Diversity among Traditional Minority Red Grape Varieties According to Their Aromatic Profile

Round 1

Reviewer 1 Report

You should pay attention on the space between writing the value and the unit (exp. Line 165). It would be much more transparent if all the detected compounds were shown in one table than it is shown by varieties in Table 4.

 

Author Response

REVIEWER 1.

We appreciate the valuable comments of the Reviewer and we have made changes following reviewer’s suggestions. We hope the paper would be now suitable for publication in Agronomy’s journal.

1. ‟ You should pay attention on the space between writing the value and the unit (exp. Line 165)”

Response: Thank you for your review in detail. We have looked over the manuscript and we hope every value is now correctly written.

2. “It would be much more transparent if all the detected compounds were shown in one table than it is shown by varieties in Table 4”.

Response: Despite we know that the whole list of compounds identified would be much more informative, there is such a big number of compounds that we decided to group them in families in terms of facilitate their treatment. The results showed in this manuscript were included in a broad research project to characterize a great number of minor grape varieties. For this reason, once we have analysed all the varieties that we have in progress of study, we expect to carry out a more subtle statistical treatment, such as a network meta-analysis, that allow us to know more in detail the role of each compound in the potential varietal differentiation.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript entitled “Relationship between traditional minor red grape varieties according to their aromatic profile” is presenting interesting approach to explore phenotypic variability of minor Spanish cultivars related to their volatile organic compounds (VOC) composition in grape juice.

Aldo the obtained results have limited direct value for scientific community, the method used for exploration of variability of local diversity of Vitis genetic resources can be consider as interesting subject. In this sense I consider that manuscript would be more appropriate for journal “Diversity”.

Even so, several manuscript has to be carefully revised, especially taking into account following things:

Title: I suggest that term “Relationship” is replaced with term “diversity” or if the specific groups of cultivar are compared (previously defined based on genetic relationships) then “discrimination” of this groups could also be an option.

Abstract:

One of the aims of this study mentioned in abstract ism (L 14-15-): “...test if this varietal diversity could provide a quality product diversification to the current globalized wine market.” This aim is not included in the research and conclusions can not be given based on results obtained...

L 21-22: “Good differentiation between varieties was achieved...using...free and bound forms..”.

First, this is not correct – groups of varieties, and not varieties are compared, and second only limited differentiation is obtained for free and not for bond forms of volatiles.

Introduction:

 L 68-69 – More precise data should be presented (based on the model used in the study presented)

Materials and Methods

L 102 – Differentiation based on the origin of the cultivars is the aim of the study, and for this reason data related to origin, and other basic information on cultivars used such as origin (later in the PCA RPP1 and RPP2 groups of varieties are presented....), berry colour and other relevant information on accession used in research (Eurisco ID or VIVC code).  

L 129-132 – Grapes? – probably berries?

Duplicates are mentioned for VOC and basic chemical analyses but are not presented in results...

Altogether 500 berries were collected, and 400 used (two 100 berries duplicates)?

L 147 – subtitle . Oenological parameters – must (Juice) basic chemical composition is more appropriate title. (same for subtitle 3.2.)

L 144 -145 – please add reference for this maturation indexes.

 Why berry skins were not used? The majority of VOC are locted in the skin of the berry. 

Statistical analysis

Effect of year and genotype x year interaction should be tested and results presented – huge differences are visible between years...

Detailed explanation of data sets used for specific analyses must be presented here i.e. % of compounds used for PCA...

In case if significant differences are confirmed between years, and especially interactions – I suggest that instead of using % of compound groups in the profile you do the data standardisation within the years and use this data set for PCA. And for correlations standardised data within cultivar should be used due to the significant differences among cultivars.

 

Results and discussion

More precise data on temperature data in three years of study, especially in growing season should be presented. I suggest new table is introduced.

 

Table 5. Huge differences among cultivars are the big problem for this analysis  - as previously suggested – data standardisation within cultivars for three years should be performed before correlation analysis.

