Mechanisms and Candidate Genes for Seed and Fruit Set in Grapevine

Mechanisms and Candidate Genes for Seed and Fruit Set in Grapevine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Mechanisms and Candidate Genes for Seed and Fruit Set in Grapevine Laura Costantini, Paula Moreno-Sanz, Chinedu Charles Nwafor, Silvia Lorenzi, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-72371/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2021 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract Background : Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like cluster compactness, bunch and berry size, berry skin to pulp ratio or seedlessness. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. Results : We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on fruit and seed set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. A number of genes potentially involved in sporogenesis and gametogenesis showed contrasting expression between Corinto Nero and Sangiovese and five missense single nucleotide polymorphisms were identified from transcriptomic data. The above findings suggest that the seedless phenotype of Corinto Nero is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. Finally, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Conclusions : Our collective results suggest that parthenocarpy and stenospermocarpy are not restricted to Black Corinth (alias Korinthiaki) and Sultanina-derived cultivars. The single nucleotide polymorphisms identified between Sangiovese and its parthenocarpic variant Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype. Plant Molecular Biology and Genetics Plant Physiology and Morphology Vitis vinifera seedlessness somatic variation reproductive development flower berry fertilization parthenocarpy stenospermocarpy single-nucleotide polymorphism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Full Text Supplementary Files Additionalfile9.pdf Additionalfile8.pdf Additionalfile7.pdf Additionalfile6.pdf Additionalfile5.pdf Additionalfile4.pdf Additionalfile3.pdf Additionalfile2.pdf Additionalfile1.xlsx Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2021 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Major revision 26 Oct, 2020 Review # 1 received at journal 22 Sep, 2020 Review # 2 received at journal 22 Sep, 2020 Editor assigned by journal 15 Sep, 2020 Reviewers invited by journal 15 Sep, 2020 Reviewer # 1 agreed at journal 15 Sep, 2020 Reviewer # 2 agreed at journal 15 Sep, 2020 Submission checks completed at journal 14 Sep, 2020 Editor invited by journal 14 Sep, 2020 First submitted to journal 04 Sep, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-72371","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":2379819,"identity":"ad86e1ca-c1fc-4b8a-9189-8c73e08b1462","order_by":0,"name":"Laura 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Ricerche","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ivana","middleName":"","lastName":"Gribaudo","suffix":""},{"id":2379829,"identity":"be5ab3d0-2581-4799-b463-a6f25f7be6bd","order_by":10,"name":"Anna Schneider","email":"","orcid":"","institution":"Institute for Sustainable Plant Protection National Research Council: Istituto per la Protezione Sostenibile delle Piante Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Schneider","suffix":""},{"id":2379830,"identity":"a5213f9c-09ed-4802-9fd2-1235d8766319","order_by":11,"name":"Maria Stella Grando","email":"","orcid":"","institution":"University of Trento: Universita degli Studi di Trento","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Stella","lastName":"Grando","suffix":""}],"badges":[],"createdAt":"2020-09-04 11:45:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-72371/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-72371/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-021-02865-2","type":"published","date":"2021-03-13T15:00:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2478323,"identity":"0f1e818a-697a-467d-8ad6-a4e9da502486","added_by":"auto","created_at":"2020-09-18 15:16:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77611,"visible":true,"origin":"","legend":"Phenotyping of variant pairs upon open-pollination. Members of the same pair (or triplet) are depicted with the same color. For each accession, a mean value was calculated from different bunches, seasons and locations. Bars correspond to standard errors. Asterisks indicate significant (P \u003c 0.05) differences between seeded and seedless variant pairs, as established by one or more test(s) among T-Student test (or Welch test in the case of unequal variances), Mann-Whitney test, and Kolmogorov-Smirnov test. Different letters indicate significant differences in the whole set of accessions (Kruskal-Wallis test followed by Dunn’s post hoc test with Bonferroni correction for multiple tests, P \u003c 0.05). Berries with apparently normal seeds were considered as seeded, whereas berries containing only rudimental seeds, seed traces or unfertilized ovules were classified as seedless. Abbreviations: CN = Corinto Nero, TN = Termarina Nera, SG = Sangiovese, Asp = Aspirant-false, Lis = Liseiret, Mosc mt = Moscato Bianco mutant, Mosc wt = Moscato Bianco, Ter rosa = Termarina Rosa, Term = Termarone, Ch ap = Chasselas apyrène, Ch rose = Chasselas Rose, Sult = Sultanina, Dast = Dastatchine-false, CB = Corinto Bianco, PX = Pedro Ximenez, K = Corinthe Noir (reference for parthenocarpy).","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure1.jpg"},{"id":2478324,"identity":"d1b4d9a9-8ffe-4ea8-b9f3-fdaa216e8b52","added_by":"auto","created_at":"2020-09-18 15:16:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81470,"visible":true,"origin":"","legend":"Comparison between Moscato Bianco wild-type (A, C) and Moscato Bianco mutant (B, D) inflorescences and leaves. The inset in (B) shows a close-up of a «star» flower.