Diversity of Pummelos (Citrus maxima (Burm.) Merr.) and Grapefruits (Citrus x aurantium var. paradisi) Inferred by Genetic Markers, Essential Oils Composition, and Phenotypical Fruit Traits

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

In this work genotypic and phenotypic diversity were assessed in 25 pummelos and 33 grapefruits (or assimilated to grapefruit or pummelo).

Aside from a few typos that I recommend addressing, the article is well written and of significant scientific interest. I believe this paper is suitable for publication.

Following, I report some suggestions to improve the overall quality of the manuscript:

In the Introduction section, when you present apomixis, please cite the characterization performed on lemon by Catalano et al., 2022 (https://doi.org/10.3390/agriculture12122020).

Figure 1 should be displayed after its citation in the text.

Formatting from page 6 need to updated according with MDPI recommendation.

“Diversity structure of the cultivars set”: Is it a subtitle?

Figure 6: please, identify Figure A and Figure B.

Figure 7 caption: “tru” is a typo.

“All grapefruit cultivars (C. maxima) are monoembryonic.”: please, review this sentence.

“Grapefruit (C. paradisi) weight and shape vary very little between cultivars”: I think there is no need to write the name of the species in this sentence. The same in the previous one.

Figure 8 caption: “ans” is a typo.

“The number of varieties representing Citrus reticulata and Citrus medica species is low iin the current study”: “iin” is a typo.

“SSR markers are not tottaly efficient markers for species diagnostic”: “totally” is a typo

“SNP markers diagnostic to ancestral species (DSNPs) have been déevelopped for citrus and employed by Kaspar genotyping technology”: “déevelopped” is a typo.

In M&M it is reported that genotypes under study were cultivated on different rootstocks: can this factor influence essential oil composition and fruit quality? Please, report this in the discussion section (e.g. rootstocks can influence susceptibility towards the two-spotted spider mite in the grafted variety, by different mechanisms involving also a different profile of Volatile Organic Compounds, and fruit quality). Also, I would suggest to stress the importance of the characterization of EO also for commercial use purposes and for their involvement in plant-environment interaction.

Author Response

We sincerely thank the reviewers for having accepted to evaluate our work and the manuscript and for having invested their time to correct and propose modifications to improve the quality of the text. You will find hereafter the answers to your questions, suggestions and remarks. We have asked MDPI to revise the English.

Reviewer 1

In this work genotypic and phenotypic diversity were assessed in 25 pummelos and 33 grapefruits (or assimilated to grapefruit or pummelo).

Aside from a few typos that I recommend addressing, the article is well written and of significant scientific interest. I believe this paper is suitable for publication.

Following, I report some suggestions to improve the overall quality of the manuscript:

Comments 1: In the Introduction section, when you present apomixis, please cite the characterization performed on lemon by Catalano et al., 2022 (https://doi.org/10.3390/agriculture12122020).

Response 1: Now this reference has been introduced in introduction section

Comments 2: Figure 1 should be displayed after its citation in the text.

Response 2: Done

Comments 3: Formatting from page 6 need to updated according with MDPI recommendation.

Response 3: Sorry, but we didn't understand this recommendation. Is it the position of the figure, the size and font of the text indentation? The manuscript has been formatted as MDPI according to the template sent to us. “Diversity structure of the cultivars set”: Is it a subtitle?

Comments 4: Figure 6: please, identify Figure A and Figure B.

Response 4: Done

Comments 5: Figure 7 caption: “tru” is a typo.

Response 5: Corrected

Comments 6: “All grapefruit cultivars (C. maxima) are monoembryonic.”: please, review this sentence.

Response 6: Grapefruit was substituted by pummelo

Comments 7: “Grapefruit (C. paradisi) weight and shape vary very little between cultivars”: I think there is no need to write the name of the species in this sentence. The same in the previous one.

Response 7: We modified by removing the Latin name in all the document

Comments 8 : Figure 8 caption: “ans” is a typo.

Response 8: Corrected

Comments 9: “The number of varieties representing Citrus reticulata and Citrus medica species is low iin the current study”: “iin” is a typo.

Response 9: Corrected

Comments 10: “SSR markers are not tottaly efficient markers for species diagnostic”: “totally” is a typo

Response 10: Corrected

Comments 11: “SNP markers diagnostic to ancestral species (DSNPs) have been déevelopped for citrus and employed by Kaspar genotyping technology”: “déevelopped” is a typo.

Response 11: Corrected

Comments 12: In M&M it is reported that genotypes under study were cultivated on different rootstocks: can this factor influence essential oil composition and fruit quality? Please, report this in the discussion section (e.g. rootstocks can influence susceptibility towards the two-spotted spider mite in the grafted variety, by different mechanisms involving also a different profile of Volatile Organic Compounds, and fruit quality).

