Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes in Korean Cucumber Inbred Lines
Round 1
Reviewer 1 Report
The manuscript entitled “Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes on the Korean Cucumber Inbred Lines” by Anarjan et al. developed a CAPS marker and an Indel marker for the genotyping of CsLRR-RPK2 and CsaMLO8, two candidates of cucumber powdery mildew resistant genes. The markers showed a strong correlation with the PM-resistance phenotype in 100 inbred lines derived from susceptive and resistant varieties. Therefore, the markers can be used for evaluation of powdery mildew resistance in cucumber. However, I do have several concerns list below.
Main:
1, L95, what are the variety/commercial names of the resistance resources A-L? In addition, there was no information provided in table 1 about the origin of the lines used for resistance phenotyping. Which parents were used for generating the lines? And more details should be provided for the evaluation of powdery mildew responses. For example, how to perform the scoring? How many replicates were used for phenotyping?
2, The figures presented in fig.3c and d were combinations of theses cropped from different pictures. The authors should put all the samples in the same gel for electrophoresis. It is not acceptable to use the marker on one gel to indicate the size of bands on another gel.
In addition, please use the variety names to replace R, S, and use the line codes to replace R1, s1… 3, The authors should provide the CsaMLO8-InDel and CsLRR-RPK2-CAPS genotypes of the susceptive and resistant parents, for further supporting the assertion
“First, PM-resistance source is originated from CsLRR-229 RPK2 gene, represented as A, J, and K. Second, ... Third, ...”
Minor:
Line56-62, incomplete sentence.
Author Response
Response to Reviewer 1 Comments:
Comments and Suggestions for Authors:
The manuscript entitled “Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes on the Korean Cucumber Inbred Lines” by Anarjan et al. developed a CAPS marker and an InDel marker for the genotyping of CsLRR-RPK2 and CsaMLO8, two candidates of cucumber powdery mildew resistant genes. The markers showed a strong correlation with the PM-resistance phenotype in 100 inbred lines derived from susceptive and resistant varieties. Therefore, the markers can be used for evaluation of powdery mildew resistance in cucumber. However, I do have several concerns list below.
Point 1: L95, what are the variety/commercial names of the resistance resources A-L? In addition, there was no information provided in table 1 about the origin of the lines used for resistance phenotyping. Which parents were used for generating the lines?
Response: We made new Table 1. The Table I include all the information related to 100 inbred lines. You may find the name of resistance susceptible line and types of resistance source. Furthermore, you can find the sources of paternal and maternal lines for crossing. (plese see the Table 1).
And more details should be provided for the evaluation of powdery mildew responses. For example, how to perform the scoring? How many replicates were used for phenotyping?
Response: We revised “evaluation of PM responses” in the “material and methods” section as following the reviewer’s comment. Please see L 106~116.
Point 2: The figures presented in fig.3c and d were combinations of theses cropped from different pictures. The authors should put all the samples in the same gel for electrophoresis. It is not acceptable to use the marker on one gel to indicate the size of bands on another gel.
Response: Following your comment, we added new gel image of the samples including resistance and susceptible lines ran on the same gel. Please see the new Figure 3.
In addition, please use the variety names to replace R, S, and use the line codes to replace R1, s1… 3,
Response: We changed the R, S, R1-R5 and S1-S5 to their inbred line names. We also added same information into the figure legend. Please see L 197 ~207 (Figure 3 and Figure 3 legend)
The authors should provide the CsaMLO8-InDel and CsLRR-RPK2-CAPS genotypes of the susceptive and resistant parents, for further supporting the assertion “First, PM-resistance source is originated from CsLRR-229 RPK2 gene, represented as A, J, and K. Second, ... Third, ...”
Response: We added genotyping data as Supplementary Figure 1.
Line56-62, incomplete sentence.
Response: We made complete sentence. Please see L 60~61.
Reviewer 2 Report
Please check the grammar and subject verb agreements in the entire text. Scientific language editing is required.
There are some problems that need to be addressed. Please see the attachment.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 2 Comments:
Comments and Suggestions for Authors:
Point 1: Please check the grammar and subject verb agreements in the entire text. Scientific language editing is required.
Response 1: We did English proofreading at a company called “Editage” You may find the certificate issued by “Editage” at end of document.
Point 2: As a result of these efforts, six temperature -dependent QTLs (chromosomes 1, 5, 6, and 7) from PI 197088 -1, five PM -resistance QTLs (PM1.1, PM1.2, PM2.1, PM2.2, and PM5.1) from S06, two QTLs (chromosome 5) from PI 250147 , four QTLs (pm5.1, pm5.2, pm5.3, and pm6.1) from K8, six QTLs (pm1.1, pm1.2, pm3.1, pm4.1, pm5.1, and pm5.2) from WI 2757, nine QTLs (pm1.1, pm1.2, pm2.1, pm3.1, pm4.1, pm5.1, pm5.2, pm5.3, and pm6.1) from CS - PMR1 and four QTLs (pm1.1, pm2.1, pm5.1, and pm 6.1) from PI 197088 [17,12,4,22,7,6,18] have been identified.
Response 2: We made complete sentence. Please see L 60~61
Point 3: Query_ Why other candidate gene based markers were not developed and not explored on the selected germplasm?
