 132] (BioRad S1000^TM ).

 Description of PCR products sequencing is poorly written [line 194-200].

 Experimental design of BSA is unclear. In the part 2.6 is mentioned crossing between P1Y9805 and P2 Y5-3-41, then only one F₁ plant and bulked DNA of 15 plants F₂ generation per bolting type. On the other way, in the part 3.4 [line 271-276] are parents Y9805 and Y5-3-14 (probably two typos?), F₁ population (?) and 224 “individual population” [line 275-276]. What does it mean? It was really tested 224 populations? If so, how it is connected with the description in the part 2.6? My conclusion is that the authors should carefully reconsider how to change the structure of the MM and Res sections in order to make it more readable.

Response: Thanks for your valuable comment. Plant materials used to test the transferability of SSR tags have been added to the method. The procedure for constructing the F₂ generation segregation population has been removed from section 3.4 and integrated into section 2.5 of the materials and methods. The original sections 2.6, 2.8, and 2.9 have been consolidated, with the relevant experimental methods amalgamated within the same section.

The redundant depiction of the PCR experiment has been excised, retaining solely the more elaborate description found in Section 2.4. The nomenclature of the thermocycler "BioRad S1000TM" has been adjusted to “BioRad S1000^TM”. The description of PCR product sequencing has been streamlined for improved clarity and simplicity.

The typographical error regarding the female parent material in the materials and methods section has been rectified to read as P₂Y5-3-14. The F₂ population was developed from a single heterozygous F₁ individual plant and the agronomic traits of 224 F₂ plants were examined. We have rectified the erroneous description about this part of the article.

In light of the “materials and methods” and “results” presented in this article, we have implemented comprehensive revisions aimed at enhancing coherence between the two sections and enhancing readability.

5) Was it necessary to wrote whole paragraph about that all traits followed normal distribution?

Response: Thanks for your valuable comment. The redundant descriptions have been rewritten.

6) Text with explanation what is recombination [line 328-333] is pointless because every geneticist already known it.

Response: Thanks for your valuable comment. Superfluous descriptions regarding recombination have been removed.

7) The manuscript should be better formatted. The authors did not remove some MDPI template notes like “Type of the Paper. . . ” [line 1], “Background:” [line 17], “;Methods” [line 20], “;Results:” [line 22-23], “Interventionary studies. . . ” [line 200-202],“6. Patents. . . ” [line 470-472] and “All authors have read . . . ” [line 192-495], respectively.

 There are other small formatting issues or typos like “DNA(RAPD), . . . ” [line 39] (space between DNA and RAPD), “bulked segregate analysis (BSA) method” [line 73] (bulked segregant analysis), “Raphanus sativus” [line 119] (Raphanus sativus), “ddH2O” [line 128] (ddH₂O), “Brasica oleracea” [line 167] (Brassica oleracea), “E. coli” [line 199] (E. coli), “. . . 82,984 SSR primers. . . ” [line 237] (. . . 82,984 SSR loci. . . ), “TM value” [line 238] (T_m value), “Morgen” [line 329] (Morgan).

Response: Thanks for your valuable comment. We have deleted unnecessary sentences from the MDPI template. Spelling and formatting errors in the article have been carefully reviewed and corrected.

8) Why are the Figures S2 and S3 uncompressed? It is better to use lzw compression for the tif format in order to minimize picture size without sacrificing resolution quality.

Response: Thanks for your valuable comment. Figures S2 and S3 have been compressed without altering their resolution.

At the same time, some minor errors were also corrected during the process of revision to the manuscript. The grammar and vocabulary of this article have been carefully revised by English-native reader.

Special thanks to you for your good comments, these comments can help us to improve our manuscript significantly. We really benefit a lot. Thank you!

Best regards to you,

Xiaolin YU

Reviewer 2 Report

Authors, screened the whole genome dataset of Brasica available in BRAD to identified SSRs and designed some primers for PCR. Authors used F2 population to develop bolting associated SSR marker.

I find the manuscript interesting and results can be used for MAS in breeding programs. However, the manuscripts writing style is not clear. I give some examples below but entire manuscript needs to be revised.