PCA1 results – Figure 2 and corresponding paragraph L 527 – 532

The correlation of VOC compounds (groups %) and F1 and F2 presented in the figure 2 must be revised. I.e. in case of aldehydes – they do not contribute at all to cultivars distribution, and Ketones that are not mentioned have significant contribution. This must be revised, but, as suggested previous standardisation of data within years should be performed. I suggest that all compounds, and not classes are used in this analysis. Other option is discriminant analysis among groups of cultivars.

 

Conclusions must be adjust according to previous suggestions and possible new outcome of the statistical analyses modifications as suggested.

 

 

 

 

 

Author Response

REVIEWER 2

We appreciate the valuable comments of the Reviewer. We have made changes following reviewer’s suggestions. We hope the paper would be now suitable for publication in Agronomy’s journal.

1. ‟ Title: I suggest that term “Relationship” is replaced with term “diversity” or if the specific groups of cultivars are compared (previously defined based on genetic relationships) then “discrimination” of this groups could also be an option¨.

Response: Certainly, it is more the difference than the relationship that we are looking for between varieties through their aromatic characterization, which is why we consider the recommendation to be very accurate and change the title into: “Diversity among traditional minority red grape varieties according to their aromatic profile”.

2. ‟ Abstract: One of the aims of this study mentioned in abstract is (L 14-15-): “...test if this varietal diversity could provide a quality product diversification to the current globalized wine market.” This aim is not included in the research and conclusions cannot be given based on results obtained...‟.

Response: It is true that although it is an objective, we could say indirect, of this study, there are no specific tests in the work with the monovarietal wines of these varieties that can test the potential quality that reflects the aromatic profile of the grapes of some of the varieties included in the study. For this reason, we rewrite the sentence, putting it as an objective of future study to check if the data obtained here are maintained in the study of wines, rather than as an objective of this study. Sentence remains as follows in the revised version: “The two main study aims were to evaluate the possibility of aromatically differentiate varieties based on their origin and to test the aromatic profile for being used as a chemotaxonomic tool. Based on the results obtained in this research, it would be also interesting to verify in future studies if this varietal diversity could translate into a diversification of quality products in the current globalized wine market”.

3. ‟ L 21-22: “Good differentiation between varieties was achieved...using...free and bound forms.” First, this is not correct – groups of varieties, and not varieties are compared, and second only limited differentiation is obtained for free and not for bond forms of volatiles‟.

Response: There are two main objectives in the study, and despite the fact that in PCA’s we label and group the varieties according to their genetic origin (RPP1 and RPP2), we also consider the statement that there is differentiation between varieties to be true, since in both PCA´s , although the explanation of the variance is not as high as might be desirable, there is no overlapping of the varieties and therefore we consider that their aromatic profile could differentiate not only between groups but also between individual varieties.

4. “Introduction: L 68-69 – More precise data should be presented (based on the model used in the study presented)”

Response: We include, in the revised version of the manuscript, more information lines (71-75).

5. “Materials and Methods L 102 – Differentiation based on the origin of the cultivars is the aim of the study, and for this reason data related to origin, and other basic information on cultivars used such as origin (later in the PCA RPP1 and RPP2 groups of varieties are presented....), berry colour and other relevant information on accession used in research (Eurisco ID or VIVC code).”

Response: Thank you for your comment. Truly, because of the numerous synonyms across the grapevine varietal denominations, a simple denomination of the varieties could lead to misunderstanding, that is why we complete the information, as recommended, with their VIVC codes, were existent.

All varieties studied in this research are red varieties, with a different range of colour intensity because of their different phenolic composition, the corresponding results were published in Díaz-Fernández et al., 2022a [21].

6. “L 129-132 – Grapes? – probably berries?”

Response: Thank you for your comment. Grapes were substituted by berries along the manuscript.

7. “Duplicates are mentioned for VOC and basic chemical analyses but are not presented in results...Altogether 500 berries were collected, and 400 used (two 100 berries duplicates)?”

