Carolina Royo

Rafael Torres-Pérez

Nuria Mauri

Nieves Diestro

José Antonio Cabezas

Cécile Marchal

Thierry Lacombe

Javier Ibanez

Manuel Tornel

Juan Carreño

Javier Ibáñez

José Miguel Martínez-Zapater

Pablo Carbonell-Bejerano

Chin Cs

Sedlazeck Fj

Concepcion Gt

O ' Malley

Cramer Gr

Delledonne M Luo

The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The reduction of seed content without altering fruit size is a major breeding goal in many fruit crops because it eases fruit eating, which increases attractiveness for consumers and improves the suitability of fruits for the food processing industry (Varoquaux et al., 2000). For centuries, seedlessness has been one of the most prized quality traits in grapevine (Vitis vinifera) berries intended for direct consumption either as fresh fruit or raisins (Ledbetter and Ramming, 1989). World demand for seedless grapes is rising and vine-growers are increasingly asking for seedless cultivars (FAO and OIV, 2016). Understanding the genetic control of seedlessness is therefore paramount to boost the success of table grape breeding and fulfill such demands.

Although seeds are the sexual propagules of angiosperm plants, they are not required for the propagation of many woody crops such as grapevine that are vegetatively multiplied. This propagation method enables the establishment of new cultivars of seedless somatic variants that appeared spontaneously along the history of grapevine domestication (This et al., 2006). Seedless grape variants can be classified into two major groups depending on the type of seedlessness (Stout, 1936;[START_REF] Pratt | Reproductive Anatomy in Cultivated Grapes -A Review[END_REF]): (i) Stenospermocarpy, in which fertilization and embryo development take place, but seed development is prematurely aborted (Stout, 1936;Ledbetter and Ramming, 1989;Kovaleva et al., 1997); (ii) Parthenocarpy, in which fruits develop in the absence of fertilization yielding small berries that completely lack seeds, which has recently been related with impaired meiosis [START_REF] Royo | Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco[END_REF] Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 5 size is less compromised, likely due to the presence of seminal rudiments or seed traces that promote fruit growth (Stout, 1936;[START_REF] Nitsch | Natural Growth Substances in Concord and Concord Seedless grapes in Relation to Berry Development[END_REF][START_REF] Pratt | Reproductive Anatomy in Cultivated Grapes -A Review[END_REF].

A stable stenospermocarpy phenotype is shown by a few ancient oriental grapevine cultivars known as 'Kishmish' and derived varieties. They include the white-berried 'Kishmish', also known as 'Sultanina' or 'Thompson Seedless' (Dangl et al., 2001), which has been the major source of seedlessness in table grape breeding programs (Adam-Blondon et al., 2001;Ibáñez et al., 2009;Ibáñez et al., 2015). Studies of stenospermocarpy have therefore focused on 'Sultanina' and 'Sultanina'-derived cultivars, which produce soft and often imperceptible seed traces generally lacking seed coat lignification. While the embryo usually remains viable, the degree of endosperm degeneration detected from 3-4 weeks after flowering (WAF) and the final size of seed traces are variable, depending on the genetic background (Pearson, 1932;Stout, 1936;Barritt, 1970;[START_REF] Pratt | Reproductive Anatomy in Cultivated Grapes -A Review[END_REF]Striem et al., 1992;[START_REF] Wang | Evolutionary and expression analysis of a MADS-box gene superfamily involved in ovule development of seeded and seedless grapevines[END_REF][START_REF] Wang | Transcriptome analyses of seed development in grape hybrids reveals a possible mechanism influencing seed size[END_REF]. Stenospermocarpy in 'Sultanina' has been associated with defects in the development of maternal seed coat tissues (Malabarba et al., 2017).

Specifically, endotesta growth and lignification does not take place in seed traces. Abnormal development of the precursor inner ovule integument has also been reported in 'Sultanina' at earlier developmental stages (Pearson, 1932).

