Intercultivar Diversity of Sour Orange (Citrus aurantium L.) Based on Genetic Markers, Phenotypic Characteristics, Aromatic Compounds and Sensorial Analysis

Round 1

Reviewer 1 Report

Manuscript by Ferrer et al is interesting, but it has not been well planned, designed and discussed. There are several aspects which must be improved before to its acceptance.

General comments: The evaluation of the paper has not been easy... I feel that the manuscript has a huge amoung of information. The reading becomes boring as I went in depth in to the manuscript. There is something which must be highlighted, there are 8 file in the suplementary materials... did authors think about preparing two manuscripts? I think that there is a lot information but only a few conclusions can be obtained...Think about it...

Not only authors used numerical expressions but also report some values in an alphabetical way. See lines 183-184 -> 39 sour oranges or Twenty-nine descriptors...

Section 2.3.3. Please include more details about the method described by Luro et al.

Further studies must be conducted in two consecutive seasons?

What is the proper harvest season of the cultivars? 

Section 2.4.1. Check-All-That-Apply (CATA) is a simple and fast sensory profiling tool. Yet, its application has been mainly focused on consumer studies. Authors report trained panelists, however no details are shown on how they were trained. Please clarify this aspect. How 29 descriptors were selected? Which reference producst were used? What was the scale of the questionaire?

Section 3.3. Authors claimed that for sensory analysis 39 regular sour oranges varieties...however results are shown about peel essential oils sensory analysis...

There a lot of controvery about the material and methods and the discussion.

 

Author Response

Thank you for agreeing to review our manuscript and for suggesting corrections to improve it. Our answers to your questions and criticisms are written in blue and text changes in italics.

General comments: The evaluation of the paper has not been easy... I feel that the manuscript has a huge amoung of information. The reading becomes boring as I went in depth in to the manuscript. There is something which must be highlighted, there are 8 file in the suplementary materials... did authors think about preparing two manuscripts? I think that there is a lot information but only a few conclusions can be obtained...Think about it...

It seems difficult to us to remove elements from the article even if we recognize that it presents a lot of information that could probably have been the subject of 2 articles. Our objective is not to make a varietal description by adding genetic and phenotypic (morphological and biochemical) characteristics but to demonstrate that mutation can generate more or less important phenotypic variations according to the traits of agronomic and industrial interest. We started with the genetic analysis to identify the origin of each variety, i.e. whether it is the product of a cross or the result of a mutation. All the varieties whose phenotypic variation is the result of mutations allowed us to evaluate the degree of variation in traits such as those used in taxonomy (morphological traits), agronomy (acidity and sugar content) and of interest to the processing industry (aromas). Despite the absence of major variations in the composition of the essential oil of fruit peel, we demonstrated a perceptible sensory aromatic variation that could give a certain typicality to a transformed product. Concerning the large number of additional files, each reader has the possibility to consult the totality of the measured values and their statistical comparisons, which were used to make general illustrations, integrated in the text.  

Since the article combines studies from several disciplines (genetics, biochemistry, sensory analysis), we admit that some readers may not find the details of all the experiments interesting. However, we have taken care to present the most informative illustrations to facilitate the understanding of the results. For ease of reading, we have reduced and modified the discussion to develop essentially the two themes of the article: the impact of mutation on phenotypic variability and the classification of sour oranges related genotypes (no true type sour oranges) according to their genetic origin.

The parts removed from the discussion are the following:

 

Given these genetic origin and morphological considerations, "Gou Tou", "Tosu", "Fraser" and "Australian" could be considered sour orange. On the other hand, "Chinensis" and "Chinensis de semis", although probably related to "Chinotto", cannot be considered sour oranges because of their direct relationship to citron and because of phenotypic characteristics that are very different from those of regular sour oranges. However, the composition of EO, especially LEO, shows that even cultivars that also result from C. reticulata/C. maxima admixtures have very different chemical profiles from regular sour oranges.

For example, in the study published by Polat et al. [22], “Alibert” and “Chinotto” were identified as hybrids, while they are in fact regular sour oranges that differ only by mutations, as demonstrated by the present work.