Line 99-102: "including 52,469,534 bp,..... " what is that including? you mentioned that "A total of 539,673,885 bp genome sequences....." and then including....  what are those sequences?

Line 166: you don't need to write "Simple Sequence Repeat" again and again.

Line 173: What is "resistant-bolting gene bulk"???

Line 173-174: What do you mean by: The result shows that maybe linked to the target character (bolting).

Line 270 to 281: You don't need to repeat the M&M

My main concern is that : in Fig 5. Among the 23 bolting resistant number 4 and 5 also look different, I am not sure the BolSSR040196 can be correctly associated with bolting. Do you have some explanation?

Line 340-341: What is that "B. oleracea resistant genes". Which genes? what is the name? Who identified those genes? etc

English style needs to be improve.

Author Response

Response letter to reviewer 2

Dear Editor-in-Chief,

The response letter to reviewers is as follows:

Reviewer 2:

Authors, screened the whole genome dataset of Brassica available in BRAD to identified SSRs and designed some primers for PCR. Authors used F2 population to develop bolting associated SSR marker.

I find the manuscript interesting and results can be used for MAS in breeding programs. However, the manuscripts writing style is not clear. I give some examples below but entire manuscript needs to be revised.

1. Line 99-102: "including 52,469,534 bp,..... " what is that including? you mentioned that "A total of 539,673,885 bp genome sequences....." and then including....  what are those sequences?

Response: Thanks for your valuable comment. The numerical value presented here pertains to the magnitude of each chromosome. The preceding inappropriate description has been rectified to: “The size of each chromosome is: 52,469,534 bp, 66,012,635 bp, 74,510,869 bp, 65,432,303 bp, 57,884,551 bp, 47,844,321 bp, 55,965,641 bp, 52,084,256 bp, and 67,469,775 bp, respectively.”

2. Line 166: you don't need to write "Simple Sequence Repeat" again and again.

Response: Thanks for your valuable comment. Repetitive words "Simple Sequence Repeat" have been deleted.

3. Line 173: What is "resistant-bolting gene bulk"???

Response: Thanks for your valuable comment. The term "resistant-bolting gene bulk" was adjusted to "resistant-bolting gene pool".

4. Line 173-174: What do you mean by: The result shows that maybe linked to the target character (bolting).

Response: Thanks for your valuable comment. The inappropriate description has been removed, and the method in this section has been rewrited.

5. Line 270 to 281: You don't need to repeat the M&M

Response: Thanks for your valuable comment. Redundant descriptions have been removed, and section of the materials and methods have been re-integrated to enhance conciseness and readability.

6. My main concern is that: in Fig 5. Among the 23 bolting resistant number 4 and 5 also look different, I am not sure the BolSSR040196 can be correctly associated with bolting. Do you have some explanation?

Response: Thanks for your valuable comment. We think that the materials corresponding to lanes 4 and 5 are heterozygotes, hence they do not represent recombination events.

7. Line 340-341: What is that " oleracearesistant genes". Which genes? what is the name? Who identified those genes? etc

Response: Thanks for your valuable comment. The inappropriate description here has been amended to "bolting-resistant locus." Furthermore, we have revised the language to enhance readability.

At the same time, some minor errors were also corrected during the process of revision to the manuscript. The grammar and vocabulary of this article have been carefully revised by English-native reader.

Special thanks to you for your good comments, these comments can help us to improve our manuscript significantly. We really benefit a lot. Thank you!

Best regards to you,

Xiaolin YU

Round 2

Reviewer 1 Report

Revised version of the manuscript was improved and most of the issues mentioned in the first review were solved. Therefore, I’ll only provide some suggestions and another list of typos or language issues.