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure2.jpg"},{"id":2478325,"identity":"dd074186-6ec2-425b-b7de-bb0523d73ffc","added_by":"auto","created_at":"2020-09-18 15:16:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56499,"visible":true,"origin":"","legend":"Bunch evaluation. Comparison between (A) Sangiovese and Corinto Nero (above and below, respectively), (B) Liseiret and Aspirant, (C) Moscato Bianco wild-type and mutant, (D) Termarone and Termarina Rosa, (E) Chasselas Rose and Chasselas apyrène, (F) Pedro Ximenez and Corinto Bianco clusters deriving from open-pollination. In each picture from B to F, the seeded cultivar is shown on the left, the seedless variant on the right.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure3.jpg"},{"id":2478326,"identity":"1f26a187-eb02-4ac5-a52b-7bf5ff6ea9ed","added_by":"auto","created_at":"2020-09-18 15:16:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37941,"visible":true,"origin":"","legend":"Berry evaluation. (A) Berry size and shape as evaluated with a digital caliper in 2017 and 2018 (for the pair Aspirant/Liseiret data were registered only in 2017). When more than 50 berries per bunch were available from one berry size category, pictures were taken from 50 berries; when there were less than 50 berries per bunch belonging to a size category, pictures were taken from all berries. The number of analyzed berries ranged from a minimum of 280 (Moscato Bianco mutant) to a maximum of 1137 (Corinto Nero). The 25-75 percent quartiles are shown with a box, the median with a horizontal line inside the box, the minimal and maximal values with short horizontal lines (“whiskers”). Asterisks indicate significant (P \u003c 0.05) differences between seeded and seedless variant pairs, as established by Mann-Whitney test. (B) Berry size as evaluated with an ad hoc aluminum sizer card in 2018. Abbreviations: CN = Corinto Nero, TN = Termarina Nera, SG = Sangiovese, Asp = Aspirant-false, Lis = Liseiret, Mosc mt = Moscato Bianco mutant, Mosc wt = Moscato Bianco, Ter rosa = Termarina Rosa, Term = Termarone.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure4.jpg"},{"id":2478327,"identity":"6d3f5658-411a-4d4f-899e-9c5dac7411d9","added_by":"auto","created_at":"2020-09-18 15:16:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54001,"visible":true,"origin":"","legend":"Seed evaluation. (A) Gradient of seed development observed in the accessions under study. Only normally developed seeds (as indicated by the arrow) were considered to estimate the percentage of seeded berries. They possess a normal testa (consisting of outer and inner integument), endosperm and embryo. The remaining structures are supposed to correspond to incomplete (“floater”) or rudimental seeds, seed traces and ovules. (B) On the left, sections of Corinto Nero berries (the rightmost berry contains a normal seed); on the right, some examples of traces extracted from the majority of Corinto Nero berries. (C) Sections of Aspirant-false berries. (D) Sections of “star-flower” Moscato Bianco berries. (E) A Chasselas apyrène berry. (F) A Sultanina berry. (G) Berries of Corinto Bianco. (H) A Corinthe Noir berry.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure5.jpg"},{"id":2478328,"identity":"655ae652-b913-499a-aa23-da468a527070","added_by":"auto","created_at":"2020-09-18 15:16:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59272,"visible":true,"origin":"","legend":"Relationship between berry size and presence of normal seeds in Sangiovese and its seedless variants. (A) Classification of berries according to size; the prevalent type of seeds or seed traces is shown at the bottom. (B) Representative berries from Corinto Nero (on the left) and Sangiovese (on the right). (C) Percentage distribution of berries according to size and seed content. The percentage of small, medium and large berries was calculated from the total number of berries per bunch, while the percentage of seeded berries was established on the total number of berries opened for seed examination (it was a representative portion of the total number of berries when this number was too big). Berries with apparently normal seeds were considered as seeded, whereas berries containing only rudimental seeds, seed traces or unfertilized ovules were classified as seedless. For each combination of accession, season and pollination treatment, from one to nine clusters were analyzed and an average value was calculated. Abbreviations: CN = Corinto Nero, TN = Termarina Nera, SG = Sangiovese in the Grinzane Cavour collection (Corinto Nero plants from two distinct parcels were analyzed in 2017); CN*, SG* = Corinto Nero, Sangiovese in the FEM collection, respectively.","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure6.jpg"},{"id":2478329,"identity":"0dedc117-3830-4ec9-bc12-639a4b656fc6","added_by":"auto","created_at":"2020-09-18 