Response 3: The genetic inheritances of powdery mildew resistance is very complex. Therefore, most of the QTL mapping analysis just revealed the PM resistance loci, because the size of chromosomal regions that control PM resistance are too big to find candidates genes. Conclusively, both complex genetic inheritance and technical limitation of QTL analysis has been made us hard to find PM resistance candidates.
Point 4: Reframe the sentence, ‘On the spring of 2020, all the seeds were germinated and grown in the greenhouse of DONGOH SEED according to their culture protocol’.
Response 4: The sentence was revised. Please see L 97-101.
Point 5: What is the incidence of natural occurrence of powdery mildew disease? How the results could be considered as authenticated. Why artificial inoculation technique is not followed?
Response Powdery mildew is one of the most serious disease in cucumber cultivation in Korea. Furthermore, most of the cucumber cultivation areas has been contaminated by the pathogens. The fact indicates that powdery mildew could be easily occurred if appropriate plant protection is not applied. There are several different ways of artificial inoculation of powdery mildew. For seedling, we usually collected spore of powdery mildew and mixed with solution and inoculate into cucumber leaf by needless syringe. This method hardly applied to mature cucumber plant, because leaf structure of mature cucumber is very hard to infiltration by needless syringe. For mature plant, we usually collect powdery mildew spore and directly spray onto the cucumber leaf. Since the natural occurrence of powdery mildew in cucumber field is very easy and very frequent event, the assay result of dusting of powdery mildew spore onto mature plant is similar to those of natural occurrence. Furthermore, the PM resistance of seedling cucumber is different to those of mature cucumber plant. Therefore, the infiltration of mature plant is very hard. Natural occurrence of PM disease is easy and very frequent event. The result of natural occurrence of PM disease is similar to those of dusting method. Therefore, we scored the disease index in filed condition with natural occurrence of powdery mildew.
Point 6: Both maker-related information was shown in the table 2 and figure 2.
Rewrite this sentence as--The sequence of primers for both markers is given in table 2 while the gene organization is presented in figure 2.
Response 6: The sentence was revised. Please see L 144~145.
Point 7: The CsLRR-RPK2-CAPS marker can amplify the flanking region of SNP (T/C) (Figures 2, 3a). The SNP of PM-susceptible line could be cut by HinfI restriction enzyme and produce double bands (224 bp and 100 bp) (Figure 3b). Reframe the sentence as:
The CsLRR-RPK2-CAPS marker amplified the flanking region of SNP (T/C) (Figures 2, 3a). The SNP of PM-susceptible line produced double bands (224 bp and 100 bp) after HinfI digestion while PM-resistant line showed absence of restriction site in the amplicon (Figure 3b).
Response 7: The sentence was revised. Please see L 177~181.
Point 8: Phenotypically identified 10 inbred lines, consisted of 5 PM-resistance and 5 PM-susceptible were also verified for successful application of CsaMLO8-InDel (Figure 3d).
Response 8: The sentence was revised. Please see L 186~188.
Point 9: Highlight about the powdery mildew race specificity for both candidate genes, probably that could help to draw better conclusion about the statement ‘Consecutive selection of PM-resistance lines from generation to generation may accumulated both PM-resistance allele pairs (CsaMLO8 and CsLRR-RPK2) in a PM-resistance lines’.
Response 9: The sentence was revised. Please see L 275~284.
Point 10: The paper needs minor scientific language editing.
Response 10: We did English proofreading at a company called “Editage”. Please see next page.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript entitled ‘Marker-assisted evaluation of two powdery mildew resistance candidate genes on the Korean cucumber inbred lines’ by Anarjan et al. evaluated the involvement of the alleles of two PM-resistance candidate genes (CsaMLO8 and CsLRR-RPK2) in one-hundred Korean cucumber inbred lines. Both of them showed a strong correlation with the PM-resistance phenotype. Although a few PM-resistance inbred lines harbored neither one of CsaMLO8 or CsLRR-RPK2 alleles, CsaMLO8 and CsLRR-RPK2 can be as candidate genes in detection of the PM-resistance resources of cucumber inbred lines. The results are trustworthy, but the content of the manuscript is less. So the authors should provide more experimental data.
Author Response
Response to Reviewer 3 Comments:
Comments and Suggestions for Authors:
Point 1:The manuscript entitled ‘Marker-assisted evaluation of two powdery mildew resistance candidate genes on the Korean cucumber inbred lines’ by Anarjan et al. evaluated the involvement of the alleles of two PM-resistance candidate genes (CsaMLO8 and CsLRR-RPK2) in one-hundred Korean cucumber inbred lines. Both of them showed a strong correlation with the PM-resistance phenotype. Although a few PM-resistance inbred lines harbored neither one of CsaMLO8 or CsLRR-RPK2 alleles, CsaMLO8 and CsLRR-RPK2 can be as candidate genes in detection of the PM-resistance resources of cucumber inbred lines. The results are trustworthy, but the content of the manuscript is less. So the authors should provide more experimental data.
Response : We revised and updated results as following all reviewers’ comment. Furthermore, we added the genotyping data of all parental lines used for making inbred lines as supplementary data. In addition, we added the names of all parental lines used for making inbred lines as Table 1. If needed more data, please specify what kinds of data would be needed, we are welcome to update the results.
Round 2
Reviewer 1 Report
The authors has addressed all my concerns and made proper revisions.
Reviewer 3 Report
The revised manuscript has been improved a lot, I don't have any further criticisms.