Concerning the genetic control of 'Sultanina'-derived seedlessness, different hypotheses were initially proposed depending on the approaches used to measure the trait and the genetic backgrounds analyzed (Bouquet and Danglot, 1996). However, a systematic analysis in several F 1 cross progenies shows that a model involving three independent recessive loci regulated by a dominant www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 6 locus could explain most segregations (Bouquet and Danglot, 1996). This dominant locus was later named SEED DEVELOPMENT INHIBITOR (SDI) (Lahogue et al., 1998). Different quantitative genetic studies located the SDI quantitative trait locus (QTL) on linkage group (LG) 18, explaining up to 70% of the phenotypic variance in seed content parameters (Cabezas et al., 2006;Mejia et al., 2007;Costantini et al., 2008;[START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Doligez et al., 2013).

Based on genetic linkage and putative homology, grapevine AGAMOUS-LIKE 11 (VviAGL11= AGAMOUS-LIKE 3, VviAG3= MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUMRESPONSE FACTOR 5, VvMADS5) was proposed as the SDI candidate gene in the absence of information for other genes in the region (Costantini et al., 2008;[START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]. This assumption was done considering its homology to the MADS-box gene AGL11, also known as SEEDSTICK (STK, At4g09960), which controls ovule morphogenesis and seed coat differentiation in Arabidopsis (Arabidopsis thaliana) [START_REF] Pinyopich | Assessing the redundancy of MADS-box genes during carpel and ovule development[END_REF][START_REF] Mizzotti | SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat[END_REF]. A role in seed morphogenesis was also shown for grapevine VviAGL11 homolog proteins (Malabarba et al., 2017). Molecular analyses identified two non-silent single nucleotide polymorphisms (SNPs) in the seedless mutant haplotype (sdi+) of VviAGL11 [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Malabarba et al., 2017). However, both amino acid substitutions were detected in homozygosity in the seeded wine cultivar 'Asyl Kara', a result that excluded them as functional dominant polymorphisms causing seedlessness (Mejía et al., 2011). Alternatively, the reduced expression of VviAGL11 in fruits of seedless sdi+ individuals compared to fruits or seeds of seeded individuals was related with the origin of seedlessness (Mejia et al., www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 7 2011; [START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF]Malabarba et al., 2017). Following this hypothesis, sequence polymorphisms observed in non-coding regions of the seedless allele of VviAGL11 were proposed as putative mutations causing misexpression and seedlessness [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Di Genova et al., 2014). In a later effort to confirm the misexpression hypothesis, [START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF] related several putative gene conversion events in the promoter region of VviAGL11 with reversion of stenospermocarpy in somatic variants of 'Sultanina' that produce regular seeds. However, these studies did not assess the question on whether the observed expression differences in VviAGL11 are causes or consequences of the seedless syndrome. The misexpression hypothesis also has limitations in explaining the dominant nature of the sdi mutation. In addition, the fact that VviAGL11 expression is decreased in seed tissues has not consistently been proven [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF][START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF]Malabarba et al., 2017).

Here we have reassessed the genetic and molecular origin of the 'Sultanina'derived seedlessness in an independent unbiased study. We used large F 1 crosses of table grape cultivars segregating for stenospermocarpy to delimit the location of the causal mutation through crossover mapping. To identify putative misexpression or coding mutations within the delimited interval, we followed a strategy that combined RNA-seq comparisons in F 1 hybrids and targeted sequencing in a large collection of seeded and stenospermocarpic grapevine cultivars. The results clearly point to a single nucleotide missense mutation in VviAGL11 as the origin of the dominant seedless phenotype.

Results

QTL mapping of seed content variation

Since we used the bred cultivar CS as the seedlessness donor [START_REF] Ramming | Crimson Seedless': A New Late-maturing, Red Seedless Grape[END_REF] in our study, we wanted to confirm the effect of the major SDI QTL in this genetic background. For this purpose we used a F 1 mapping population derived from a RG ('Red Globe') by CS cross hybridization. As an indicator of seed lignification that is useful to discriminate stenospermocarpy (Bouquet and Danglot, 1996), we analyzed the variation in seed dry weight per berry (SDW).

