Fine Mapping and Functional Analysis of Major QTL, CRq for Clubroot Resistance in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Round 1

Reviewer 1 Report

I have attached the document herewith.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

Dear,

I would like to extend my gratitude to the respected reviewers, who spend your valuable time to review our manuscript. Your valuable comments were necessarily important for substantial improvement of our manuscript. The given comments and suggestions which we try to address point-by- point as follows:

 

Please add line numbers to the manuscript.

Response 1: Thanks for your comments. We have already corrected that.

 

Line 2-3: Title: Please use capital letters for highlighted letters in the title "Fine Mapping and Functional Analysis of Major QTL, CRq for clubroot resistance in Chinese cabbage (Brassica rapa ssp. pekinensis)".

Response 2: We have already corrected that.

 

Use Plasmodiophora brassicae when you use this scientific name for the first time, and then use P. brassicae

Response 3: We have corrected.

 

Line 109: You have mentioned that there are 50 (each from highly resistant and susceptible) plants. However, there are 30 in Table S1. In addition, we also used 50 each of the highly resistant and susceptible plants from F₂ population to analyze the possible mutations in the candidate resistant gene (Table S1).

Response 4: We are sorry for the mistake and thanks for your careful and rigorous, we have corrected with 30 plants each from highly resistant and susceptible.

 

Line 128: The equation is unclear. Explain all variables in the equation (n, N, T).  The disease index (DI) was calculated according to the formula: DI = [(n₁ + 3n₃ +… + 7n₇)/N_T× 7] ×100, where the number of plants with the symptom of I and N_T is the total number of plants tested. The DI for each F₂ individual was calculated from the mean grade of 2 replicates. Response 5: Thanks for your comments. The revised manuscript has added references here.“Fluazinam positively affected the microbial communities in Clubroot cabbage rhizosphere.”

 

Line 265/270: Results section 3.2: Please be careful to explain the methods you used in the materials and methods section and elaborate on what you find in the study in the results section. This is one example, and this sentence belongs to materials and methods. “We also performed chi-square test for the genotypic distribution in each window to determine significant deviation of SNP-index [22].” Please check 3.2 Another example:  “The SNP indices sites ≤ 0.3 or ≥ 0.7 in both pools were also removed. Finally, we retrieved 182,425 SNP for analysis.” First sentence explains experimental method. These two sentences can be merged to make meaningful result oriented sentence. Suggestion: After filtering the SNP indices sites ≤ 0.3 or ≥ 0.7 in both pools, we retrieved 182,425 SNP for analysis.

Response 6: Thanks for your comments, we have already corrected that.

 

Line 449: Please rephrase this part and avoid citing tables and figures in the discussion section. “The CRq gene was amplified and sequenced from resistant and susceptible lines using specific primers (Table S6). Sequence alignment showed 72 bp insertion between 2326 bp in the third exon of susceptible line (Figure 5A). A functional marker Br-insert1was designed, and 8 extremely resistant materials and 8 extremely sensitive materials were tested in F₂ population. The results showed that Br-insert1 co-segregated with clubroot phenotype in F₂ population (Figure 5C). In summary, base insertion in the LRR region may lead to frameshift mutation, which destroys the structure of the domain and leads to the loss of CRqprotein.”

Response 7: Thanks for your comments. We modified that sentence to "In our study, sequence alignment showed 72 bp insertion between 2326 bp in the third exon of susceptible line. A functional marker Br-insert1 was co-segregated with clubroot phenotype in F₂ population. In summary, base insertion in the LRR region may lead to frame shift mutation, which destroys the structure of the domain and leads to the loss of CRq protein".

 

Line 281: Please be consistence when use Mb. “Results indicated that the range of candidate region was successfully narrowed to 24.35-24.43 MB (0.08MB) (Figure 2B).”

Response 8: We have already corrected that.

 

Line 398: Figure 6. Add an explanation for black arrows

Response 9: Thanks for your comments. The black arrows indicate the degree of root enlargement.

 

Line 413: Use different word choice “Although the results of BSA-seq were not perfect, we used SNP-index curve and chi-square distribution to quickly locate the clubroot resistance gene on A03.”

Response 10: Thanks for your comments, we've replaced quickly with rapidly.

 

Line 409: Please use published manuscript. “This phenome-non also occurred in other studies in our laboratory [25] and another laboratory (the Chinese Cabbage Research Group of Northwest A&F University).”

Response 11: Thanks for your comments, we deleted “and another laboratory (the Chinese Cabbage Research Group of Northwest A&F University”.