Response 12: We added the following paragraph into the discussion section (the references were put on the MDPI format in the revised manuscript):

“Can we suspect a rootstock effect on the variations observed between cultivars? The two rootstocks are Carrizo citrange or Pomeroy trifoliate orange (supplemental file 1). Numerous studies have demonstrated that rootstock can modify scion fruit quality traits, such as made by Castle et al. (2010), where the performance of Valencia orange was studied on 12 genetically very different rootstocks. The differences observed in this study between Carrizo citrange and trifoliate orange were very small and often insignificant. A study of the effect of rootstock on the composition of orange essential oils revealed only slight variations between citrange and trifoliate orange rootstocks (Ferrer et al. 2022). Nevertheless, the secondary metabolite composition of a scion can be strongly modified according to the type of rootstock, especially in situations where the trees are under stress. Sour orange rootstock comparatively to Cleopatra mandarin, enhanced tolerance to spider mites of the scion by inducing flavonoids synthesis (Agut et al. 2014). Among the grapefruit accessions in our study, three are grafted on Carrizo citrange (Hassaku, Pink Ruby and Ray Ruby) and none showed significant differences from grapefruits grafted on trifoliate orange, irrespective of the traits studied. The aim of our study is not to evaluate precisely the minimal differences between cultivars, but to reveal a specific phenotypic profile in relation with the genetic divergence.”

Castle, W.S.; Baldwin, J.C., Muraro, R.P.; Litell, R. Performance of ‘Valencia’ Sweet Orange Trees on 12 Rootstocks at Two Locations and an Economic Interpretation as a Basis for Rootstock Selection. HortScience, 2010, 45, 523–533.

Ferrer, V.; Paymal, N.; Quinton, C.; Costantino, G.; Paoli, M.; Froelicher, Y.; Ollitrault, P.; Tomi, F.; Luro, F. Influence of the Rootstock and the Ploidy Level of the Scion and the Rootstock on Sweet Orange (Citrus sinensis) Peel Essential Oil Yield, Composition and Aromatic Properties. Agriculture, 2022, 12, 214. https://doi.org/10.3390/agriculture12020214

Agut, B; Gamir, J.; Jacas, J. A.; Hurtado M.; and Flors V. Different metabolic and genetic responses in citrus may explain relative susceptibility to Tetranychus urticae. Pest Manag. Sci. 2014, 70:1728-1741. DOI 10.1002/ps.3718

Comments 13: Also, I would suggest to stress the importance of the characterization of EO also for commercial use purposes and for their involvement in plant-environment interaction.

Response 13: We added the following paragraph into the discussion section:

“Citrus essential oils are mainly used in the cosmetics and perfume industries. Their composition determines their aromatic properties. It can be assumed that two EOs of identical composition will have very similar aromatic properties, and can therefore be used interchangeably. Many factors can modify the EO composition, such as growing location, cultivars (genetic variability), ripening stages, storage conditions and extraction methods (Gaff et al. 2020). Biotic and abiotic stress are also factors that induce the synthesis of new volatile compounds or modify the EO composition (Loreto & Schnitzler, 2010). The terpenoids are family of volatile organic compounds (VOC) abundantly emitted by stressed plants (Feussner and Wasternack, 2002; Dudareva et al., 2004). Some of these compounds, are involved in plant-environment interactions, either as pathogen inhibitors, chemical endogenous signal inducing abiotic-responsive genes or chemical messengers for plant-to-plant relation (Yamauchi et al. 2015). All the cultivars of our collection are healthy and under cultivating conditions avoiding any stress (Luro et al. 2017).  We can therefore conclude that the variations observed between pummelo accessions and their hybrids are indeed in relation with their genetic constitution. On the other hand, the phenotypic peculiarities observed in our study for a group of cultivars or isolated varieties are the result of a combination of interactions between genetics and environment.”

Gaff, M.; Esteban-Decloux, M.; Giampaoli, P. Bitter orange peel essential oil: a review of the different factors and chemical reactions influencing its composition. Flavour and Fragrance Journal, 2020, 35, 247-269. https://doi.org/10.1002/ffj.3570

Loreto, F.; and Schnitzler, J.-P. Abiotic stresses and induced BVOCs. Trends Plant Sci. 2010, 15, 154–166. doi:10.1016/j.tplants.2009.12.006

Feussner, I.; & Wasternack, The lipoxygenase pathway. Annu. Rev. Plant Biol. 2002, 53, 275–97. DOI: 10.1146/annurev.arplant.53.100301.135248

Dudareva, N.; Pichersky, E.; Gershenzon, J. Biochemistry of plant volatiles. Plant Physio. 2004, 135, 1993-2011. https://doi.org/10.1104/pp.104.049981