Both progenitors showed extreme divergence in their seed content (Fig. 1). RG berries had an average of 3.2±0.2 fully developed seeds with a mean SDW of 33.2±9.3 mg, while CS berries had an average of 2.2±0.7 seed traces with a mean SDW of 0.43±0.2 mg (Supplemental Table 1). SDW distribution in F 1 progeny was asymmetric and bimodal and did not fit a normal distribution in any of the analyzed years (Fig. 1 and Supplemental Table 1). Values for SDW were highly correlated over the three seasons analyzed (0.86<r<0.93) in agreement with a high broad sense heritability (0.80-0.91) of the trait.

To map the QTLs responsible for SDW in RG×CS progeny, we built a total of three linkage maps corresponding to each progenitor as well as a consensus map for the cross. Linkage maps comprised both simple sequence repeat (SSR) and SNP markers up to a total of 191 markers in the case of RG, 227 for CS and 290 for the consensus map. QTLs for SDW were analyzed in every linkage map and every season. The results confirmed the detection of a major QTL for seedlessness in LG 18 linked to the microsatellite marker VMC7F2 both in the CS and the consensus maps (Table 1). This QTL was consistently identified in the three years with a very high Logarithm of Odds (LOD) value and (Doligez et al., 2002;Cabezas et al., 2006;Mejia et al., 2007;Costantini et al., 2008;[START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Doligez et al., 2013). Another QTL detected in all the analyzed seasons for SDW was located on LG 2 (Table 1). Rather than contributing to stenospermocarpy, the genotype in this QTL marker (VVIB23)

correlates with the variation for seed number per berry (Supplemental Table 1)

and likely co-localizes with the sex locus as has been described previously (Costantini et al., 2008;Battilana et al., 2013;Doligez et al., 2013). Finally, two other minor QTLs for SDW were located on LGs 5 and 14, but they showed environmental interactions since they were not detected in every season (Table 1).

Fine mapping of the sdi mutation

A genetic fine mapping strategy was developed to delimit the location of the mutation that underlies the SDI QTL. For every RG×CS F 1 individual, the seedlessness phenotype de visu-determined during at least three years was fully linked to the genotype in the VMC7F2 marker, with no recombinant individuals or false detection observed (Supplemental Table 1). Every F 1 individual inheriting the sdi+ seedless allele (198-bp) or the sdi-seeded allele (200-bp) from the CS progenitor showed a seedless or seeded phenotype, respectively, confirming the dominant nature of the sdi mutation (Lahogue et al., 1998;Cabezas et al., 2006;Karaagac et al., 2012;Bergamini et al., 2013). This been remapped between the VVIN16 and SNP-25.16 markers (Canaguier et al., 2017). A total of 29 recombinants were detected, 14 and 15 of them corresponding to recombination in upstream and downstream sequences relative to VMC7F2, respectively (Fig. 2). Additional inner markers were genotyped to map the meiotic recombination breakpoint in these individuals. In this manner, the sdi mutation was delimited to an interval of 1.69 Mb (chr18:25,246,936,376 positions in the 12X.0 reference genome)

flanked by markers SNP-25.24 and SNP-26.93 (Fig. 2 and Supplemental Table 2). Supporting its continuity in the grapevine reference genome, the delimited mapping interval remained intact in the re-assembled 12X.v2 version (Canaguier et al., 2017).

To further delimit the location of the sdi mutation, additional recombinants within the interval delimited in RG×CS progeny were searched for in another seeded × seedless F 1 mapping population derived from an 'Imperial Napoleon' (IN) by CS cross. Inheritance of seedlessness was fully linked to the presence of the sdi+ allele for the CAPS-26.88 marker in this mapping population, which co-localizes with VviAGL11 (VIT_18s0041g01880), in close proximity to the VMC7F2 marker (Fig. 2). The IN×CS progeny was screened using markers (Fig. 2 and Supplemental (http://genomes.cribi.unipd.it/), the delimited SDI locus comprises 14 genes, although the two genes at each extreme were only partially included (Fig. 2).

