Integrative Transcriptomic and Metabolomic Analyses of the Mechanism of Anthocyanin Accumulation and Fruit Coloring in Three Blueberry Varieties of Different Colors
, Qianhui Du †, Aizhen Li, Guiting Liu, Hexin Wang, Qingqing Cui, Zhichao Liu, Haixia Liu, Yani Lu, Yanqiong Deng and Guohui Xu *Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors and Editors,
I am pleased to inform you that I have completed the review of the manuscript with the full title: “Integrative Transcriptomic and Metabolomic to Analyze the Mechanism of Anthocyanin Accumulation and Fruit Coloring through Three Blueberry Varieties of Different Colors” (manuscript ID: horticulturae-2748189) by Liwei Chu, Qianhui Du, Aizhen Li, Guiting Liu, Hexin Wang, Qingqing Cui, Zhichao Liu, Haixia Liu, Yani Lu, 5 Yanqiong Deng and Guohui Xu.
Blueberries are grown all over the world and are very popular with consumers because of their unique taste and high antioxidant content. Blueberries have been found to contain high levels of vitamins A and C, dietary fiber, magnesium and bioactive phenolic compounds such as anthocyanins.
In recent decades, many studies have shown that the anthocyanins in blueberries have a positive effect on neurodegenerative diseases, cardiovascular disorders, diabetes and cancer. Anthocyanins are known to occur naturally in various tissues of fruits, flowers and vegetables, where they produce the colors red, purple and blue. The fruit color of blueberries is a very important agronomic characteristic and of great importance for the commodity value and competitiveness of the fruit on the market.
The cultivar factors can play an important role in modulating the expression of individual anthocyanin glcosides, such as malvidin, cyanidin, peonidin, petunidin and delphinidin. The aim of this study was to identify and quantify anthocyanins during the ripening of blueberry fruit of three different varieties “Pink Popcorn”, “Chandler” and “Black pearl”.
The cultivar factors can play an important role in modulating the expression of individual anthocyanin glcosides, such as malvidin, cyanidin, peonidin, petunidin and delphinidin. The aim of this study was to identify and quantify anthocyanins during the ripening of blueberry fruit of three different varieties.
The results of this study indicate that the most important pigment in the formation of blueberry fruit color is delphinium. The pigmentation analysis showed that the dark fruit color of “Black pearl” and “Chandler” was due to the highest content of delphinidin and cyanidin. The transcription level of the anthocyanin genes correlates with the color of the fruit. According to the results of this study (phylogenetic tree, HeatMAP and WGCNA analysis), the transcription factor VcMYBA plays an important role in regulating the expression level of the structural genes of the anthocyanin biosynthetic pathway and thus in the formation of fruit color in blueberries. The most important candidate gene responsible for the pink color of “Pink popcorn” fruits is VcANS.
The manuscript is overall of good quality. In agreement with the authors, the article represents a significant current knowledge on the mechanism of anthocyanin accumulation and fruit coloration by three blueberry varieties. Important information published in this manuscript are key candidate genes for the regulation of fruit color in different blueberry cultivars. Given the relationship between fruit color and fruit quality as well as antioxidant properties, these results form the basis for genomic editing and selection of this popular fruit variety.
Thank you for inviting me to participate in the journal's manuscript review process. As the authors will see from the comments below, this manuscript may become acceptable for publication in Horticulturae after they perform a minor revision.
Kind regards,
Following the review, I propose minor changes to the wording and grammar of the manuscript as well as the following annotations:
Please use the names of these two varieties consistently (“Pink Popcorn” and “Black pearl”). Decide whether the second word in the variety name should be capitalized or not, and make this consistent throughout the text of the manuscript.
MATERIALS AND METHODS
- Line 135: Please replace the word “Blueberry” with “blueberry”.
- Line 146: Please complete the existing text with details of the device used - centrifuge: manufacturer, city, country (as you have already written for the RNA extraction kit in line 174).
- Line 152: Please complete the existing text with details of the water bath (manufacturer, city, country).
- Line 153: Please complete the existing text with details of the spectrophotometer (manufacturer, city, country).
- Line 161: Please replace the text “http://www.metware.cn/” with details of the MetWare (manufacturer, city, country).
- Line 164: Please complete the existing text with details of the ultrasound (manufacturer, city, country).