The diagnostic SNP markers provided information about the origin of the parents of the regular sour orange. Indeed, five C. maxima specific alleles (out of 18) are absent whereas only one is missing from the C. reticulata diagnostic SNPs. This observation suggests that the mandarin parent of sour orange is closely related to the mandarin panel used to design the diagnostic SNP markers, contrary to the pummelo parent, which probably belong to a different subgroup of pummelos distant from our subset. The subset of pummelos from the INRAE‐Cirad citrus collection is composed mainly of sweet pummelos, whereas sour and bitter pummelos have already been identified and could be more related to the pummelo parent of sour orange [51].

 

Not only authors used numerical expressions but also report some values in an alphabetical way. See lines 183-184 -> 39 sour oranges or Twenty-nine descriptors...

The count values have been written alphabetically throughout the text

Section 2.3.3. Please include more details about the method described by Luro et al.

We have completed the paragraph with the following information:

Gas chromatography analyses were performed on a Clarus 500 gas chromatograph (PerkinElmer, United states of America) equipped with flame ionization detector and equipped with 2 fused silica gel capillary columns (50 m, 22 mm id, film thickness 0.25 μm), BP-1 (polydimethylsiloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 to 220°C at 2°C/min and then held isothermal at 220°C for 20 minutes, with injector temperature 250°C, detector temperature 250°C, carrier gas hydrogen (1.0 mL/min), and split 1/60. The relative proportions of the oils constituents were expressed as percentages obtained by peak area normalization, without using correcting factors. Retention indices were determined relatives to the retention times of a series of n-alkanes (C7-C28) with linear interpolation (“Target Compounds” software of Perkin Elmer).

 

Gas chromatography coupled with mass spectrometry were conducted with a TurboMass quadrupole detector (PerkinElmer, United states of America), directly coupled to a Autosystem XL (PerkinElmer, United states of America), equipped with a fused silica gel capillary column  (50 m, 0.22 mm id, film thickness 0.25 μm), (BP-1 polydimethylsiloxane). Carrier gas, helium at 0.8 mL/min; split 1/75; injection volume 0.5 μL; injector temperature, 250°C; energy ionization, 70 eV; electron ionization mass spectra were acquired over the mass range 40-400 Da. The identification of components  was based: on comparison of their gas chromatography retention indices on polar and apolar columns, determined relative to the retention times of a series of n-alkanes with linear interpolation with those of authentic compounds and literature data; on computer matching against National Institute of Standards and Technology (NIST) commercial mass spectral library and by comparison of spectra with literature data. For further information were refer to the publication by Luro et al. 2019 [23].

 

Further studies must be conducted in two consecutive seasons?

We do not understand to which studies this remark is addressed. Is it the composition of the essential oils, sensorial analysis or the morphological characteristics of the fruit? Concerning the aromatic compounds, the results obtained for the essential oil of the fruit are in accordance with the results of the work done by Lota et al. 2001, 20 years earlier. The variability of the composition of leaf essential oils was first observed during the sampling of leaves carried out in June 2019. To confirm this variation, we conducted a second analysis in 2020 from batches of leaves corresponding to each of the 3 trees of the 6 selected varieties, collected in March 2020. This variability in LEO composition was thus observed over 2 years and statistically verified during the second sampling.

The morphology of the fruit and the composition of the juice had the same objective, i.e. to evaluate the degree of variability of the characteristics which made it possible to select the various forms (or varieties) of sour orange trees. Some characteristics are independent of the environment such as the shape of the fruit and the number of segments as well as the high content or absence of acidity. These characteristics do not vary over several years and the proof is given by the equivalent values found between the sugar content and acidity of some common varieties between two studies carried out in 2010 (Luro et al. 2011) and 2019 (the present manuscript). 

Mobilizing a panel of sensory analysts is not very easy and analyzing more than 40 samples is very time consuming. The objective of this work was not to describe the aromatic profile of each variety but to explore the possibility that there may be variability. For this purpose, it is not necessary to repeat this work a second year.