Response: 500 berries were picked for each variety. In the laboratory, two replicates of 100 berries were selected for VOC and another two replicates of 100 berries for the estimation of the must physicochemical parameters. Those berries that presented poor sanitary health were eliminated. The analysis of both replicas was carried out to estimate the must physicochemical compounds and since no significant differences were obtained, the mean of both data obtained is shown. In the case of VOCs, only one of the replicas was analysed per year, reserving the other for analysis in case of methodological problems during the extraction procedure, as we were more interested in having three different year samples than different replicas of the same year.

8. “L 147 – subtitle: Oenological parameters – must (Juice) basic chemical composition is more appropriate title. (same for subtitle 3.2.)”

Response: Thank you for your recommendation. Consequently, both subtitles have been changed.

9. “L 144 -145 – please add reference for this maturation indexes.”

Response: Reference has been added for both maturation indexes – Ref. [24]-Togores, J. H. Chapter 3: Vendimia. Recepción de uva en la bodega. In: Tomo I: Tratado de Enología; 2ª ed. Eds. Mundi-Prensa, Madrid, 2010.

10. “Why berry skins were not used? The majority of VOC are located in the skin of the berry.”

Response: We agree with you. Most primary volatile compounds are present in the skin and under glicosidically form. In our research group we have some published manuscript about this subject. However, the aim of this research was not evaluating the different distribution of VOC among skin and pulp, we want to determine the aroma potential of each grape variety. For this reason, we crush the hole berry (without pedicel and seeds) to obtain the corresponding must in the same analytical conditions, and then we evaluated free and bound compounds in each must.

11. “Statistical analysis- Effect of year and genotype x year interaction should be tested and results presented – huge differences are visible between years...”

Response: Thank you for your comment. Nevertheless, our main objective was to difference among varieties considering three different years data to have more significant results and not so much considering the specific year effect, that is why we used aromatic profiles, it is said, aromatic compounds family percentages´ instead of using raw data and the reason why we neither calculate the year effect.  

12. Detailed explanation of data sets used for specific analyses must be presented here i.e. % of compounds used for PCA...

Response: Thank you for your interest. In terms of not making the manuscript too long, we decided to include two tables with percentages data in a supplementary file, we attached it together with this cover letter in answer to the reviewers´ suggestions.

13. In case if significant differences are confirmed between years, and especially interactions – I suggest that instead of using % of compound groups in the profile you do the data standardisation within the years and use this data set for PCA. And for correlations standardised data within cultivar should be used due to the significant differences among cultivars.

Response: Due to the great influence that different environmental factors have on the concentration of aromatic compounds in the grape and in agreement with studies in other type of compounds, such as phenolic ones, in which they stand out the influence of non-genetic factors, such as environmental ones, in the content of polyphenolic compounds to a much greater degree than their qualitative profile (Dimitrovska et al., 2011; Mattivi et al., 2006; Pomar et al., 2005), we use family compounds percentages´ in the PCA to compare varieties among them in terms of diminishing the influence of the external factors in their aromatic composition and try to limit the results as much as possible as the ones triggered by their genetic influence, following with the methodology line previously carried out in [23] with aromatic characterization and in [21] with the phenolic characterization, studies developed in the same line of research.

We were more interested in seeing how PCA analysis characterized and related the different varieties to each other rather than the three different years of each variety independently. For this reason, we decided to use the average value of the proportion of their volatile families, since we consider these data to be more representative with this objective than the separate annual data.