- Line 166: Please complete the existing text with details of the membrane filter (city, country).
- Line 167: Please complete the existing text with details of the UPLC-ESI-MS/MS system.
- Line 176: Please complete the existing text with details of the bioanalyzer (city, country).
- Line 177: Please complete the existing text with details of the UV spectrophotometer NanoDrop (manufacturer, city, country).
- Line 192: Please complete the existing text with details of the kit for purification of cDNA (manufacturer, city, country).
- Line 198: Please complete the existing text with details of the sequencer (city, country).
- Line 201: I am of the opinion that the link should be deleted.
Line 234: Please complete the existing text with details of the reverse transcription kit (city, country).
Line 238: Please complete the existing text with details of the Real-Time PCR (city, country).
Line 240: Please complete the existing text with details of SYBR (manufacturer, city, country).
RESULTS
- Lines 254-255: Please change the sentence, because your results do not refer to morphological comparisons, but to comparisons of the color of the fruits of three blueberry varieties in three phases of their growth and development.
- Line 256: Please replace the word “stage2” with “Stage 2”.
- Line 258: Please replace the word “stage3” with “Stage3”.
- Line 268: Please replace the word “stage1” with “Stage1”.
MDPI recommends that research results from the last 5 years be taken more into account. In view of the fact that recent literature is represented in your bibliography with 37%, I suggest that you increase this proportion by supplementing the discussion with more recent publications.
Author Response
Reviewer #1
Dear Authors and Editors,
I am pleased to inform you that I have completed the review of the manuscript with the full title: “Integrative Transcriptomic and Metabolomic to Analyze the Mechanism of Anthocyanin Accumulation and Fruit Coloring through Three Blueberry Varieties of Different Colors” (manuscript ID: horticulturae-2748189) by Liwei Chu, Qianhui Du, Aizhen Li, Guiting Liu, Hexin Wang, Qingqing Cui, Zhichao Liu, Haixia Liu, Yani Lu, 5 Yanqiong Deng and Guohui Xu.
Blueberries are grown all over the world and are very popular with consumers because of their unique taste and high antioxidant content. Blueberries have been found to contain high levels of vitamins A and C, dietary fiber, magnesium and bioactive phenolic compounds such as anthocyanins.
In recent decades, many studies have shown that the anthocyanins in blueberries have a positive effect on neurodegenerative diseases, cardiovascular disorders, diabetes and cancer. Anthocyanins are known to occur naturally in various tissues of fruits, flowers and vegetables, where they produce the colors red, purple and blue. The fruit color of blueberries is a very important agronomic characteristic and of great importance for the commodity value and competitiveness of the fruit on the market.
The cultivar factors can play an important role in modulating the expression of individual anthocyanin glcosides, such as malvidin, cyanidin, peonidin, petunidin and delphinidin. The aim of this study was to identify and quantify anthocyanins during the ripening of blueberry fruit of three different varieties “Pink Popcorn”, “Chandler” and “Black pearl”.
The cultivar factors can play an important role in modulating the expression of individual anthocyanin glcosides, such as malvidin, cyanidin, peonidin, petunidin and delphinidin. The aim of this study was to identify and quantify anthocyanins during the ripening of blueberry fruit of three different varieties.
The results of this study indicate that the most important pigment in the formation of blueberry fruit color is delphinium. The pigmentation analysis showed that the dark fruit color of “Black pearl” and “Chandler” was due to the highest content of delphinidin and cyanidin. The transcription level of the anthocyanin genes correlates with the color of the fruit. According to the results of this study (phylogenetic tree, HeatMAP and WGCNA analysis), the transcription factor VcMYBA plays an important role in regulating the expression level of the structural genes of the anthocyanin biosynthetic pathway and thus in the formation of fruit color in blueberries. The most important candidate gene responsible for the pink color of “Pink popcorn” fruits is VcANS.
The manuscript is overall of good quality. In agreement with the authors, the article represents a significant current knowledge on the mechanism of anthocyanin accumulation and fruit coloration by three blueberry varieties. Important information published in this manuscript are key candidate genes for the regulation of fruit color in different blueberry cultivars. Given the relationship between fruit color and fruit quality as well as antioxidant properties, these results form the basis for genomic editing and selection of this popular fruit variety.