 

Line 338: Some information are repeatedly use in results and discussion sections. “CRqgene we reported is likely to be the allele of the CRbgene, but the CRqgene contains two introns and three exons, while the CRbgene contains three introns and four exons.” “However, our reported CRqgene contained two introns and three exons, whereas CRbcontains three introns and four exons.”

Response 12: Thanks for your comments, we deleted the duplicates in results.

Line 294: Replace “may be” with potential candidate genes “Functional annotation anal-ysis showed that Bra019409, Bra019410, Bra019412 and Bra019413 might be candidate genes, which are encoded TIR-NBS-LRR protein (Table 2).”

 

Response 13: We have already corrected that.

 

Please format Table 2. It is hard to follow the row. Suggestion: 

Gene Name	Gene Position on A03	A. thaliana homolog	Gene function
Bra019413	24350950:24353977	Unknown	NB-ARC domain
Bra019412	24370531:24371199	AT5G11250	Encodes an atypical TIR-NBSLRR protein that is involved in stress responses. Loss of function alleles overproduce stress hormones JA, SA, ABA, and ET.

Response 14:We have already corrected that.

 

Line 530-645: References:

 Remove one of two numbers as highlighted.

1. Ren, W., Li, Z., Han, F., Zhang, B., Li, X., Fang, Z., et al. Utilization of Ogura CMS germplasm with the clubroot resistance gene by ferti…

Response 15: We have already corrected that.

 

Please add DOI information if it is available. The complete author list is not available for most references in the manuscript.

Response 16: We have already corrected that.

 

Additional periods: 9. Kuginuki, Y., Ajisaka, H., Yui, M., Yoshikawa, H., Hida, K.I., and Hirai, M. RAPD markers linked to a clubroot-resistance locus in Brassica rapa L. Euphytica. 1997, 98, 149-154.

Response 17: We have already corrected that.

 

Suggestions:

1. Line 26, 28, 31, 36, 37, 39 Abstract: I want to suggest what is in highlighted letters.

Clubroot disease caused by Plasmodiophora brassicae, is one of the major threats to Brassica crops.

• New clubroot resistant varieties of Chinese cabbage ( rapa ssp. pekinensis) have been developed through breeding, but still the underlying genetic mechanism of clubroot resistance is still unclear.
• After sequence analysis, the expression vector was constructed by gateway technology and transferred into Arabidopsis thaliana for functional characterization.
• In addition, we found 72-bp insertion in the third exon of CRq in the susceptible line.

We developed and verified the functional marker Br-insert1, by which the genotyping results showed that 72-bp insertion may might lead to the destruction of the LRR region of Y177-47, resulting in loss of resistance to clubroot.

• Therefore, CRq is a candidate gene of for clubroot disease resistance in Chinese cabbage, which could be used as a reference for elucidating the disease resistance mechanisms and the marker assisted breeding of clubroot resistant varieties.

Response 18: Thanks for your Suggestions, we have already corrected all of them.

 

2. Line 60, 62, 75, 76, 77, 81, 84, 85, 88, 90, 93, Introduction:
   • Plasmodiophorabrassicae is a biotrophic soil-borne pathogen that causes clubroot disease and affected the production of …
   • Bulked segregant analysis (BSA) is another important technique, which separates the extreme phenotypes into two groups based on associated markers are used to identify disease resistance genes [6].
   • TheBbulked segregant analysis is another important technique, which separates the extreme phenotypes into two groups based on associated markers that are used to identify disease resistance genes [6].
   • Although Crr1 was initially thought to be a single locus, but it was subdivided into two loci, Crr1a and Crr1b, where Crr1a plays a major role and Crr1b plays a minor role in clubroot resistance [10,11].
   • CRa was identified from Brassica host ECD02 of European Clubroot Differentials (ECDs), which was reported to carry a single dominant gene for resistance against race 2 of brassicae using differential sets of cultivars [12,13].
   • An F2 population derived from the cross of CR cultivars ‘Akiriso’ and ‘CR

Shinki’ conferred that CRb is a single dominant gene conferring resistance to the P. brassicae group 3 pathotype based on the differential cultivar sets [15].