Santana Vieira, D.D.S.; Giovanni, E.; Michelozzi, M.; Centritto, M.; Luro, F, Morillon, R, Loreto, F.; Gesteira, A. and Maserti, B. Polyploidization alters constitutive emission of volatile organic compounds (VOC) and improves membrane stability under water deficit in Volkamer lemon (Citrus limonia Osb.) leaves. Environmental and Experimental Botany 2016, 126. http://dx.doi.org/10.1016/j.envexpbot.2016.02.010

Yamauchi, Y. ; Kunishima, M. ; Mizutani, M. ; Sugimoto Y. Reactive short-chain leaf volatiles act as powerful inducers of abiotic stress-related gene expression. Sci. Rep. 2015, 5, 8030. https://doi.org/10.1038/srep08030

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is complex in the reading, but it sounds interesting. The authors are highly encouraged to revise many points, in the form (mislabellings – alos, tru, ans, othere, tottaly, déevelopped, speciesi, Marsh, uppercase/lowercase in the text and in the figure, … - the list is not exhaustive) and the substance, by reading carefully all the text, including supplementary files. It seems that the authors did not read the files before proceeding with the submission, considering the numerous inconsistencies. I suggest a revision by a mother tongue.

Abstract:

• It is not essential to report the double identification of sweet orange and grapefruit (( sinensis or C. x aurantium var. sinensis) … (C. paradisi or C. x aurantium var. paradisi)). Choose one of them. For grapefruit, I suggest you use the same as you indicated in the title, and I encourage authors to also maintain it in the manuscript.
• Replace paradisi with grapefruit if it is not essential to cite the species (…and very low levels for the group of mutants (C. paradisi)). The same logic must be applied to C. maxima and pummelo. Not only in the text, but also in the captions.
• … no other compound is specific to a hybrid genotype: which one?
• Once you have defined your acronyms, use them from the beginning to the end of your manuscript (LEO, PEO, ….)
• It is unclear in which way the results can be useful for the breeding program.

Introduction:

• I suggest using “pummelo” instead of “pummelos” as incipit. And also along the text, I think that the use of the singular form sounds better, and, if deemed appropriate, the authors can use the form “pummelo varieties/accessions”. Moreover, the authors also use pomelos. Please, use just one form.
• Probably Citrus, after the first time, can be reported as C.?
• The section between “The composition of citrus volatiles has been intensively investigated ….” and “…and the other is that varietal identification is sometimes erroneous.” It is extremely detailed for the introduction. I suggest reporting only the most interesting and essential elements that represent the prerequisite to establish the concept of your study. If the authors consider useful, they can find in this part elements supporting the discussions.
• “The pummelo cultivars in this study have multiple Eastern and Western origins, and can thus serve as a basis for comparison with few studies carried out on Asian pummelos.” Probably this sentence can be moved to M&M or moved to another part, but surely deleted from the actual position.

Results:

• Most of the results are sometimes confusing, with several errors.
• I propose to replace the titles of each section with more explanatory ones.
• It is quite difficult to follow the discussion if the supplementary materials are named wrong.
• “Other genotypes (WHICH ONE?) assimilated to grapefruit and pummelo species (ACCORDING TO WHAT?) are also close to grapefruit and orange (IN FIGURE 1?). To elucidate the genetic origin of these genotypes, which differ from pomelo and grapefruit, and to verify their taxonomy, we determined the origin of the alleles at each locus, defining their presence in each species.” To make the reading easier (the names of the accessions are uncommon), I suggest indicating with an asterisk (for example) the accession in the dendrogram.
• If authors write that “The Cuban cultivar is related to the citron and lemon group”, why in Table 1 it seems that it is related to pummelo and citron?
• Who are the “crosses” in the part below of the table 1?
• Probably the interpretation that I do of Figure 2 is wrong, because I don’t understand why the authors write that Cuban is made up by 50% of citron and 50% of pummelo. I see that Cuban variety 100% of yellow bar (citron) and more or less 80% of light blue (heterozygous pummelo). Or not? Similar doubts are related to the other varieties that the authors comment on.
• “The pummelo group is subdivided into 3 distinct subgroups (1, 2, and 3).” Make sure all three subgroups are correctly reported (one of the subgroups reported as 3 must be 1).
• “The most variable characters in grapefruit (C. paradisi) are fruit mass, number of seeds per fruit and pulp color (h), whereas in grapefruits all characters are variable except the number of embryos per seed and color indices.” Grapefruit and grapefruit?
• “All grapefruit cultivars (C. maxima)”. Grapefruit OR pummelo.
• “The , while the..” ????