Thank you for inviting me to participate in the journal's manuscript review process. As the authors will see from the comments below, this manuscript may become acceptable for publication in Horticulturae after they perform a minor revision.
Response: Thank you very much. We have corrected many issues in our article.
Please use the names of these two varieties consistently (“Pink Popcorn” and “Black pearl”). Decide whether the second word in the variety name should be capitalized or not, and make this consistent throughout the text of the manuscript.
Response: Thank you very much. After confirmation, we have changed "Black pearl" to "Black Pearl" in our article.
MATERIALS AND METHODS
Line 135: Please replace the word “Blueberry” with “blueberry”.
Response: Thank you very much. We have corrected this issue in our manuscript.
Line 146: Please complete the existing text with details of the device used - centrifuge: manufacturer, city, country (as you have already written for the RNA extraction kit in line 174).
Response: Thank you very much. We have added some details about the centrifuge in the materials and methods.
Line 152: Please complete the existing text with details of the water bath (manufacturer, city, country).
Response: Thank you very much. We have added some details about the water bath in the materials and methods.
Line 153: Please complete the existing text with details of the spectrophotometer (manufacturer, city, country).
Response: Thank you very much. We have added some details about the spectrophotometer in the materials and methods.
Line 161: Please replace the text “http://www.metware.cn/” with details of the MetWare (manufacturer, city, country).
Response: Thank you very much. We changed some details of MetWare in our materials and methods.
Line 164: Please complete the existing text with details of the ultrasound (manufacturer, city, country).
Response: Thank you very much. We have added some details about the ultrasound in the materials and methods.
Line 166: Please complete the existing text with details of the membrane filter (city, country).
Response: Thank you very much. We have added some details about the membrane filter in the materials and methods.
Line 167: Please complete the existing text with details of the UPLC-ESI-MS/MS system.
Response: Thank you very much. We have added some details about the UPLC-ESI-MS/MS system in the materials and methods.
Line 176: Please complete the existing text with details of the bioanalyzer (city, country).
Response: Thank you very much. We have added some details about the bioanalyzer in the materials and methods.
Line 177: Please complete the existing text with details of the UV spectrophotometer NanoDrop (manufacturer, city, country).
Response: Thank you very much. We have added some details about the UV spectrophotometer NanoDrop in the materials and methods.
Line 192: Please complete the existing text with details of the kit for purification of cDNA (manufacturer, city, country).
Response: Thank you very much. We have added some details about the kit for purification of cDNA in the materials and methods.
Line 198: Please complete the existing text with details of the sequencer (city, country).
Response: Thank you very much. We have added some details about the sequencer in the materials and methods.
Line 201: I am of the opinion that the link should be deleted.
Response: Thank you very much. We have deleted the link.
Line 234: Please complete the existing text with details of the reverse transcription kit (city, country).
Response: Thank you very much. We have added some details about the reverse transcription kit in the materials and methods.
Line 238: Please complete the existing text with details of the Real-Time PCR (city, country).
Response: Thank you very much. We have added some details about the Real-Time PCR in the materials and methods.
Line 240: Please complete the existing text with details of SYBR (manufacturer, city, country).
Response: Thank you very much. We have added some details about the SYBR in the materials and methods.
RESULTS
Lines 254-255: Please change the sentence, because your results do not refer to morphological comparisons, but to comparisons of the color of the fruits of three blueberry varieties in three phases of their growth and development.
Response: Thank you very much. We have changed these in our article.
Line 256: Please replace the word “stage2” with “Stage 2”.
Response: Thank you very much. We have replaced the word “stage2” with “Stage 2” in our article.
Line 258: Please replace the word “stage3” with “Stage3”.
Response: Thank you very much. We have replaced the word “stage3” with “Stage3” in our article.
Line 268: Please replace the word “stage1” with “Stage1”.
Response: Thank you very much. We have replaced the word “stage1” with “Stage1” in our article.
MDPI recommends that research results from the last 5 years be taken more into account. In view of the fact that recent literature is represented in your bibliography with 37%, I suggest that you increase this proportion by supplementing the discussion with more recent publications.
Response: Thank you very much. We have cited and added some research results from the last five years.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study identified that anthocyanidin content determines the color of different blueberry varieties, with specific genes influencing these variations, providing insights for future genetic editing in blueberry breeding.