• Rcr1 was identified from Pak choy ( rapa ssp. chinensis) as a single dominant resistance gene of P. brassicae pathotype 3 based on Williams’s classification [16].
• CRb is a useful locus to improve the durability of resistance, because of its effectiveness against brassicae pathotype group 3, in where Crr1 and Crr2 were not so effective [15].
• CRa and CRb are the same allele of clubroot resistance found in a previous mapping study [18].
• Although so many resistance loci have been reported, but only three resistance genes; CRa [19], CRb [20]an d Crr1a [11] have been cloned.
• In 2015, we used F₂ population derived from the cross of resistant Chinese cabbage line Y635-10 and susceptible line Y177-47 for constructing a preliminarily map of resistance gene

Response 19: Thanks for your Suggestions, we have already corrected all of them.

 

3. Materials and Methods

The verb does not agree with the subject. 

Line 105: A set of 340 QJF₂ population were developed by crossing between clubroot resistantline Y635-10 (derived from hybrid ‘Qiulihuang’) and susceptible line Y177-47 (derived from hybrid ‘Jianchun’) for mapping.

Response 20: Thanks for your comments, we've replaced were with was.

 

• Add an article

Line 108: Plasmodiophora brassicae race 4 was collected from the roots of infected Chinese cabbage field and characterized by William’s system [13].

Response 21: Thanks for your Suggestions, we have already corrected that.

 

 

(2.2, 2.3, 2.4, 2.5) rephrase these sentences

Line 113: Ten days old seedlings were inoculated with Plasmodiophora brassicae by application of 2 mL of resting spore (10⁷ spores/mL) suspension at the bottom of the stem base of each seedling [35].

Response 22: Thanks for your comments, we modified that sentence to “The suspension of 2ml of p. brassica resting spores (10⁷ spores/mL) were injected at the Bottom of stem base of 10-day-old seedlings”

Line 117: The infected roots were collected on 30 days post=inoculation as previously described.

Response 23: Thanks for your comments, we modified that sentence to “The infected roots were observed 30 days after inoculation.”

 

Line 119: Thirty day later, the incidence of clubroot disease was determined by combining the pathogenesis of the diseased plant and the size of the root.

Response 24: Thanks for your comments, we deleted it.

 

Add an article Line 133, 135, 138, 139, 142, 165, 178, 184, 201, 205, 224

DNA was extracted from the leaves of parents and F2 (Y635- 10×Y17747) plants by modified cetyltrimethyl ammonium bromide (CTAB) method [38].  

For BSA-seq, two DNA pools were constructed, of which one contained DNA from 50 highly resistant (DI = 0; R-pool) and other contained DNA from 50 highly susceptible (DI = 5-7; S-pool) F₂ plants.

In order to ensure the reliability of reads, raw data (raw reads) were first processed through a series of quality control (QC) procedures and removed low quality paired reads and adapter contamination [39].

SAM tools were used to convert alignment files to SAM/BWA files and to conduct SNP calling.

Linkage groups were established with a minimum logarithm of odds (LOD) threshold of 4.0 and genetic linkage map was constructed following the Kosambi mapping function [43,44].

Physical and chemical properties of protein were determined following Expasy (http://web.expasy.org/protparam/).

First strand cDNA was synthesized in a 20 μL volume containing approximately 7 μg RNA and oligo (dT) primers and used TransScript One-Step gDNA Removal (Trans, Beijing, China).

Each qRT-PCR experiment was performed in 3 technical replications and mean value was used for qRT-PCR analysis [18].

Whereas, attP1 and attP2 sites were added at both ends of donor carrier.

In LR reaction, same principle of BP reaction was followed to transfer the target gene from the entry vector into the expression vector by means of gene recombination through the catalysis of LR reactivity enzyme [49].

The Arabidopsis flower buds were soaked in the dip solution for 30 s and then wrapped in plastic wrap, incubated in the dark for 24 h and then cultured in normal light.

Response25: Thanks for your Suggestions, we have already corrected all of them.

 

4. Results Line 254, 260, 265, 274, 279, 311, 384
   • Figure 1: incorrect capital letter- 0: had no tumor; 1: There was a small tumor in the lateral …
   • Table 1. 7^th column header- χ^2(0.05) – χ²p-value at 0.05
   • We filtered the predicted SNP set by removing SNPs that had mass value ≤ 20.

Add an article “With the results of SNP-exponential curve and Chi-square test, we identified three poten-tial candidate regions on chromosome A03 (Table S3) located in the 23.80-28.10 Mb region of chromosome.”

• CRqgene from ‘Qiulihuang’ was mapped between GC30-FW/RV and BGA06 with a genetic distance of 0.2cM and 0.4cM, respectively (Figure 2A).
• Therefore, we assumed that Bra019412 and Bra019413 are the most likely the candidate genes regulating clubroot resistance.
• Add articles- “After screening, transgenic Arabidopsis thaliana of T₁ generation were grown in growth chamber.”