 

What is the proper harvest season of the cultivars? 

There is no one proper season for harvesting because it depends on the fruit use. As it is not a fresh consumption fruit, there is no well-defined season of maturity. In the framework of the multiplication of the rootstocks by sowing, it is essential to harvest the fruits at physiological maturity, when the seeds present an optimal rate of germination. The harvesting of sour orange fruits for seed extraction in our BRC is done between January and March every year.

In the industry for the transformation of the fruit (jam, candied fruits, sodas…) there are several harvest periods. For example in Savona (Italy) the Chinotto fruit is harvested at different periods (immature and mature) of its development to take advantage of the variability of color and aroma of its essential oils. We have chosen to make all the observations when the fruit has reached full maturity (from January onwards) and above all to carry out each type of observation over the shortest possible period in order to observe the variations linked to the variety and not to the environment or the stage of maturity. For example, for the extraction of essential oil, the fruit was harvested in one week for all varieties.

Section 2.4.1. Check-All-That-Apply (CATA) is a simple and fast sensory profiling tool. Yet, its application has been mainly focused on consumer studies. Authors report trained panelists, however no details are shown on how they were trained. Please clarify this aspect. How 29 descriptors were selected? Which reference producst were used? What was the scale of the questionaire?

As Cointreau manufactures liqueurs based on essential oils of citrus fruits, it has its own team of sensory analysts who are trained in the description of orange and bitter orange aromas. We have added in the text the method whose the 29 descriptors were chosen by the panelists: 

Twenty-nine descriptors were selected during consensus training sessions of the Cointreau Expert panel, on a few randomly selected samples of bitter orange essential oils. These descriptors are terms that the Expert panel already uses for Cointreau distillates already identified during CG-O sessions on the distillate (Table S6). The panelists (ten women and eight men aged between 22 and 55) are people from the laboratory who control the quality of the liquids during the manufacturing process and participate in daily tastings……The question asked to the panelists is the following: Which of the twenty-nine descriptors characterize the aroma of the sample?

Section 3.3. Authors claimed that for sensory analysis 39 regular sour oranges varieties...however results are shown about peel essential oils sensory analysis...

We have modified the sentence in the material and method by adding PEO of thirty-nine regular sour oranges.

There a lot of controvery about the material and methods and the discussion.

We checked the numbers of samples analyzed, the type of material analyzed, between the Material and Methods and Discussion sections. We have made the necessary modifications.

Reviewer 2 Report

In this manuscript authors studied the impact of morphological selection and genetic mechanisms of varietal diversification on the aromatic and odor variability of sour orange essential oils. In my opinion, the manuscript is well written, and the conclusions are sound. I recommend publication of a version of the manuscript that has been revised according to the following comments:

• According to IUPAC nomenclature it should be written “hex-2-enal” instead of "2-hexenal", "(E)-dodec-2-enal" instead of "E-2-dodecenal" (line 388), and "trans-dec-2-enal" instead of “trans-2-decenal" (line 532)
• Labels "p", "Z" and "E" should be written in italic (lines 398, 442 and 460)

Author Response

Thank you for agreeing to review our manuscript and for suggesting corrections to improve it. Our answers to your questions and criticisms are written in blue and text changes in italics.

 

In this manuscript authors studied the impact of morphological selection and genetic mechanisms of varietal diversification on the aromatic and odor variability of sour orange essential oils. In my opinion, the manuscript is well written, and the conclusions are sound. I recommend publication of a version of the manuscript that has been revised according to the following comments:

• According to IUPAC nomenclature it should be written “hex-2-enal” instead of "2-hexenal", "(E)-dodec-2-enal" instead of "E-2-dodecenal" (line 388), and "trans-dec-2-enal" instead of “trans-2-decenal" (line 532)
• Labels "p", "Z" and "E" should be written in italic (lines 398, 442 and 460)

We made the changes they are highlighted in yellow in the text

Round 2

Reviewer 1 Report

Manuscript has been improved and explanations have clarified many aspects.