1) There are some typos:
Maybe term “loci” instead of “sites” is more suitable [line 21].
“byPCR” [line 46] (by PCR)
“. . . food reserves..” [line 63] (. . . food reserves.)
Unexplained abbreviation SLAF-BSA (site-specific amplified fragment BSA) [line 84]
“. . . whole . . . ” [line 94] (different font type)
“. . . and total genomic DNA were extracted. . . ” [line 127] (. . . DNA was extracted)
“[13].PCR amplification. . . ” [line 127] ([13]. PCR amplification. . . )
“. . . male parent (P₁ Y9805) and bolting-easy female parent (P₂ Y5-3-14). . . ” [line 138] (It should be more consistent either (P₁ Y9805) and (P₂ Y5-3-14) or (P₁ Y9805) and (P₂ Y5-3-14). “. . . , then an F₂ population. . . ” [line 139] (. . . , then F₂ population)
“. . . chromosomes are also observed. . . ” [line 211] (. . . chromosomes were also observed. . . )
“Meanwhile, the ratio of transfer to AA, BB, AABB, AACC, and BBCC are . . . ” [line 236-237] (Meanwhile, the ratio of transfer to AA, BB, AABB, AACC, and BBCC is . . . )
“. . . spanning P₁ , P₂ , and F₂ generations. . . ” [line 306] It is better to use “. . . spanning parental and F₂ generations. . . ”, because P₁ nor P₂ generation does not exist (it notification of parent’s plants).
“. . . repeats (29.2%). [8].” [line 345] (. . . repeats (29.2%) [8].)
“. . . published to date. [27].” [line 353] (. . . published to date [27].)
“. . . Brassicaceae Database.In . . . ” [line 382] (. . . Brassicaceae Database. In . . . )
“. . . gene pool .” [line 404] (. . . gene pool.)
“According to Gao et al.’s findings. . . ” [line 421] (According to Gao et al. findings. . . )

2) It is questionable whether 10 randomly selected primer pairs out of 82984 designed primer pairs have something to say about “the success of our previously designed SSR primers.” [line 224-227]. It represents only ~0.012% of all designed primers. Moreover, I’m not sure what the authors tried to tell
reader in this paragraph.

3) The information about tested species is now duplicated [line 157-159] and [line 231-233]. Text in the Result section should be modified.

4) The authors should take into consideration some sort of statistical testing (e.g. via χ2 test) for haplotype analysis in order to support their conclusion in a more formal way.

5) The name of BLAST bioinformatic tool or algorithm is usually capitalized because BLAST is abbreviation [line 319]. Moreover, it should by cited (given citation depends on used algorithm). It is unclear what software was used for alignment of sequenced alleles of the BolSSR04016 marker (Figure 6) [line 322].

I still found some strange sentences, although whole manuscript is now more readable. The authors should check it again.
“Moreover, BolSSR040196 exhibits (CT₉ ) in bolting-easy plant,. . . ” [line 29-30]
“Boudry et al. successfully mapped the gene associated with bolting gene B to two markers. . . ” [line 71-72]
“. . . gene map comparable to the genes that control the vernalization . . . ” [line 77]
“PCR detection of the amplification system . . . ” [line 130-131]

Author Response

Response letter to reviewer 1

Dear Editor-in-Chief,

The response letter to reviewer is as follows:

Reviewer 1:

Revised version of the manuscript was improved and most of the issues mentioned in the first review were solved. Therefore, I’ll only provide some suggestions and another list of typos or language issues. 1) There are some typos: Maybe term “loci” instead of “sites” is more suitable [line 21].

 “byPCR” [line 46] (by PCR)

“. . . food reserves..” [line 63] (. . . food reserves.)

Unexplained abbreviation SLAF-BSA (site-specific amplified fragment BSA) [line 84]

“. . . whole . . . ” [line 94] (different font type)

“. . . and total genomic DNA were extracted. . . ” [line 127] (. . . DNA was extracted)

“[13].PCR amplification. . . ” [line 127] ([13]. PCR amplification. . . )

“. . . male parent (P₁ Y9805) and bolting-easy female parent (P₂ Y5-3-14). . . ” [line 138] (It should be more consistent either (P₁ Y9805) and (P₂ Y5-3-14) or (P₁ Y9805) and (P₂ Y5-3-14). “. . . , then an F₂ population. . . ” [line 139] (. . . , then F₂ population)