These 14 genes were considered as SDI QTL candidates in subsequent analyses.

Transcriptome analysis reveals the activation of salicylic acid (SA)dependent defenses along with the repression of seed morphogenesis during stenospermocarpic seed abortion

Transcriptome comparisons were addressed to understand the processes involved in stenospermocarpic seed abortion and to search for candidate mutations underlying the SDI locus. To these aims, RNA-seq was used to compare seedless and seeded RG×CS F 1 individuals. To minimize genetic background effects, different F 1 individuals were analyzed as independent biological replicates. Individuals were selected to contrast in the allele of the SDI locus inherited from CS (sdi+, seedless or sdi-, seeded; Supplemental Table 1). From pea size fruits collected at 4 WAF, seeds or seed traces were extracted to specifically analyze gene expression in the affected organ. In this manner, we identified 2,888 differentially expressed genes (DEGs) with adjusted p-value ≤0.05 in edgeR and ≥2-fold change, most of which (76%) were upregulated in seed traces of seedless F 1 progeny (Fig. 3A, and Supplemental

Table 3). To understand the biological meaning underlying these differences, a functional enrichment analysis was carried out. Remarkably, several pathogen response-related functions were over-represented among genes upregulated in seed traces, which coincided with the activation of senescence/catabolism processes (Fig. 3B and Supplemental Nevertheless, 55% of these DEGs coincided with 4 WAF DEGs and seedlessupregulated genes predominated this dataset as well (86.6% of DEGs identified at 2 WAF), showing a consistency between the two experiments in spite of ontogeny, tissue and inter-annual differences (Fig. 3A and Supplemental Table 3). Also in agreement with 4 WAF results, genes downregulated in seedless fruits at 2 WAF were over-represented in seed differentiation related functions such as 'lignin metabolism' (Fig. 3B and Supplemental increase in SA levels was detected in seed traces when sdi+ and sdi-RG×CS F 1 individuals were compared (Fig. 3C), suggesting a role of SA in sdi-mediated seed abortion. A greater difference (33-fold) was observed between the seedless progenitor CS, with small seed traces, and RG, with big seeds. When the active gibberellins GA 1 and GA 4 were measured, no significant difference was observed in F 1 progeny (Fig. 3C), suggesting a minor contribution of this phytohormone to stenospermocarpy, at least in the studied developmental stage.

Stenospermocarpy does not associate with misexpression mutations

To identify candidate misexpression mutations, we inspected the transcriptome dataset for the presence of DEGs within the SDI fine mapping interval.

Supporting the continuity of this interval in the grapevine reference genome (Jaillon et al., 2007), we found that it is almost fully covered by one contig (000279F) in the genome assembly that was independently produced in the 'Cabernet-Sauvignon' cultivar (Chin et al., 2016). In addition, the same gene models are annotated in that interval of the reference genome according to V1 and V2 gene predictions (Vitulo et al., 2014) and no additional missing gene was supported by our RNA-seq read alignments (PRJNA418130 in NCBI Sequence Read Archive). While no DEG was detected in the RNA-seq assay,

VviAGL11 was the only one out of the 14 genes within the interval that was differentially expressed in the microarrays of whole berries 2 WAF, showing a 3.4-fold repression in sdi+ fruits (Supplemental Considering the mean allelic frequency at heterozygous SNP positions (Fig. 4A), the expression between sdi+ and sdi-alleles was balanced in the seedless RG×CS F 1 individuals analyzed (Fig. 4B). In fact, the seedless individual 669-60