Discussion:

• “Values of the proportion of markers with an allele common to the genotype whose origin we are trying to determine, of between 80 and 100%, could be the consequence of these characteristics of SRR markers and of the underestimation of the total allelic diversity of the group by too restricted varietal representation.” I suggest rewriting that, because it is long and difficult to comprehend.
• “In this case, the pummelo x mandarin combination as the origin of Bali is more likely than that of Alanoek, even with an equal proportion of markers supporting the two hypotheses (88%), as it relies on the possibility of varietal under-representation of the two parental species.” Please, rewrite also this one.
• “The genetic data do not support the sweet orange or sour orange parentage and they can discarded to the candidatest”. Candidatest for what?

Material and method

• “or assimilated to grapefruit or pummelo”. Are they the outgroups?
• “SSR genotyping”. Move as title
• “All the criteria measured are quantitative and are of interest to breeders and producers: size, shape, fruit mass, external and internal color, skin thickness, number of seeds per fruit, sugar content and acidity. We added two criteria which are the number of segments per fruit and the average number of embryos per seed. The first is a criterion used in the taxonomic classification of citrus [1] and the second characterizes a variety and is of interest to citrus seed curators. Usually juiciness (juice content) is used for marketing citrus fruits but this criterion was not considered because its method of evaluation is subject to experimenter variation.”. It doesn’t sound like M&M. Or reduce, or move to the discussion, to better explain the corresponding part.
• “The color is determined by three ….. [35]” is Please, delete.
• In the title of section 4.4 delete “analysis” because you reserved 4.5 to them. Report “analysis” in the title 4.5.2, similarly to the others.

Conclusion:

• “… for all pummelo and grapefruit accessions”. I suggest to include “under evaluation”.
• Which is a probable application of this study and in which way (as the authors reported in the abstract) “These results will be useful for breeding programs by selecting cultivars according to traits of interest”?

Figure:

• Reading Table S1 (I suppose it is “Citrus list”), if sour orange and sweet orange are just one accession for each one, why not report only the name, as for the rest? in addition to the fact that the use of plural is wrong. Moreover, I suggest making a circle also to indicate the grapefruit varieties. Diversely, the comment in the result section makes it difficult to follow.
• I suggest moving Figure 2 to the end of section 2.1.
• Figure 5: What is the meaning of the red arrows? And what of the red brackets?
• Figure 6: Why is the figure split in two? Limonene refers to? “Means other” refers to?
• Figure 7: What is the meaning of the varieties reported in green and yellow?
• Figure 9: The authors are encouraged to write a unique, reasonable title, and separate details in the two figures reported (not top and lower, but left and right – better if they are separated in a) and b)).

Supplementary files:

• First of all, I don’t find the captions of the supplementary files.
• They are numbered starting from M&M, even though the first supplemental document is cited from the results. So please, revise the numbering.
• It is unclear if the Supplemental document cited is a figure or a table, if the authors write Supplemental S1...
• In Table S1 some genotypes are reported in red. What are they?
• To uniform the supplemental figure 8, I suggest splitting it into two (instead of 8a and 8b), and in “8a”, put the name directly into the photo. Moreover, rename the caption adequately.

Author Response

We sincerely thank the reviewers for having accepted to evaluate our work and the manuscript and for having invested their time to correct and propose modifications to improve the quality of the text. You will find hereafter the answers to your questions, suggestions and remarks. We have asked MDPI to revise the English.

Reviewer 2

The manuscript is complex in the reading, but it sounds interesting. The authors are highly encouraged to revise many points, in the form (mislabellings – alos, tru, ans, othere, tottaly, déevelopped, speciesi, Marsh, uppercase/lowercase in the text and in the figure, … - the list is not exhaustive) and the substance, by reading carefully all the text, including supplementary files. It seems that the authors did not read the files before proceeding with the submission, considering the numerous inconsistencies. I suggest a revision by a mother tongue.

Abstract

• Comments 1: It is not essential to report the double identification of sweet orange and grapefruit (( sinensis or C. x aurantium var. sinensis) … (C. paradisi or C. x aurantium var. paradisi)). Choose one of them. For grapefruit, I suggest you use the same as you indicated in the title, and I encourage authors to also maintain it in the manuscript.

Response 1: We thought that by associating the former taxonomy with the new one (Ollitrault et al. 2020), our article would be better understood by readers used to the taxonomies of Tanaka or Swingle & Reece. As recommended by the reviewer, we have kept the most recent taxonomy. 

• Comments 2: Replace paradisi with grapefruit if it is not essential to cite the species (…and very low levels for the group of mutants (C. paradisi)). The same logic must be applied to C. maxima and pummelo. Not only in the text, but also in the captions.