-          Dimitrovska, M., Bocevska, M., Dimitrovski, D. & Murkovic, M. (2011). Anthocyanin composition of Vranec, Cabernet Sauvignon, Merlot and Pinot Noir grapes as indicator of their varietal differentiation. European Food Research and Technology, 232, 591-600. http://dx.doi.org/10.1007/s00217-011-1425-9

-                Mattivi, F., Guzzon, F., Vrhowsek, U., Stefanini, M. & Velasco, R. (2006). Metabolite profiling of grape: flavonols and anthocyanins. Journal of Agriculture and Food Chemistry, 54, 7692-7702. https://doi.org/10.1021/jf061538c

-                Pomar, F., Novo, M. & Masa, A. (2005). Varietal differences among the anthocyanin profiles of 50 red table grapes cultivars studied by high performance liquid chromatography. Journal of Chromatography A, 1094, 34-41. https://doi.org/10.1016/j.chroma.2005.07.096

14. Results and discussion - More precise data on temperature data in three years of study, especially in growing season should be presented. I suggest new table is introduced.

Response: Thank you for your comment. In terms of not duplicating information, we introduced Figure 1 as we thought it was enough illustrative to show the biggest differences in temperature and rainfall among years. Moreover, we include a new reference [23], in which a more detail table could be seen in Supplementary Table 1, which shows seasonal as well as annual temperatures. We replicate it below.

Table S1: Yearly (2015, 2016 and 2017) bioclimatic indices and climatic data in the experimental plot site in north-west Spain (EVEGA, Ourense) – [23].

Index	2015		2016		2017
	Value	Viticulture climate	Value	Viticulture climate	Value	Viticulture climate
HI: Heliothermal	2664.15	Warm (HI+2)	2522.11	Warm (HI+2)	2678.62	Warm (HI+2)
CI: Night cold (ºC)	12.24	Cool nights (CI+1)	11.74	Very cool nights (CI+2)	11.13	Very cool nights (CI+2)
Parameter	Seasonal	Annual	Seasonal	Annual	Seasonal	Annual
Max Tª (ºC)	29.31	23.32	28.85	22.81	29.81	23.87
Mean Tª (ºC)	18.92	14.26	18.39	13.92	19.05	14.03
Min Tª (ºC)	10.30	7.23	9.97	7.23	10.35	6.81
Rainfall (mm)	231.20	659.20	382.40	1211.80	228.60	869.00
Days with mean T > 35 ºC	29	29	46	46	28	29

15. Table 5. Huge differences among cultivars are the big problem for this analysis - as previously suggested – data standardisation within cultivars for three years should be performed before correlation analysis.

Response: Thank you for your appreciation. Contrary to what it happened with the PCAs, in this case there is no standardisation process of the variables which correlations were shown, that is why we repeat the correlations but in this case with the annual concentrations data. No quite differences were obtained, reason why we maintain the manuscript text, attaching in this letter the new correlations table estimated just to compare and allow having all the information required.

Variables	Max T (°C)	Mean T (°C)	Min T (°C)	Rainfall (mm)	Days with T>35 °C
F Alcohols	0,315	-0,134	0,199	-0,070	-0,269
F Acids	-0,002	0,122	0,239	-0,142	-0,196
F Aldehydes	-0,047	0,181	0,251	-0,184	-0,213
F C6	0,173	-0,093	0,216	0,007	-0,151
F Thiols	-0,398	0,530	0,145	-0,387	-0,174
F Esters	0,691	-0,503	0,005	0,090	-0,244
F Phenols	-0,032	0,100	0,114	-0,100	-0,105
F Ketones	-0,059	0,034	-0,225	-0,030	0,085
F Lactons	0,158	-0,053	0,371	-0,013	-0,216
F Terpenes	-0,031	0,094	0,031	-0,105	-0,079
F Norisoprenoids	-0,296	0,223	-0,460	-0,123	0,204
F PAH´S	0,106	-0,011	0,137	-0,063	-0,147
F Sesquiterpenes	-0,197	0,134	-0,556	-0,101	0,211
A.P. Alcohols	0,145	-0,261	-0,139	0,228	0,162
A.P. Acids	0,121	0,070	0,197	-0,189	-0,273
A.P. Aldehydes	-0,265	0,109	-0,456	0,018	0,299
A.P. C6	-0,306	0,172	-0,348	-0,019	0,249
A.P. Thiols	0,397	-0,144	0,265	-0,123	-0,371
A.P. Esters	-0,347	0,256	-0,458	-0,123	0,227
A.P. Phenols	-0,273	0,305	-0,301	-0,242	0,044
A.P. Ketones	-0,371	0,370	-0,497	-0,277	0,132
A.P. Lactons	0,000	0,058	0,059	-0,078	-0,082
A.P. Terpenes	-0,065	0,103	-0,020	-0,094	-0,036
A.P. Norisoprenoids	-0,422	0,197	-0,674	0,002	0,437
A.P. PAH´S	-0,419	0,243	-0,574	-0,053	0,356
A.P. Sesquiterpenes	-0,093	0,080	0,109	-0,013	-0,008