Line 125 (Methods), it is not clear how many blueberry fruits were used for one biological replications; the authors should provide more information on it.
Line 130, please insert the exact geographical coordinates (latitude) and altitude of the experiment station for absolute geological information.
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- Line 145, modify '200uL' to include a space between the value and the unit, changing it to '200 uL'.
-
Line 201, the font size is disproportionately large compared to the adjacent text. Please adjust it to match the surrounding font size.
-
In Figures 4 and 5, the text is too small to read comfortably. Please increase the font size for better legibility.
- In Figure 4A and B should be vertically displayed, I suggest
-
Author Response
Reviewer #2
The study identified that anthocyanidin content determines the color of different blueberry varieties, with specific genes influencing these variations, providing insights for future genetic editing in blueberry breeding.
Response: Thank you very much. We have corrected many issues in our article.
Line 125 (Methods), it is not clear how many blueberry fruits were used for one biological replications; the authors should provide more information on it.
Response: Thank you very much. We have added descriptions about this part in manuscript. All materials had three biological replicates sampled, each replicate consisted of 10g samples mixed with more than three individuals.
Line 130, please insert the exact geographical coordinates (latitude) and altitude of the experiment station for absolute geological information.
Response: Thank you very much. We have added descriptions about this part in manuscript.
Line 145, modify '200uL' to include a space between the value and the unit, changing it to '200 uL'.
Response: Thank you very much. We have corrected this issue in our manuscript.
Line 201, the font size is disproportionately large compared to the adjacent text. Please adjust it to match the surrounding font size.
Response: Thank you very much. We have corrected this issue in our manuscript.
In Figures 4 and 5, the text is too small to read comfortably. Please increase the font size for better legibility.
Response: Thank you very much. We have changed these in Figures 4 and 5.
In Figure 4A and B should be vertically displayed, I suggest.
Response: Thank you very much. We have changed these in Figure 4.
Reviewer 3 Report
Comments and Suggestions for AuthorsOverview:
In the manuscript the authors performed multiple omics approaches to analyze the differences in coloring mechanisms among three colors of blueberries with the aim to understand the ABP regulatory mechanisms in blueberry. Although the anthocyanin biosynthetic process and its regulation is well known and documented in many species, the approach of comparing three blueberry varieties with different color intensity is interesting. Therefore the current study is on a topic of relevance and general interest to the readers of the journal. But a different approach in terms of data analysis and results interpretation needs to be followed. In fact, many important issues in the methodology and in the results interpretation and discussion are present and often the data shown are not in line with the results description and discussion. Therefore, the manuscript must be reconsidered after a major revision process.
Major comments:
Both results and discussion sections contain information that deeply differ from the data coming from the analysis and shown in the figures and tables. Especially when considering gene expression data coming for RNA seq and real-time PCR validation. Just to make an example, expression data of the structural genes for anthocyanin biosynthesis says that the entire biosynthetic pathway is switched of in the “pink popcorn” variety and not just few genes. Furthermore, this results might lead to a down expression of the MYB transcription factor, that is directly correlated with anthocyanin biosynthesis and accumulation. Your results and discussion contain different information. Please re-elaborate results and discussion according the data obtained by the analysis.
Materials and methods need to be better described in some sections (2.3, 2.6, 2.8).
You consider structural and regulatory genes for ABP as candidate genes for anthocyanins accumulation. These genes are well known and documented so it’s incorrect to define them as candidate genes.
No information about DEG analysis (sampled compared), DEG’s annotation and functional enrichment are reported in the results section and discussed.
An important literature about ABP regulation exists (https://doi.org/10.1021/acs.jafc.5b01123). Please refer to this knowledge when discussing the results.
Minor comments
Lines 37-39: please add references to the studies cited
Lines 113-114: “Pink is an extremely rare fruit color mutant in blueberry varieties with very low anthocyanin content”: There’s something known about this mutation. Have you screened the expression level of something linked to this mutation?
Lines 131-134: this paragraph is not a material or method. Better in the introduction section.
Section 2.3: please add the formula for anthocyanin quantification with the spectrophotometric assay.
Section 2.4: please change the title of the section
Section 2.6: How the transcripts annotation have been obtained?