15:16:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":57821,"visible":true,"origin":"","legend":"Scatter plots of traces’ length against traces’ width for the reference cultivars for parthenocarpy and stenospermocarpy, Corinthe Noir and Sultanina, respectively (A), and for the other seedless accessions under investigation (B). Reported measures refer to traces extracted only from the smaller berries (with the exception of Sultanina having berries of a unique size). In (C) scatter plot of the length against the width of the ovules/traces of Sangiovese (SG) and Corinto Nero (CN) measured at six stages from flowering (stage 1) to pepper-corn size (stage 6), as detailed in Additional file 7: Figure S12. The intensity in the color filling the diamonds/dots increases with the stages. Ovules from stages 1 and 2 of Corinto Nero could not be measured because they were destroyed during extraction from the ovary due to their reduced size and fragility.","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure7.jpg"},{"id":2478330,"identity":"fc4c51d7-6a30-47aa-a34e-4c536902a739","added_by":"auto","created_at":"2020-09-18 15:16:45","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":40856,"visible":true,"origin":"","legend":"Evaluation of pollen functionality and morphology. (A) Pictures of some Sangiovese, Corinto Nero, Pedro Ximenez and Corinto Bianco pollen grains subjected to the viability (on the left) and germination (on the right) in vitro tests, as observed at the microscope (200X). (B) Mean values (± standard error) of pollen viability and germination percentage per accession; N is the number of replicates. The total number of observed pollen grains per accession ranged from a minimum of 1040 to a maximum of 4528, in relation to the available inflorescences. To detect differences between each seeded variety and its seedless variant, the non-parametric Kolmogorov-Smirnov test has been performed. (C) Box plots representing the polar and equatorial axis lengths measured on fifty randomly selected pollen grains for each genotype in each season.","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure8.jpg"},{"id":2478331,"identity":"cd5b9dea-09bb-49b4-ad5e-c84e27d51906","added_by":"auto","created_at":"2020-09-18 15:16:45","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":166439,"visible":true,"origin":"","legend":"Clusters (A), seeds and traces (B) derived from open-pollination (control), self-pollination (self) and emasculation. Abbreviations: CN = Corinto Nero, SG = Sangiovese, EMS+ST = emasculated (without stigma removal), SP = self-pollinated, stage I = stage E-L 15, stage II = stage E-L 18 of the modified Eichhorn-Lorenz scheme [226]. Red arrows indicate apparently normal seeds among several rudimental seeds. (C) Seedlings derived from occasional normal seeds extracted from emasculated bunches of Gamay (on the left) and Sangiovese (on the right). Pictures were taken 75 days after sowing.","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1/Figure9.jpg"},{"id":13532784,"identity":"4183f189-2f1a-4078-8525-7562b53ed000","added_by":"auto","created_at":"2021-09-17 01:18:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1196266,"visible":true,"origin":"","legend":"","description":"","filename":"Maintext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-72371/v1_covered.pdf"},{"id":2478332,"identity":"4577d4c9-3a2d-4aa3-8b20-7c5e5cbf0ea8","added_by":"auto","created_at":"2020-09-18 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PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vitis vinifera, seedlessness, somatic variation, reproductive development, flower, berry, fertilization, parthenocarpy, stenospermocarpy, single-nucleotide polymorphism","lastPublishedDoi":"10.21203/rs.3.rs-72371/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-72371/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like cluster compactness, bunch and berry size, berry skin to pulp ratio or seedlessness. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on fruit and seed set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. A number of genes potentially involved in sporogenesis and gametogenesis showed contrasting expression between Corinto Nero and Sangiovese and five missense single nucleotide polymorphisms were identified from transcriptomic data. The above findings suggest that the seedless phenotype of Corinto Nero is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. Finally, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our collective results suggest that parthenocarpy and stenospermocarpy are not restricted to Black Corinth (alias Korinthiaki) and Sultanina-derived cultivars. The single nucleotide polymorphisms identified between Sangiovese and its parthenocarpic variant Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.