showed ca 50% more counts for the sdi+ allele (Fig. 4A), which indicates that no cis-acting regulatory mutation inhibiting the expression of VviAGL11 was present in the sdi+ haplotype. Similar balanced expression between the two sdi-VviAGL11 alleles was observed in seeded F 1 individuals (Fig. 4C). In agreement with RNA-seq results, the absence of allelic imbalance in VviAGL11

was confirmed when allele-specific primers were used to compare the expression between the two alleles in 'Sultanina' seed traces at fruit set stage 18 by reverse transcription quantitative PCR (RT-qPCR) (sdi+/sdi-allelic expression ratio = 0.91±0.1 SD). According to RNA-seq data, allelic imbalance was also absent in all other expressed genes located within the SDI interval (Supplemental Table 5 and GSE107014 entry of Gene Expression Omnibus (GEO) database).

We also compared absolute expression of VviAGL11 in additional seedless and seeded accessions and different fruit tissues using RT-qPCR. This assay showed that at fruit set and pea size stages, the expression of VviAGL11 was restricted to seed or seed traces and was not present in fruit pericarp (Fig. 4D).

Remarkably, when sdi+ seed traces and sdi-developing seeds were directly compared, VviAGL11 transcript levels were similar or even higher in several seedless accessions. Altogether, the balanced allelic expression of VviAGL11 in seedless sdi+/sdi-heterozygous accessions, along with the lack of correlation of VviAGL11 absolute expression in seed organs and seedlessness phenotype, reject the hypothesis of misexpression mutations in this gene as the origin of stenospermocarpy.

Transcriptome sequence analysis identifies sdi candidate coding mutations

Once the presence of causal misexpression mutations was ruled out, we investigated the RNA-seq dataset for candidate sdi mutations in coding sequences. Variant calling of RNA-seq data detected 68 variants specific to the sdi+ haplotype within the fine mapping interval and all of them were single nucleotide variants (SNVs). While these SNVs affected the sequence of six genes within the interval, no high effect (loss of start or gain of stop codon) was predicted for any of them (Supplemental Table 5). Nonetheless, four of these genes collectively harbored six missense substitutions specific to the sdi+ haplotype: two located in the putative pantetheine-phosphate adenylyltransferase VviPPAT2 (VIT_18s0041g01870) gene, two in VviAGL11

(VIT_18s0041g01880), one in an unknown protein (VIT_18s0041g01890) gene, and one in a translation elongation factor (VIT_18s0041g01900) gene (Table 2).

An analysis using PROVEAN software predicted that three of these missense substitutions impact the biological function of the affected proteins: Arg151Cys (chr18:26,859,228) and Gln195Leu (chr18:26,871,891) in VviPPAT2,and Arg197Leu (chr18:26,889,437) in its consecutive downstream gene VviAGL11

(Table 2). These three missense SNVs were thereby tested as candidate sdi mutations in subsequent phenotype-association genotyping analyses.

Candidate missense substitutions in VviPPAT2 do not associate with seedlessness

VviPPAT2 is homolog to a gene coding for an enzyme involved in coenzyme A biosynthesis and lipid storage in Arabidopsis seeds [START_REF] Rubio | The coenzyme a biosynthetic enzyme phosphopantetheine adenylyltransferase plays a crucial role in plant growth, salt/osmotic stress resistance, and seed lipid storage[END_REF]. We named this gene in that manner because a VviPPAT1 paralog is present in the grapevine reference genome (VIT_04s0023g01990). To assess for any possible connection between the two predicted deleterious amino acid substitutions in VviPPAT2 and seedlessness determination, amplicons containing each SNV were sequenced in 20 and 73 sdi+ seedless and sdi-seeded cultivars, respectively (Table 3 andSupplemental Table 6) Blanc' and 'Verdil', Table 3 andSupplemental Table 6). These results do not support a role for candidate variants in VviPPAT2 as major seedlessnessresponsible dominant mutations.