“. . . chromosomes are also observed. . . ” [line 211] (. . . chromosomes were also observed. . . )

“Meanwhile, the ratio of transfer to AA, BB, AABB, AACC, and BBCC are . . . ” [line 236-237] (Meanwhile, the ratio of transfer to AA, BB, AABB, AACC, and BBCC is . . . )

“. . . spanning P₁ , P₂ , and F₂ generations. . . ” [line 306] It is better to use “. . . spanning parental and F₂ generations. . . ”, because P₁ nor P₂ generation does not exist (it notification of parent’s plants).

“. . . repeats (29.2%). [8].” [line 345] (. . . repeats (29.2%) [8].)

“. . . published to date. [27].” [line 353] (. . . published to date [27].)

“. . . Brassicaceae Database.In . . . ” [line 382] (. . . Brassicaceae Database. In . . . )

“. . . gene pool .” [line 404] (. . . gene pool.)

“According to Gao et al.’s findings. . . ” [line 421] (According to Gao et al. findings. . . )

Response: Thanks for your valuable comment. We have corrected the typos mentioned above one by one, and thoroughly reviewed the whole article again.

The term "sites" in line 21 has been substituted with "loci".

A space has been inserted between the two words "by PCR" in line 46.

The phrase "site-specific amplified fragment BSA" has been included in line 84.

Different fonts have been corrected in line 94.

Line 127 has been corrected.

The names of male parent and female parent has been rectified.

The sentence tenses in line 211 have been modified.

The sentence in line 236 has been corrected as follows: “the ratio of transfer to AA, BB, AABB, AACC, and BBCC is…”

The phrase “spanning P₁, P₂, and F₂ generations” in line 306 have been modified to “spanning parental and F₂ generations”.

Extraneous punctuation marks have been removed from lines 345, 353, and 404.

A space has been inserted between the two sentences in line 382.

The sentence in line 421 was revised.

2) It is questionable whether 10 randomly selected primer pairs out of 82984 designed primer pairs have something to say about “the success of our previously designed SSR primers.” [line 224-227]. It represents only ~0.012% of all designed primers. Moreover, I’m not sure what the authors tried to tell reader in this paragraph.

Response: Thanks for your valuable comment. In this study, we have firstly conducted a preliminary test to assess the reliability of the primer design using 10 primer pairs. We revised the word of “success” with the word of “availability”. Subsequently, we randomly selected 54 primer pairs, 6 SSR loci per chromosome, from the designed SSR primers based on the B. oleracea whole genome, and the transferability of these 54 pairs of primers was validated by 7 cruciferous species. Finally, a comprehensive set of 336 SSR markers were employed to identify the SSR markers linked with bolting loci in cabbage. In general, the SSR primers designed in this study are successful and available.

3) The information about tested species is now duplicated [line 157-159] and [line 231-233]. Text in the Result section should be modified.

Response: Thanks for your valuable comment. We have deleted the repeated descriptions in the results and tried to make the sentences more concise.

4) The authors should take into consideration some sort of statistical testing (e.g. via χ² test) for haplotype analysis in order to support their conclusion in a more formal way.

Response: Thanks for your valuable comment. We conducted a chi-square goodness-of-fit test on the outcome and evaluated the data.

5) The name of BLAST bioinformatic tool or algorithm is usually capitalized because BLAST is abbreviation [line 319]. Moreover, it should by cited (given citation depends on used algorithm). It is unclear what software was used for alignment of sequenced alleles of the BolSSR04016 marker (Figure 6) [line 322]

Response: Thanks for your valuable comment. The word BLAST has been converted to uppercase and supplemented with a reference. The utilization of MEGA-X software for sequence alignment has been incorporated into Section 2.7 of the materials and methods.

At the same time, some minor errors were also corrected during the process of revision to the manuscript. The improper descriptive sentences in the article have been revised.

Special thanks to you for your good comments, these comments can help us to improve our manuscript significantly. We really benefit a lot. Thank you!

Best regards to you,

Xiaolin YU