\u003c/p\u003e","manuscriptTitle":"Mechanisms and Candidate Genes for Seed and Fruit Set in Grapevine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-18 15:16:14","doi":"10.21203/rs.3.rs-72371/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-10-26T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-09-22T12:00:00+00:00","index":1,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nManuscript by Costantini et al. approaches the analyses of eight grapevine somatic variants affected in seed formation in parallel to their original varieties. Although a few of these variants have previously been analysed in other reports, the manuscript contains potentially interesting new information on their comparative phenotypic analysis from gamete viability to bunch features. The work also includes molecular analyses of the parthenocarpic phenotype of Corinto Nero, a variant of cultivar Sangiovese, mostly based on previous published transcriptomic data of the same authors. Part of these results can be considered purely speculative and do not contribute to develop a clear molecular hypothesis on the origin of the observed phenotype. The manuscript is too long with multiple tables and figures both in the main text and as supplementary materials. The information provided by some of them is not relevant and they could be deleted. In case of publication the manuscript could require a deep reorganization and reduction.\nGeneral comments\nAs a general comment, the text is inflated with explanations and references that in many instances could be reduced. Almost any section would need to be summarized and reduced to the essential information, deleting or simplifying accessory information. The organization of the manuscript could be improved As mentioned below, a few experiments with dubious results could be deleted.\nOne of the complexities of the phenotypic analyses is that genetic materials are analysed in two different locations and in several years that are not the same for all the traits. Different traits in different genotypes are analysed in different years and the number of year repetitions can be different for different traits and genotypes. This is better explained in supplementary tables but not always in the main text. Some traits like fruit set or berry weight can display important environmental interactions and it is not recommended deriving conclusions from a single year of analyses.\nSpecific comments\nTitle:\nThe title is too ambitious since the manuscript does not approach the mechanisms and genes involved in seed development and fruit set. Only one gene (VviAGL11) and one possible mechanism (gamete non-reduction) contributing to variation in these processes are convincingly shown. A more adequate title could be \"Somatic variants for seed and fruit set in grapevine\"\nAbstract:\nThe first conclusion is not correct, since other parthenocarpic variants, as Corinto Nero and Corinto Bianco, and stenospermocarpy variants, as Chasselas Apyrene, have at least been described in grapevine at different levels.\nResults:\nAll results sections need to start with a short description of the specific goal pursued followed by a justification of the experiments performed before the description of the results and the final short conclusion obtained.\nGenotype of variant pairs. Needs introductory sentence. Can be summarized. Why distinguishing two subsections? SSRs and SNPs can be included together since their results are the same. Figure S1 could not be required, since a comment in the text can be enough. If potentially different SNPs are not confirmed in any case what is the use of Table S2?\nPhenotype of variant pairs. Needs an introduction explaining the purpose and justifying the analysed traits.\nIt is not clear to me what is the purpose of checking whether the distributions of the traits under open pollination or self-pollination are normal. Why expecting normality in traits distribution in a sample of seven selected cultivars and their seed-formation variants? Figure S2 is not useful.\nThe most relevant trait to be analysed in these variants is seed formation. Thus, seed formation should be the first trait to be considered. The level of seed formation can impact on fruit set (or nor) and berry size and those impact on bunch development, bunch weight, size and density.\nFlower number estimation using VitisFlower could be shortly commented in the Methods section in place of the Results.\nDistinction between ovules and seed traces should be clearly established for the variants along the text.\nPlease include in the text information on the years analysed for each trait. In the case of fruit set there is only one year of analyses and this will condition the conclusions. Explain.\nMechanisms responsible for the seedless phenotype\nSanitary status description does not clarify whether viruses affect all plants used for each accession or in different locations. Their distribution could be commented to make it more clear. Table S8 is not very explicative. This section could go within Materials.\nThe main purpose of self and cross pollination studies is providing further evidence on pollen viability as well as primary information on embryo sac viability. I do not see too much interest on analysing berry and bunch traits in self and cross pollinations, given the interference that the emasculation process and the bagged conditions produce on bunch development. These experiments could be eliminated.\nIt is a pity that experiments on male gamete viability were not performed for all variants and experiments on female gamete viability and the potential causes of gamete non-functionality were only conducted in Corinto Nero. It would be interesting to analyse whether Sangiovese and Corinto Nero plants used in the ploidy level experiments could be chimeric for ploidy.\nGenetic basis of the seedless phenotype\nThe section could be better titled as: Molecular basis…since there are no genetic experiment.