Response 2: We have replaced the Latin species name with the common group name throughout the text and in figure captions wherever necessary.

• Comments 3:… no other compound is specific to a hybrid genotype: which one?

Response 3: For greater clarity, we have modified the sentence as follows: The 7 interspecific hybrids involving pummelo as one parent, show particular LEO profiles, but without specific compounds with the exception of p-cymenene present only in Wheeny.

• Comments 4: Once you have defined your acronyms, use them from the beginning to the end of your manuscript (LEO, PEO, ….)

Response 4: After the first citation in the text, we have replaced the terminology with the acronym

• Comments 5: It is unclear in which way the results can be useful for the breeding program.

Response 5: How can our results be useful for citrus breeding? Sexual crossing is the basis of many breeding programs. The degree of polyembryony is used in our programs by choosing as maternal parent a monoembryonic citrus fruit or with a low embryony rate (< 2) to give the hybrid a better chance of development. This trait was measured in our study. For all other traits, it is necessary to characterize the varieties/cultivars to know if they can be used as parents in a cross and transmit to their progeny a trait of agronomic interest.

Nevertheless, we have changed this last sentence of the abstract to emphasize the diversity of a taxonomic group whose representatives have been genetically attested:

“With genetic verification, the chemical and phenotypic diversity of the two species, pummelo and grapefruit, revealed in this study, can be used as a reference for behavior in a specific environment.”

Introduction:

• Comments 6: I suggest using “pummelo” instead of “pummelos” as incipit. And also along the text, I think that the use of the singular form sounds better, and, if deemed appropriate, the authors can use the form “pummelo varieties/accessions”. Moreover, the authors also use pomelos. Please, use just one form.

Response 6: We modified by removing pummelos and pomelos by pummelo

• Comments 7: Probably Citrus, after the first time, can be reported as C.?

Response 7: We agree and modified Citrus by C. after the first time

• Comments 8: The section between “The composition of citrus volatiles has been intensively investigated ….” and “…and the other is that varietal identification is sometimes erroneous.” It is extremely detailed for the introduction. I suggest reporting only the most interesting and essential elements that represent the prerequisite to establish the concept of your study. If the authors consider useful, they can find in this part elements supporting the discussions.

Response 8: We reduced this section by maintaining only the essential information to introduce our study.

“The composition of citrus volatiles has been intensively investigated and found to be mainly composed of monoterpenes and sesquiterpenes (Dugo et al. 2011). Pummelo leaf essential oils (LEO) often vary in composition according to variety. The proportion of major compounds are highly variable with b-pinene (10 to 46%), (E)-b-ocimene (5 to 31%), limonene (3 to 19%) and neral (less 1% to 15%)[20]. The grapefruit LEO composition can be roughly described as sabinene (50%), (E)-b-ocimene (10%), linalool (8%) and b-pinene (3%). Nevertheless, some studies mention also the punctual presence of γ-terpinene (up to 56.1%), β-pinene (up to 30.9%) and p-cymene (up to 12.5%) [21]. The composition of pummelo’s peel essential oil (PEO) varies drastically according to varieties and studies. Some authors describe a large dominance of limonene (81 to 96%), [24,25,26,27] while others observe lesser relative quantities (46 - 54%) [28], (15 - 48%) [29], or 64 % [30]. The composition of grapefruit PEO is less controversial as it is largely dominated by limonene, from 86% to 98% of total compounds [27, 31] Dugo et al. 2011.”

• Comments 9: “The pummelo cultivars in this study have multiple Eastern and Western origins, and can thus serve as a basis for comparison with few studies carried out on Asian pummelos.” Probably this sentence can be moved to M&M or moved to another part, but surely deleted from the actual position.

Response 9: We moved this sentence to the Material a& Method section

Results:

• Most of the results are sometimes confusing, with several errors.
• Comments 10: I propose to replace the titles of each section with more explanatory ones.

Response 10:  We added more explanatory sub-titles for each section

• Comments 11: It is quite difficult to follow the discussion if the supplementary materials are named wrong.

Response 11: It's true that the numbering of the additional files was not in the right order. We have corrected it.

• Comments 12: “Other genotypes (WHICH ONE?) assimilated to grapefruit and pummelo species (ACCORDING TO WHAT?) are also close to grapefruit and orange (IN FIGURE 1?). To elucidate the genetic origin of these genotypes, which differ from pomelo and grapefruit, and to verify their taxonomy, we determined the origin of the alleles at each locus, defining their presence in each species.” To make the reading easier (the names of the accessions are uncommon), I suggest indicating with an asterisk (for example) the accession in the dendrogram.