Response 26: Thanks for your Suggestions, we have already corrected all of them.

 

5. Discussion: Line 420

• Space and please try to avoid repeating same word multiple times within the sentence: In previous reports, three CR genes (CRa, CRband Rcr1) of B. rapa are located at around 23–24 Mb on chromosome A03 of B. rapa and CRa and CRb are a pair of alleles …

Response27: Thanks for your Suggestions, we have already corrected it.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors

I hope your great job opens windows to future work in order to improve Chinese cabbage resistance toward more diseases.

Please refer to comments to improve your manuscript presentation.

Regards;

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

Dear,

I would like to extend my gratitude to the respected reviewers, who spend your valuable time to review our manuscript. Your valuable comments were necessarily important for substantial improvement of our manuscript. The given comments and suggestions which we try to address point-by- point as follows:

 

Line 309: “The expressionlevels of Bra019412 and Bra019413 in 8DAI,13DAI,22DAI and Y635-10 were higher han those of Y177-47 and showed a stable trend (Figure 3C, 3D).” missing spaces

Response 1: Thanks for your comments, we have already corrected that.

• The names of 11 annotated genes in section C are merging together and they are unreadable.

Response 2: Thanks for your comments, the fonts were too small to read, so we deleted them.

• Figure 3. By choosing black color for your columns, their error bars are basically invisible. MDPI Publisher encourage authors to use colors in their illustration so please do not refrain from using suitable colors and bigger sizes in your illustrations.

Response 3: Thanks for your comments. The error bars are too small to see.

• Line 319: Table 2. Gene Function column:

Have you functionally characterized those genes? If not, then please insert your reference number in each cell.

Response 4: Thanks for your comments. The Gene Annotations were all obtained from the Brassicaceae Database. http://brassicadb.cn/#/

• Line 388/390: “were cultured to maturity for T₂seeds. In order to further validate the role of CRq geneinthe process of clubroot resistance. We infected wild and T2 seeds of Arabidopsis thaliana with clubroot spores (10⁷spores/mL)” missing spaces

Response 5: Thanks for your comments, we have already corrected that.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The authors fine-mapped the CRq gene to an 80kb interval following BSA-seq and InDel linkage analyses. And found the 72-base insertion occurred in the third exon of CRq gene. Furthermore, genetic transformation results confirmed that CRq gene played a vital role in resistance response.

1. since there are previous studies showed that the clubroot resistance was controlled by multiple genes, and the authors charaterize the plants following the scale, so did you try the QTL mapping using the disease index?
2. In key “words” and “conclusion”, the authors mentioned RNA-seq, “Due to the conserved nature of R gene, we obtained the resistance CRq gene by RNA-seq and RACE techniques”. However, the experiment was not mentioned in the “materials” and “results”.

Author Response

Response to Reviewer 3 Comments

 

Dear,

I would like to extend my gratitude to the respected reviewers, who spend your valuable time to review our manuscript. Your valuable comments were necessarily important for substantial improvement of our manuscript. The given comments and suggestions which we try to address point-by- point as follows:

 

1, since there are previous studies showed that the clubroot resistance was controlled by multiple genes, and the authors charaterize the plants following the scale, so did you try the QTL mapping using the disease index? 

Response 1: Thanks for your comments, we haven't used disease index to map QTL.

In our study, in spring, out of 340 F₂ plants 259 were identified as resistant and 81 as susceptible. In autumn, out of 340 F₂ plants 266 were identified as resistant and 74 as susceptible (Table 1). The F₂ population showed a 3:1 separation in both spring and autumn, respectively confirmed that the resistance trait is under Mendelian inheritance and governed by a single dominant gene. Then the tandem repeats of the NBS-LRR gene may promote the unequal recombination of paralogs, thus changing the gene number and producing new resistance gene recognition specificity. We assumed that CRq may be formed by the duplication and crossover of Bra019412 and Bra019413 genes.

 

2, In key “words” and “conclusion”, the authors mentioned RNA-seq, “Due to the conserved nature of R gene, we obtained the resistance CRq gene by RNA-seq and RACE techniques”. However, the experiment was not mentioned in the “materials” and “results”.

Response 2: Thanks for your comments, the data of RNA-seq has been deposited at NCBI under the accession numbers SRR3571023 and SRR3571024.

 

Author Response File: Author Response.docx