Stenospermocarpic seedlessness is specifically linked to one missense substitution in VviAGL11

Genotyping was also conducted to assess the role in stenospermocarpy for the candidate missense SNV detected in VviAGL11. Given that previous studies proposed several sdi candidate mutations in this gene [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Di Genova et al., 2014;[START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF]Malabarba et al., 2017), the whole gene plus the 2-kb upstream sequence were targeted for re-sequencing in a collection of 110 grapevine accessions using Illumina next-generation sequencing (NGS). Variant calling from NGS data identified 537 polymorphic sites in the 110 accessions, which included 448 SNPs and 89 insertiondeletions (INDELs) (Supplemental Table 7). For 146 polymorphic sites ( 124SNPs and 22 INDELs), the variant allele compared to the reference genome was shared in all five 'Sultanina'-derived seedless accessions studied (Fig. 5A). 2). The A nucleotide allele was present and absent, respectively, in every stenospermocarpic and seeded accession (Fig.

5A and Supplemental Table 7). This amino acid substitution is located in the Cterminal domain characteristic of MIKC-type MADS-box genes (Fig. 5B).

Although this domain is the most variable in MIKC genes (Kaufmann et al., 2005), the Arg residue at this position is generally conserved in AGL11 homologs of dicotyledonous species as well as in the related AGAMOUSlineage SHATTERPROOF 1 (SHP1) and SHP2 proteins, (Fig. 5B 2014)), which suggests that this residue could be functionally relevant.

Other candidate variants in VviAGL11 were discarded in the re-sequencing analysis. For the SNP at position chr18:26,889,399 (C:T) corresponding to the second amino acid substitution in the sdi+ haplotype (Thr210Ala), the C nucleotide detected in the sdi+ haplotype was also present in 'Aubun', 'Cornichon Blanc' and 'Verdil' seeded cultivars (Fig. 5A and Supplemental Table 7). Similarly, the poly-GA insertion candidate allele (8×TC according to the sequence in the plus strand) proposed in intron 1 (Di Genova et al., 2014) was detected in 17 seeded cultivars. In the putative promoter, the AG and GA sdi+ alleles of VMC7F2 and p3_VvAGL11 markers, respectively, were detected in 44 and 33 seeded cultivars (Fig. 5A and Supplemental Table 7), which does not support the role in seedlessness determination proposed by Mejía et al., 2011 .

To validate the association of the Arg197Leu substitution in VviAGL11 with stenospermocarpic seedlessness, 16 stenospermocarpic and one seeded cultivars were specifically genotyped following PCR and Sanger sequencing.

The full association for position chr18:26,889,437 (A:C) was again confirmed in these accessions (Supplemental Table 6). This study included 'Asyl Kara', a seeded cultivar that in our analysis was homozygous for the seeded allele (C:C) in that position despite that it was described as A:A homozygous in a previous study [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF] 6). Again, this genotype contrasts with that reported by [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF], suggesting a likely sample mistaking by these authors. In summary, the putative deleterious Arg197Leu substitution in VviAGL11 is the only detected mutation within the SDI fine mapping interval that is fully linked to the stenospermocarpy phenotype with no false detection in our comprehensive approach, which strongly suggests that it could be the sdi causal mutation.

Concurrent somatic variation in the SNV resulting in the VviAGL11

Arg197Leu substitution and seedlessness trait supports a causal effect Somatic or post-zygotic mutants occasionally appear during the characteristic vegetative propagation of grapevine cultivars (Torregrosa et al., 2011). In that manner, seeded somatic variants of the seedless cultivar 'Sultanina' have been reported previously and are known as 'Sultanina Monococco' (Adam-Blondon et al., 2001;Torregrosa et al., 2011;[START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF]. We compared postzygotic variants of the 'Sultanina' cultivar differing in their ability to develop seeds to evaluate the dominant effect of candidate sdi mutations.