Response 12: To make the reading easier, we propose to modify the description of tree diversity as follow: 

“Analysis of genetic relationships based on genetic distances (Figure 1) highlights 3 main clusters corresponding to the 3 ancestral species (mandarin, pummelo and citron) made up of varieties with small genetic distances between them. The pummelo cluster comprises 20 genotypes or 21 varieties, since Eilat and Eingedi are synonyms of the same genotype. The two genotypes of orange and grapefruit (a batch of 18 accessions) are localized on the same sector of the genetic diversity tree in agreement with their phylogenetic relation. Eleven other genotypes are distributed between these taxonomic groups. Of these, Oroblanco (or Sweetie) is the result of a breeding program involving a pummelo and a grapefruit as parents. The 10 other genotypes have unknown origins and, contrary to their initial classification, can be considered neither pummelo nor grapefruit. These 10 genotypes are therefore considered as non-true types (NTT). The NTN genotypes corresponding to Yama, Menara, Triumph, Royal, Hog, Wheeny, Alanoek and Gold are linked to the orange and grapefruit branch, while Cuban is linked to the lemon and citron branch and Asahikan to the pummelo branch (Figure 1). “

We don't think reading would be made any easier by using asterisks or numbers to identify unknown varieties.  Nevertheless, since they are not real grapefruits or grapefruits, we have grouped them together as non-true-types varieties (NTT).

• Comments 13: If authors write that “The Cuban cultivar is related to the citron and lemon group”, why in Table 1 it seems that it is related to pummelo and citron?

Response 13: From the dendrogram of genetic diversity and relationships between accessions (Figure 1), the Cuban variety is linked (branched) to the citron and lemon branch, even though the point of attachment is quite distant from them (fairly high genetic distance of around 0.5 with the citron varieties).  Table 1 describes the percentage of common alleles between each hybrid of unknown origin and the genetic groups (or species). In the case of the Cuban variety, 95% of SSR loci have an allele in common with pummelo group and 93% with citron group (and only 76% with lemon’s alleles). Only the combination of a cross (pummelo x citron) offers a percentage of over 90% of loci able to reproduce the same genotype that of Cuban. This makes the genetic origin of Cuban the most plausible hypothesis. The question that could be asked is: why don't we have 100% values? The number of representatives of the two citrus species (pummelo and citron) is not exhaustive, and we don't claim to have all the parent varieties present in our varietal sample (this information is included in the discussion). The same type of analysis on genetic markers was carried out in the following references:

Curk Franck, Frédérique Ollitrault, Andres Garcia-Lor, François Luro, Luis Navarro and Patrick Ollitrault (2016) Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers Annals of Botany Doi:10.1093/aob/mcw005

Demarcq B., M. Cavailles, L. Lambert, C. Schippa, P. Ollitrault and F. Luro 2021. Characterization of Odor-Active Compounds of Ichang Lemon (Citrus wilsonii Tan.) and Identification of Its Genetic Interspecific Origin by DNA Genotyping. Journal of Agricultural and Food Chemistry, 69, 10, 3175-3188. https://doi.org/10.1021/acs.jafc.0c07894

Ferrer, V.; Costantino, G.; Paoli, M.; Paymal, N.; Quinton, C.; Ollitrault, P.; Tomi, F.; Luro, F.* 2021. Intercultivar Diversity of Sour Orange (Citrus aurantium L.) Based on Genetic Markers, Phenotypic Characteristics, Aromatic Compounds and Sensorial Analysis. Agronomy, 11 (6), 1084. https://doi.org/10.3390/agronomy11061084

• Comments 14: Who are the “crosses” in the part below of the table 1?

Response 14: Crossings are indicated in the lower part of the table by the names of the possible parental types: 'pum x orange' is a cross between a pummelo and an orange tree. We added in caption the description of the abbreviations ('pum' for pummelo 'grapef' for grapefruit and ‘mand’ for mandarin). The hypothesis is based on the presence in the taxonomic groups of the alleles of the non-true-types varieties, in the knowledge that the varieties representing each ancestral species are limited in number. We therefore make no claim to identify the parent varieties, but we can put forward a hypothesis as to their species membership.

• Comments 15: Probably the interpretation that I do of Figure 2 is wrong, because I don’t understand why the authors write that Cuban is made up by 50% of citron and 50% of pummelo. I see that Cuban variety 100% of yellow bar (citron) and more or less 80% of light blue (heterozygous pummelo). Or not? Similar doubts are related to the other varieties that the authors comment on.