\nThe section is not clearly described. It needs a short introduction of the hypotheses and justification of the experiments performed in every subsection.\nTable S12 could be dispensable since the information in the table is already given in the text.\nVviAGL11 expression experiment shows different expression levels in the variety and the seedless variant. The experiment is performed on berry RNA and measures the expression of this gene only expressed in the seed exocarp normalized with respect to other genes likely expressed in the whole berry. Previous reports showed that the described point mutation does not alter its expression. If analysed in seeds or seed traces their no differences between seeded and seedless genotypes. Thus, this experiment can be confusing and could be deleted together with Figure S16\nValidated SNPs between Sangiovese and Corinto Nero. The most relevant mutations are those present in Corinto Nero but not in Sangiovese and it would be interesting to find whether those mutations affect conserved sites or domains in the same protein from other species or they correspond to variable sites in the proteins. Predicted impact value on protein function could be estimated with PROVEAN application.\nDifferentially expressed genes (DEG) between Sangiovese and Corinto Nero. This section revisits the data published by the authors in a previous paper on transcriptional analysis comparison between Sangiovese and Corinto Nero. In this case, the goal seems to be the identification of potential candidate genes for the Corinto Nero variant phenotype based on DEGs. Depending on the flower organs and developmental stage analysed it can be very difficult to distinguish DEGs that are causal to the phenotype from DEGs showing the consequences of a previous mutation. In my opinion this is not a useful exercise, finding more than 220 candidates is like not having a single one. This section is not useful for the manuscript and could be deleted.\nDiscussion\nDiscussion is too long and should be reduced. Apart from some interesting comments it contains non-relevant information as well as more speculative sections.\nI think the first sentence is misleading in the same sense as the title of the manuscript (see above). The goal of the work cannot be clarifying the mechanisms underpinning seed and fruit development but just understanding the basis of some of the variation affecting these traits in grapes. The mechanisms are much more complex and involve much more genes and most of them do not show variation.\nInformation on the different varieties and their variants (pages 22-25) cannot be considered as real discussion. This information could be briefly given in the introduction or in the materials section, explaining what is relevant for understanding and interpreting the results.\nSection on the use of molecular variants to differentiate somatic variants is irrelevant for this work and has been treated with ad hoc experiments in many other reports.\nNature of the reproductive structure traces observed in the seedless genotypes (pages 26-27) is something that should be made clear in the results section. This paragraph in the discussion does not make sense.\nSection of the effect of seed content on bunch/berry features is mostly confirmatory of what has been analysed in many previous works and it could also be reduced.\nSection on the occurrence of berry set after emasculation has several interesting considerations on the ability to set fruit in the absence of fertilization. It could be un intrinsic property of grapevine but could also exist natural variation for parthenocarpic development of the fruit. Unfortunately, there is no confirmatory experiments to distinguish these hypotheses. On the other hand, the simplest and likely hypothesis for seed formation in emasculated bagged flowers is the existence of some mature pollen traits left within the bag.\nSection on mechanisms responsible for the seedless phenotype is unclear and also too long. It does not make sense to mention again the problems with sanitary status and it is not clear how star flowers would affect seed formation in Moscato Bianco. On the other hand, the cases of Sultanina and Aspirant are widely described in the literature in the first case and derive from the phenotype and the detection of the same mutation in the second one. It is not commented but it is very curious finding the same mutation in the same gene, what could suggest that a very specific mutation is required for stenospermocarpy based on VviAGL11 gene. Other sources of this type of seedlessness should exist since both Chasselas Apyrene and Iordan do not carry that mutation, although other mutations in the same gene have not been discarded in this work. The rest of the section should be dedicated to the putative parthenocarpic variants. Corinto Nero and Corinto Bianco seem to be those with more information from previous experiments and from this work. Just mention what experiments support the functional problem of embryo sac and pollen grains in them and in the Moscato Bianco and Termarina. It is likely that, if both male and female gametes are affected, a single mutation could be responsible for both effects.\nSection on potential causes of gamete non-functionality is too speculative when discussing possible altered genes. There is no evidence from the analysed variant sequences. This section should be strongly reduced.