Development of Yein-Early, a Unique Fruit-Color and Leaf-Shape Mutant of Citrus unshiu, and Its Specific Selection Marker

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Ko et al., report “Citrus unshiu Marc. cv. Miyagawa-wase….” They conducted mutation breeding using gamma irradiation to develop new varieties of C. unshiu. Yein-early, which has a redder peel, greater hardness, and higher sugar content compared longer longitudinal length, shorter transverse length, stronger curling, and longer petiole length. Genome resequencing of Yein-early and the WT uncovered significant single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). They developed an allele-specific PCR marker

They show significant data of SNP but the report lack in data to relate the phenotypic observations and SNP data. I recommend the ms to be accepted as major revision.

 

Major

1.     WT that is used in this report is the same with the reference. In Table 5, the statistics of SNPs and In/Dels between WT and the reference should be provided, too. So how many spontaneous SNPs occur between them.

2.     The report presented SNPs of gamma irradiated mutant and showed several phenotypic differences such as fruit colors and leaf shapes. They should show what kind of mutations occur to those genes related to those phenotypic differences.   

3.     Hunter color values showed a higher red index for Yein-early fruits (Yein-early: 33.33±2.23; WT: 25.79±1.84), and fruit color of Yein-early is red. Search genes pathways such as flavonoid or anthocyanin related to color and show SNP among those genes.

4.     It is not clear how the authors designed Yein-SNP5 primer. Is it from the genes in Table S5.

If so show the nucleotide sequences of WT and Yein gene and primer position.

 

Minor

Table 1 legend, paraphrase for G for the first appearance

Figure 3 What is difference between A and B. Explain it in detail.

Comments on the Quality of English Language

Check the manuscript and improve the readability.

Author Response

First of all, we are grateful to reviewer 1 and editor for the valuable comments on this article and thank you for the time and consideration given in this regard. The response to reviewer 1 is as follows:

 

Major

Comments 1: WT that is used in this report is the same with the reference. In Table 5, the statistics of SNPs and In/Dels between WT and the reference should be provided, too. So how many spontaneous SNPs occur between them.

Response 1 : The SNP and In/Del statistics between WT and reference are provided in Table S1 and Table S3

 

Comments 2:. The report presented SNPs of gamma irradiated mutant and showed several phenotypic differences such as fruit colors and leaf shapes. They should show what kind of mutations occur to those genes related to those phenotypic differences.  

Comments 3: Hunter color values showed a higher red index for Yein-early fruits (Yein-early: 33.33±2.23; WT: 25.79±1.84), and fruit color of Yein-early is red. Search genes pathways such as flavonoid or anthocyanin related to color and show SNP among those genes.

Response 2, 3 : While it is also important to identify mutations in genes related to phenotypic differences, we are currently conducting transcriptome analysis to select candidate genes associated with the Yein-early trait. This will be combined with SNP and InDel data, and the findings will be submitted for publication.

 

Comments 4: It is not clear how the authors designed Yein-SNP5 primer. Is it from the genes in Table S5. If so show the nucleotide sequences of WT and Yein gene and primer position

Response 4 : The Yein-SNP primers were selected from the SNP results, where both Yein-early and WT were homozygous. After filtering based on depth, primers for AS-PCR were designed from the top 10 SNPs.

 

 

Minor

 

Comments 1: Table 1 legend, paraphrase for G for the first appearance

Response 1: We added as “G: force of gravity”

 

Comments 2: Figure 3 What is difference between A and B. Explain it in detail.

Response 2: A is a comparison with our other citrus mutant variants, and B is a comparison with commercially available citrus varieties. We added in Fig 3 as “A, J, G: our other citrus variants; CG, GP, KH: commercially available citrus varieties”

Reviewer 2 Report

Comments and Suggestions for Authors

The article concerns the molecular and -limited to some characters- morpho-physiological characterization of the new Citrus unshiu variety Yein-early, obtained through gamma ray irradiation of the -early ripening- cv. Miyagawa-wase, in turn studied as the reference cv (WT). The work is therefore very complex, enclosing a considerable temporary span going from the obtaining and multiplication of the first mutated cuttings, to the verification of the fixation of the -preliminary- character of interest, to the characterization of the fruits and leaves, to the identification of genomic mutations (SNPs and InDels), always carrying out the comparison with the reference cv. In addition, apart from being interesting at a scientific level, it implies a very promising practical impact as the new cv presents aesthetic as well as textural and sensorial characteristics that are attractive for the market. As such, I have only a few observations to submit to the authors.

-First of all an aesthetic curiosity: despite the climate can impact this evidence, the fruit of the cv. Miyagawa-wase has a characteristic yellowish-green color when it reaches the market (as an edible fruit), while it becomes orange with ripening (mature fruits often visible on ornamental plants). Is this your case -are they intended to be eaten when yellow-green?- or are they typically orange? Obviously, in the former case the redder peel would not be an added value. But, however it is, I wonder if some differences in color are evident even at the previous stages (early ripening).

-The description of the samplings are quite vague in some points: “more than 10 fruits were harvested from at least five Yein-early trees and five WT trees each year over two years” please provide the overall number of collected fruits per cultivar

or

“Three WT and three Yein-early individuals” how many leaves per individual?

or

“Genomic DNA was extracted from each three leaves of Yein-early and WT plants” What do you mean with “each three leaves”? How many leaves?