Response 15: The reviewer's remark is pertinent, as the sum of the proportions of ancestral genomes is never equal to 100%, as sometimes markers have not amplified well and the origin of their alleles could not be identified with certainty.  We have therefore corrected the percentages by including markers of unknown origin as follow:

To verify the hypothesis of genetic origins and the identification of potential parental species, diagnostic SNP markers from the three ancestral species, C. medica, C. maxima and C. reticulata, were used to calculate the proportion of ancestral genomes and the frequency of homozygous and heterozygous alleles (Figure 2). The Cuban genome was constituted by 50% citron,45% pummelo and 5% citrus of unknown origin. The frequency of heterozygous markers of C. reticulata and C. maxima were high (100% and 90% respectively). This profile confirms the interspecific hybrid status of Cuban genotype detected with the SSR markers. The frequency of pummelo’s homozygous markers for Menara and Royal (44% and 38% respectively) was conform with the pummelo x sweet orange origin hypothesized by the SSR markers. With approximatively 48% of C. reticulata, 38% of C. maxima and 14% of citrus of unknown origin and a very high proportion of heterozygous markers (95%), Bali and Yama are probably direct hybrids between a mandarin and a pummelo. The genomes of Alanoek, Hog, Wheeny and Asahikan were constituted approximatively in the same proportion by mandarin and pummelo and share a significant proportion (15 - 20%) of C. reticulata and C. maxima homozygous markers. Due to the low frequency of homozygous markers, those profiles are not compatible with a pummelo x sweet orange origin and probably belonging from crosses between unknown interspecific (mandarin x pummelo) hybrids.

• Comments 16: “The pummelo group is subdivided into 3 distinct subgroups (1, 2, and 3).” Make sure all three subgroups are correctly reported (one of the subgroups reported as 3 must be 1).

Response 16: We have corrected the error and put the description of the 3 sub-groups in the right order.

• Comments 17: “The most variable characters in grapefruit (C. paradisi) are fruit mass, number of seeds per fruit and pulp color (h), whereas in grapefruits all characters are variable except the number of embryos per seed and color indices.” Grapefruit and grapefruit?

Response 17: We have replaced the second citation of “grapefruits” by “pummelo”: The most variable characters in grapefruit are fruit mass, number of seeds per fruit and pulp color (h), whereas in pummelo all characters are variable except the number of embryos per seed and color indices.”

• Comments 18: “All grapefruit cultivars (C. maxima)”. Grapefruit OR pummelo.

Response 18: We have changed the word “grapefruit” by “pummelo” and deleted (C. maxima)

• Comments 19: “The , while the..” ????

Response 19: It was corrected by suppressing “The ,”

Discussion:

• Comments 20: “Values of the proportion of markers with an allele common to the genotype whose origin we are trying to determine, of between 80 and 100%, could be the consequence of these characteristics of SRR markers and of the underestimation of the total allelic diversity of the group by too restricted varietal representation.” I suggest rewriting that, because it is long and difficult to comprehend.

Response 20: We propose replacing this incomprehensible sentence with the following text:

For these reasons, the frequency of markers with common alleles between ancestral species and non-true-type varieties is often less than 100%.

• Comments 21: “In this case, the pummelo x mandarin combination as the origin of Bali is more likely than that of Alanoek, even with an equal proportion of markers supporting the two hypotheses (88%), as it relies on the possibility of varietal under-representation of the two parental species.” Please, rewrite also this one.

Response 21: We propose the following text to replace this sentence and make the meaning more understandable

Despite an equal frequency (88%), the cross-breeding hypothesis at the origin of Bali is more likely than that of Alanoek. In fact, due to the under-representation of ancestral species diversity, the probability of a pummelo x mandarin cross at the origin of Bali, is still high. On the other hand, the pummelo x orange hypothesis at the origin of Alanoek is less likely, as there is no diversity between orange varieties with SSR markers. So, a single variety represents the allelic diversity of the entire orange group. For two SSR markers, Alanoek and orange had no alleles in common. We can therefore conclude that one of Alanoek's parents is a pummelo, but the other is not the orange, but an unknown citrus variety with a genotype close to that of the orange.

• Comments 22: “The genetic data do not support the sweet orange or sour orange parentage and they can discarded to the candidatest”. Candidatest for what?

Response 22: We have replaced the sentence with: “Unfortunately, the genetic data rules out the possibility that the orange and the sour orange are one of the parents.”

Material and method

• Comments 23: “or assimilated to grapefruit or pummelo”. Are they the outgroups?

Response 23: Yes, the assimilated to grapefruit or pummelo are finally the non-true-type varieties.

• Comments 24: “SSR genotyping”. Move as title

Response 24: Done

• Comments 25: “All the criteria measured are quantitative and are of interest to breeders and producers: size, shape, fruit mass, external and internal color, skin thickness, number of seeds per fruit, sugar content and acidity. We added two criteria which are the number of segments per fruit and the average number of embryos per seed. The first is a criterion used in the taxonomic classification of citrus [1] and the second characterizes a variety and is of interest to citrus seed curators. Usually juiciness (juice content) is used for marketing citrus fruits but this criterion was not considered because its method of evaluation is subject to experimenter variation.”. It doesn’t sound like M&M. Or reduce, or move to the discussion, to better explain the corresponding part.