Line 245: Is there a reference for using GADPH as reference gene for Real-time validation?
Section 3.1: there is no reference in the text to the supplementary table 2
Line 321: please correct figure 3a with figure 4a
Figure 5: figure 5a and b represent the same data, please chose one
The quality of the figures needs to be improved. They are difficult to read
Author Response
Reviewer #3
In the manuscript the authors performed multiple omics approaches to analyze the differences in coloring mechanisms among three colors of blueberries with the aim to understand the ABP regulatory mechanisms in blueberry. Although the anthocyanin biosynthetic process and its regulation is well known and documented in many species, the approach of comparing three blueberry varieties with different color intensity is interesting. Therefore the current study is on a topic of relevance and general interest to the readers of the journal. But a different approach in terms of data analysis and results interpretation needs to be followed. In fact, many important issues in the methodology and in the results interpretation and discussion are present and often the data shown are not in line with the results description and discussion. Therefore, the manuscript must be reconsidered after a major revision process.
Response: Thank you for your patience and suggestions. We have improved the manuscript.
Major comments:
Both results and discussion sections contain information that deeply differ from the data coming from the analysis and shown in the figures and tables. Especially when considering gene expression data coming for RNA seq and real-time PCR validation. Just to make an example, expression data of the structural genes for anthocyanin biosynthesis says that the entire biosynthetic pathway is switched of in the “pink popcorn” variety and not just few genes. Furthermore, this results might lead to a down expression of the MYB transcription factor, that is directly correlated with anthocyanin biosynthesis and accumulation. Your results and discussion contain different information. Please re-elaborate results and discussion according the data obtained by the analysis.
Response: Thank you very much. Our result was carefully discussed and consistent with the conclusion. Our result show that the entire biosynthetic pathway is not switched of in the “pink popcorn” variety but only the low expression of VcANS may lead to the pink fruit color. MYB transcription factor-VcMYBA may be the candidate genes between the difference“Chandler” and “Black Pearl” but not Chandler” and“Pink Popcorn”.
Materials and methods need to be better described in some sections (2.3, 2.6, 2.8).
Response: Thank you very much. Materials and methods have been improved in this study.
You consider structural and regulatory genes for ABP as candidate genes for anthocyanins accumulation. These genes are well known and documented so it’s incorrect to define them as candidate genes.
Response: Thank you very much. “Candidate genes” in this study means the key gene which may led to the difference of fruit colour in blueberry but not the candidate genes for anthocyanins accumulation.
No information about DEG analysis (sampled compared), DEG’s annotation and functional enrichment are reported in the results section and discussed.
Response: Thank you very much. The DEG analysis (sampled compared), DEG’s annotation and functional enrichment you mean are only the basic analysis of RNA-Seq. But the target is clear in this study. We should pay more attention on the intersection between DEG and key genes of ABP to find the key gene.
An important literature about ABP regulation exists (https://doi.org/10.1021/acs.jafc.5b01123). Please refer to this knowledge when discussing the results.
Response: Thank you very much. We have added this reference in our manuscript.
Minor comments
Lines 37-39: please add references to the studies cited
Response: Thank you very much. We have added references about this part in manuscript.
Lines 113-114: “Pink is an extremely rare fruit color mutant in blueberry varieties with very low anthocyanin content”: There’s something known about this mutation. Have you screened the expression level of something linked to this mutation?
Response: Thank you very much. Our study is talking about the expression level of candidate genes linked to this mutation. The RNA-Seq and qRT-PCR result show the possible reason about this mutation.
Lines 131-134: this paragraph is not a material or method. Better in the introduction section.
Response: Thank you very much. We have changed these in our article.
Section 2.3: please add the formula for anthocyanin quantification with the spectrophotometric assay.
Response: Thank you very much. There is no formula because what we measured was the relative content of anthocyanin. The absorbance reading at 530 nm for each 0.1 mol/L sample was used as the unit of measurement for the relative anthocyanin content.
Section 2.4: please change the title of the section
Response: Thank you very much. We have changed these in our article.
Section 2.6: How the transcripts annotation have been obtained?
Response: Thank you very much. We used HISAT to compare the clean reads to the blueberry reference genome (https://purr.purdue.edu/publications/3123/versions?v=1). Gene annotation was performed by NR(Non-Redundant Protein Sequence Database) and NT(Nucleotide Sequence Database)
Line 245: Is there a reference for using GADPH as reference gene for Real-time validation?