 

 

 

16. PCA1 results – Figure 2 and corresponding paragraph L 527 – 532. The correlation of VOC compounds (groups %) and F1 and F2 presented in the figure 2 must be revised. I.e. in case of aldehydes – they do not contribute at all to cultivars distribution, and Ketones that are not mentioned have significant contribution. This must be revised, but, as suggested previous standardisation of data within years should be performed. I suggest that all compounds, and not classes are used in this analysis. Other option is discriminant analysis among groups of cultivars.

Response: Thank you very much for your comment. Truly, aldehydes had not such an important role in the varietal differentiation while other families have it. Sentence remain like follows: “the aromatic profile of 'Mouratón', 'Mencía', 'Evega 6', 'Corbillón' and 'Merenzao' is mainly determined by thiol, ketones, PAHs and alcohols families while the one of 'Evega 3', 'Espadeiro', 'Zamarrica', 'Albarín Tinto', 'Ferrón' and 'Pedral' is mainly defined by ester, norisoprenoid and C₆ families”. Once again, we maintained PCAs trying to follow same methodology line as followed in previous work [23].

17. Conclusions must be adjust according to previous suggestions and possible new outcome of the statistical analyses modifications as suggested

Response: Respect to the conclusions, a modification in reference to the second reviewer commentary was carried out as it follows: “Moreover, in this study is verified that there are minority grapevine varieties that are interesting for their higher aromatic potential, even more than some varieties already included in the community PDOs. They could contribute richness and diversity when making wines with differential qualities. This latter fact should still be corroborated through further studies of their corresponding monovarietal wines”.

• Other commentaries:

Research design must be improved

Response: Many thanks. We will take it into account in future research.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have sent us a manuscript to verify the differentiation between red grapevine varieties using as variables total free and bound forms. Volatile profile was established grouping aroma compounds into families. The main goal of this research is the evaluation of aromas to diferentiate grapevines, coupling this with genetic population classification.

The manuscript is well written, but some suggestions can be done.

Replace "." in mg L by "x"

Ta? This is not a correct acronym for temperature. Use international units. "T"

Galician region, Galicia, northern Iberia,... Please, use consistently i.e. Galicia, Galiza, or any other else.

p-propyl. Italize "p", if it comes from para

 

Author Response

REVIEWER 3

We appreciate the valuable comments of the Reviewer. We have made changes following reviewer’s suggestions. We hope the paper would be now suitable for publication in Agronomy’s journal.

1. “Replace "." in mg L by "x" “

Response: Thank you for your recommendation, units have been reviewed, hope everything its right now.

2. Ta? This is not a correct acronym for temperature. Use international units. "T"

Response: Thank you for your recommendation, units have been reviewed, hope everything its right now.

3. Galician region, Galicia, northern Iberia,... Please, use consistently i.e. Galicia, Galiza, or any other else.

Response: Thank you for your comment. We were looking to avoid repeat the same denomination, but it may be more confusing. There is now unified.

4. p-propyl. Italize "p", if it comes from para

Response: Thank you for your appreciation, Table 4 compounds writing was reviewed hoping it is everything correct now.

 

 

Author Response File: Author Response.docx