 -There is a point in the count of the SNPs found that is not totally clear to me: you wrote that the comparative genomic analysis between the WT and Yein-early revealed 650,257 SNPs in 26,339 genes and 105,817 InDels in 14,889 genes. I imagine that these genes are where you were searching for the possible cause of the redder peel. Then, in the annotation of non-synonymous mutations in Yein-early (3.5), you found 607,749 genes with SNPs and 490,151 genes with InDels (interestingly divided per function); did you check for non-shared non-silent SnIPs/InDels in the comparative analysis with Miyagawa-wase? This would have reduced the number of candidate genes for the different fruit color/leaf shape (despite it is known that silent mutations may not be so silent).

 -Eventually, a spontaneous reddish mutant of Miyagawa Wase mandarin has been analyzed at transcriptome level in a previous study, maybe you could find some correspondences with the findings in the article for identifying some mutated candidate genes (Suh, S.J., Lee, SH., Lee, DH. et al. Transcriptome analysis of a spontaneous reddish mutant in Miyagawa Wase Satsuma mandarin. J Korean Soc Appl Biol Chem 56, 391–399 (2013). https://doi.org/10.1007/s13765-013-3085-x)

Minor checks:

You missed the “Conclusions” section

Line 43: Defining mutation breeding “innovative” (although only related to Citrus) is quite strange

Lines 95 and 197: please write  C. unshiu in italics

Line 123: merged words: “usingan”

Line 129: please change “These DNA” into “These DNAs”

Author Response

First of all, we are grateful to reviewer 2 and editor for the valuable comments on this article and thank you for the time and consideration given in this regard. The response to reviewer 2 is as follows:

 

Comments 1: First of all an aesthetic curiosity: despite the climate can impact this evidence, the fruit of the cv. Miyagawa-wase has a characteristic yellowish-green color when it reaches the market (as an edible fruit), while it becomes orange with ripening (mature fruits often visible on ornamental plants). Is this your case -are they intended to be eaten when yellow-green?- or are they typically orange? Obviously, in the former case the redder peel would not be an added value. But, however it is, I wonder if some differences in color are evident even at the previous stages (early ripening).

Response 1: The change in pericarp color between Yein-early and WT shows no difference at the break-stage in October, but from November onwards, the pericarp of Yein-early appears redder compared to WT. In Korea, farms typically harvest and sell the fruits in December when grown in open fields.

 

Comments 2: The description of the samplings are quite vague in some points: “more than 10 fruits were harvested from at least five Yein-early trees and five WT trees each year over two years” please provide the overall number of collected fruits per cultivar

Response 2: We changed as “…, each 65 and 50 fruits were harvested from…..”

 

Comments 3: Three WT and three Yein-early individuals” how many leaves per individual?

Response 3: We changed as “Fifteen leaves from each of the three WT individuals and three Yein-early individuals were used to analyze leaf morphology, and the analysis was based on nine leaf characteristics,….”

 

Comments 4: “Genomic DNA was extracted from each three leaves of Yein-early and WT plants” What do you mean with “each three leaves”? How many leaves?

Response 4: We changed as “Leaves were harvested from three Yein-early and three WT individuals, and genomic DNA was extracted using…”

 

Comments 5: There is a point in the count of the SNPs found that is not totally clear to me: you wrote that the comparative genomic analysis between the WT and Yein-early revealed 650,257 SNPs in 26,339 genes and 105,817 InDels in 14,889 genes. I imagine that these genes are where you were searching for the possible cause of the redder peel. Then, in the annotation of non-synonymous mutations in Yein-early (3.5), you found 607,749 genes with SNPs and 490,151 genes with InDels (interestingly divided per function); did you check for non-shared non-silent SnIPs/InDels in the comparative analysis with Miyagawa-wase? This would have reduced the number of candidate genes for the different fruit color/leaf shape (despite it is known that silent mutations may not be so silent).

Response 5: We did not check for non-shared non-silent SnIPs/InDels in the comparative analysis with Miyagawa-wase. We will do check for non-shared non-silent SnIPs/InDels and the main objective of this paper is to develop molecular markers to distinguish the newly developed mutant citrus cultivar from other citrus varieties and secure intellectual property rights. We are currently conducting transcriptome analysis to select candidate genes associated with the Yein-early trait. This will be combined with SNP and InDel data, and the findings will be submitted for publication.

 

Comments 6: Eventually, a spontaneous reddish mutant of Miyagawa Wase mandarin has been analyzed at transcriptome level in a previous study, maybe you could find some correspondences with the findings in the article for identifying some mutated candidate genes (Suh, S.J., Lee, SH., Lee, DH. et al. Transcriptome analysis of a spontaneous reddish mutant in Miyagawa Wase Satsuma mandarin. J Korean Soc Appl Biol Chem 56, 391–399 (2013). https://doi.org/10.1007/s13765-013-3085-x)

Response 6: We will refer to the above paper for the transcriptome analysis we are currently conducting.

 

Minor checks:

Comments 1: You missed the “Conclusions” section

Response: I understand that the "Conclusions" section is optional.

 

Comments 2: Line 43: Defining mutation breeding “innovative” (although only related to Citrus) is quite strange

Response 2: We removed the “innovative”

 

Comments 3: Lines 95 and 197: please write  C. unshiu in italics

Response 3: We changed as italics

 

Comments 4: Line 123: merged words: “usingan”

Response 4: We changed as “using an”

 

Comments 5: Line 129: please change “These DNA” into “These DNAs”

Response 5: We changed as “These DNAs”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

They moderately addressed raised questions.