Response: Thank you very much. Here is the reference for using GADPH as reference gene for Real-time validation in blueberry.
Valenzuela, F., D’Afonseca, V., Hernández, R., Gómez, A., & Arencibia, A. D. (2022). Validation of reference genes in a population of blueberry (Vaccinium corymbosum) plants regenerated in colchicine. Plants, 11(19), 2645.
Section 3.1: there is no reference in the text to the supplementary table 2
Response: Thank you very much. The supplementary table 2 is mentiond in line398.
Line 321: please correct figure 3a with figure 4a
Response: Thank you very much. But Figure 3a was the seven delphinidin-based anthocyanins identified through an LC-ESI-MS/MS system. Figure 4a was the expression profiles of ABP structural genes during three development stages in fruit of ‘Black Pearl’, ‘Chandler’ and ‘Pink Popcorn’ analyzed by RNA-Seq. How to correct figure 3a with figure 4a?
Figure 5: figure 5a and b represent the same data, please chose one
Response: Thank you very much. Figure 5a and b represent totally different data.
Figure 5b represent the relative expression level by RNA-Seq but Figure 5c represent the relative expression level by qRT-PCR. If you mean figure 5b and c.
The quality of the figures needs to be improved. They are difficult to read
Response: Thank you very much. We have changed these in our figures.
Reviewer 4 Report
Comments and Suggestions for AuthorsComments for author File: 
Comments.pdf
The authors should be carefully checking their written English.
Author Response
Reviewer #4
Review on Blueberry fruit color
12/05/2023
This manuscript was well written, reporting the relationship between blueberry fruit colors and
anthocyanin accumulation and candidate gene expression during different fruit development stages among three varieties. The experiments were well designed. Data were properly analyzed. The conclusions were drawn from solid analyzed data. The authors also projected some gene networks even some results were not confirmed by gene knockout or transformation complementation, but the information is useful for blueberry research community. After minor revisions, I think the results can be published on the journal. To enhance the quality of this manuscript, I have the following comments and suggestions:
Response: Thank you for your patience and suggestions. We have improved the manuscript.
Major revisions:
- For the materials and methods, the authors should briefly provide some sample collection information (such as soil conditions, weather conditions, and management conditions) because they are important for blueberry fruit quality.
Response: Thank you very much. We have added some more details in Materials and Methods.
- Improve the quality of Figure 4 and make it readable. Even at 150%, I could not read some of the labels.
Response: Thank you very much. We have changed these in Figure 4.
- In the discussion, the authors should discuss whether the fruit sizes have some effect on the fruit quality from their chemical analysis. We observed that there was a clear difference in fruit sizes among three varieties they used in the experiment.
Response: Thank you very much. We also identified the relative anthocyanin contents in only peel of the three blueberry varieties. The result is similar to Figure 2B in this study. So, the fruit size had no significant effect on the anthocyanin content that we were trying to study.
Minor revisions:
- The authors should be carefully checking their written English. For example, fruits and fruit.
Stage1 and Stage 2, and so on.
Response: Thank you very much. We have changed these in our article.
- For the references, keep the journal names spelling consistency. For example, using abbreviations and capital letters. “Trends in plant science” should be “Trends in Plant Science”. Frontiers in plant science” should be “Frontiers in Plant Science” and so on. Spell out “Int J Mol Sci” as “International Journal of Molecular Sciences”.
Response: Thank you very much. We have changed these in our references.
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
thank you for addressing my comments and concerns. Before the final acceptance there is a last comment that needs to be clarified: to which figure do the results described between line 326 and 341 refer? I guess is figure 4 and not figure 3 as reported in the text, since you are describing the expression levels of ABP structural genes and not the quantification of anthocyanins in fruit.
Author Response
Thank you for addressing my comments and concerns. Before the final acceptance there is a last comment that needs to be clarified: to which figure do the results described between line 326 and 341 refer? I guess is figure 4 and not figure 3 as reported in the text, since you are describing the expression levels of ABP structural genes and not the quantification of anthocyanins in fruit.
Response: Thank you for your patience and suggestions. We have changed these in our article.