Response 25: This part was removed

• Comments 26: “The color is determined by three ….. [35]” is Please, delete.

Response 26: OK

• Comments 27: In the title of section 4.4 delete “analysis” because you reserved 4.5 to them. Report “analysis” in the title 4.5.2, similarly to the others.

Response 27: Done

Conclusion:

• Comments 28: “… for all pummelo and grapefruit accessions”. I suggest to include “under evaluation”.

Response 28: Done

• Comments 29: Which is a probable application of this study and in which way (as the authors reported in the abstract) “These results will be useful for breeding programs by selecting cultivars according to traits of interest”?

Response 29: We completed the conclusion by putative applications in breeding programs and in perfume and cosmetic industry:

The results of a phenotypic characterization of genetic resources is an expectation of breeders, as it enables them to choose the varieties they will be able to combine by sexual crossing or somatic hybridization according to their traits and characteristics. The monoembryonic trait, for example, is useful for selecting maternal parents, as polyembryony is a handicap for obtaining offspring due to competition between embryos for germination and growth. The composition of essential oils is also of interest in the search for new aromatic profiles, and for identifying molecules that can be used as identifiers of varieties or of the environment in which the trees were grown (tracers of geographical origin).

Figure:

• Comments 30: Reading Table S1 (I suppose it is “Citrus list”), if sour orange and sweet orange are just one accession for each one, why not report only the name, as for the rest?

Response 30: We don’t report the varietal name of sour orange and sweet orange in the text and figures, because they represent all the cultivars of these two citrus groups. Indeed, they have evolved only by mutation selected by breeders, and so there is no SSR marker polymorphism between cultivars. Consequently, just one cultivar represents all these two groups/ species and its name no has importance to make a genetic relationship analysis. in addition to the fact that the use of plural is wrong. Moreover, I suggest making a circle also to indicate the grapefruit varieties. Diversely, the comment in the result section makes it difficult to follow.

• Comments 31: In addition to the fact that the use of plural is wrong.

Response 31: Not really wrong for the reason explained just before, but we removed the s for all the taxonomic groups

• Comments 32: Moreover, I suggest making a circle also to indicate the grapefruit varieties. Diversely, the comment in the result section makes it difficult to follow.

Response 32: We added the circle and we changed the comments of the figure to be easier to understand but the figure is sufficiently clear with few varieties for a reader to easily understand.

• Comments 33: I suggest moving Figure 2 to the end of section 2.1.

Response 33: OK

• Comments 34: Figure 5: What is the meaning of the red arrows? And what of the red brackets?

Response 34: The red arrows and brackets indicate the position of the non-true-type varieties supposed to be pummelo’s hybrids. This information was added to the figure caption

• Comments 35: Figure 6: Why is the figure split in two? Limonene refers to? “Means other” refers to?

Response 35: We split the figure in two parts by extracting Limonene proportions from the 6 majors PEO compounds to improve the visibility of percentage variations of less abundant compounds such as Germacrene-D or a-pinene. By removing limonene, the axis represents more precisely the small proportions.    

• Comments 36: Figure 7: What is the meaning of the varieties reported in green and yellow?

Response 36: We added in the figure caption this information about color of varietal names: in green, the name of the pummelo, in yellow, the name of the non-true-type varieties supposed to be pummelo hybrids

• Comments 37: Figure 9: The authors are encouraged to write a unique, reasonable title, and separate details in the two figures reported (not top and lower, but left and right – better if they are separated in a) and b)).

Response 37: The correction was done as suggested by the reviewer

Supplementary files:

• Comments 38: First of all, I don’t find the captions of the supplementary files.

Response 38: The caption of supplementary materials is listed just after the Conclusion

• Comments 39: They are numbered starting from M&M, even though the first supplemental document is cited from the results. So please, revise the numbering.

Response 39: We have modified the numbering of the supplementary materials

• Comments 40: It is unclear if the Supplemental document cited is a figure or a table, if the authors write Supplemental S1...

Response 40: Ok, we have added in the text the type of supplementary file

• Comments 41: In Table S1 some genotypes are reported in red. What are they?

Response 41: It was to indicate the non-true-type varieties, but finally we wrote them in black.

• Comments 42: To uniform the supplemental figure 8, I suggest splitting it into two (instead of 8a and 8b), and in “8a”, put the name directly into the photo. Moreover, rename the caption adequately.

Response 42: We modified the figure according to the suggestion of the reviewer

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I want to thank the authors for having revised the manuscript and improved the document.