\nSection on genes possibly underlying the seedless phenotype is again two long and too much subdivided. Discussion on VviAGL11 expression does not make sense, as mentioned above. Section on the genes with validated SNPs could be focused on those SNPs that are specific of Corinto Nero with more possibilities of causing the phenotype. Could you explain how the same variant in VIT_11s0016g05820 could produce a parthenocarpy in Corinto Nero associated with gamete reduction and stenospermocarpy in Chasselas Apyrene associated to seed exocarp development?\nSection on DEGs is fully speculative and could be deleted.\nMethods\nDescription of the phenotyping experiments do not always indicate the years and the locations used for the sampling. In general, the phenotyping experiments are not systematic in terms of number of plants used, number of bunches or berry number and description of experiments is not clear cut. In some cases, the number of samples is too low (below 10) and in other cases the analyses have only been performed one year. This makes difficult to obtain the same values in repeated experiments.\nThe evaluation of sanitary status should also confirm whether all the plants used in the experiments were analysed because they could show differential infection profiles within the same genotype.\n\nFigures and tables\nFigure 2 is not relevant and could be deleted\nFigure 3 should improve naming and scales in all the plates\nFigure 4B does not make sense\nFigure 5 is very heterogeneous in terms of pictures and scales. Variety names could be included in the figure plates.\nFigure 6A and B should be improved in terms of scales. Figure 6B does not seem of enough quality and the Corinto Nero berry could be improved.\nFigure 9C could be deleted.\nSupplementary information\nTable S2. Does not make sense when none of the putative variants are confirmed\nTable S8. Should indicate the number of plants analysed\nTable S12. The results could easily be mentioned in the text since the table is not very informative\nFigure S1. Not really required\nFigure S2. It does not make too much sense for the number of genotypes and variants analysed\nFigure S16. These results are not consistent given the use of whole berries for the RNA extraction\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **No**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **No**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **No**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorInvitedReview","content":"","date":"2020-09-22T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reject\nForm responses:\n---\n\nComments to Author:\n---\nManuscript: PBIO-D-20-00972\n\"Mechanisms and candidate genes for seed and fruit set in grapevine\" by Constantini et al.\nThe length of the manuscripts is much over the reasonable limits of a research paper:\n- Main text (references excluded): 51 pages\nBackground pag3 to 8 (6 pages); Results page 8 to 21 (13 pages); Discussion page 21 to page 43 (22 pages); Conclusions: page 43; Methods page 44 to 54 (10 pages).\n- Number of references: 240\n- Supplementary files: 14 tables and 16 supplemental figures.\nAll the sections are unnecessary long and make difficult the reading and the evaluation of the manuscript. In particular, the results section is very descriptive and the huge amounts of figures and supplementary data do not help to extract the relevant information.\nSingle figures occupied several pages instead of been in a single page format.\nAs a summary, the results are mostly descriptive and do not provide relevant information in the molecular mechanisms or genes involved in seed and fruit set in grapevine.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. 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Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **No**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **No**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **No**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorAssigned","content":"","date":"2020-09-15T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-09-15T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-09-15T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-09-15T12:00:00+00:00","index":2,"fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-14T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-14T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-09-04T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0d0ccda-28a9-4b0c-acb7-01983ae91d16","owner":[],"postedDate":"September 18th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":536058,"name":"Plant Molecular Biology and Genetics"},{"id":536059,"name":"Plant Physiology and Morphology"}],"tags":[],"updatedAt":"2021-03-14T15:00:47+00:00","versionOfRecord":{"articleIdentity":"rs-72371","link":"https://doi.org/10.1186/s12870-021-02865-2","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2021-03-13 15:00:29","publishedOnDateReadable":"March 13th, 2021"},"versionCreatedAt":"2020-09-18 15:16:14","video":"","vorDoi":"10.1186/s12870-021-02865-2","vorDoiUrl":"https://doi.org/10.1186/s12870-021-02865-2","workflowStages":[]},"version":"v1","identity":"rs-72371","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-72371","identity":"rs-72371","version":["v1"]},"buildId":"wLkW0s4AflPzk-lpfg-fK","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}