We confirmed the seeded variant phenotype in two 'Sultanina Monococco' accessions (Fig. 6A). Mean SDW increased by more than 30 mg compared to a reference stenospermocarpic 'Sultanina' (Fig 6B), reaching values comparable to seeded individuals in other genetic backgrounds (Fig. 1 and Supplemental Amplicons containing the three candidate deleterious SNVs in VviAGL11 and VviPPAT2 were sequenced both in seedless and seeded post-zygotic variants of 'Sultanina' (Supplemental Table 6). Remarkably, the genotype obtained for the four 'Sultanina Monococco' accessions was identical to that in 'Sultanina'

with the exception of the VviAGL11 SNV at position chr18:26,889,437 (Fig. 6C and Supplemental Table 6), which in fact is the only polymorphism that was fully associated with seedlessness in a large collection of cultivars (Fig. 5A). At this

Discussion

Multiple lines of evidence converge on a VviAGL11 missense mutation as the cause of stenospermocarpy

In this study we carried out genome-wide approaches, which were devised independently of previous hypotheses, in an unbiased attempt to identify the mutation responsible for stenospermocarpic seedlessness in grapevine. Our data converge on a missense SNV resulting in an Arg197Leu substitution in VviAGL11 as the sdi mutation that is exploited worldwide for the production of commercial seedless grapes.

In the first place, crossover mapping in F 1 recombinants allowed us to delimit the sdi mutation to a 323-kb segment (Fig. 2). These recombinants involve physical/genetic distance ratios between 166 and 555 kb/cM in this chromosomal region depending on the mapping population (Fig. 2). These results are in line with average ratios between 198-390 kb/cM estimated in genetic maps produced in other grapevine crosses (Doligez et al., 2006;Houel et al., 2015;Teh et al., 2017) and therefore, they do not support the recombination hotspot proposed by [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF] in the vicinity of the SDI locus. Crossover mapping would be required to validate the smaller 92-kb confidence interval considered by these authors for the screening of the SDI gene.

In the second place, RNA-seq, allele-specific RT-qPCR and genotyping studies conducted here do not support the presence of cis-acting misexpression mutations within the sdi fine mapping interval (Fig. 4, Fig. 5, Supplemental authors to the cause of stenospermocarpy, the lower proportion of seed-related tissues in developing stenospermocarpic fruits is probably the origin of the differential expression that is detected for this gene when whole seeded and seedless fruits are compared [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF][START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF]Malabarba et al., 2017). The relatively high expression of VviAGL11 that we detected in seed traces of 'Sultanina' (Fig. 4D) is also contradictory with the absence of expression that Malabarba et al. ( 2017) reported in sections of developing flowers and fruits of this cultivar following in situ hybridization. In agreement with our results, some degree of VviAGL11 expression has repeatedly been detected in developing flowers and fruits of sdi+ accessions using diverse approaches [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF][START_REF] Wang | Evolutionary and expression analysis of a MADS-box gene superfamily involved in ovule development of seeded and seedless grapevines[END_REF][START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF][START_REF] Wang | Transcriptome analyses of seed development in grape hybrids reveals a possible mechanism influencing seed size[END_REF]Malabarba et al., 2017). Considering the fact that 80% of the probe used by Malabarba et al. (2017) for the in situ hybridization does not match with VviAGL11 transcripts synthesized by 'Sultanina' but with a specific allele of 'Pinot Noir', deficient probe-transcript hybridization instead of lack of expression would be a likely cause for the absence of signal in 'Sultanina' on that assay.

In line with the inconsistent difference in VviAGL11 expression between seeds and seed traces (Fig. 4 and Supplemental Table 3), candidate sdi polymorphisms that were previously proposed in regulatory regions of VviAGL11 [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Di Genova et al., 2014;[START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF] can be specifically discarded according to the dominant nature of the sdi mutation and the presence in seeded accessions of the alleles linked in the sdi+ haplotype (Fig. 5A and Supplemental Table 7; [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF]Karaagac et al., 2012;Bergamini et al., 2013). Importantly, irrespective of the presence of polymorphisms in regulatory regions of VviAGL11, we found lack of allelic expression imbalance in seed traces of sdi+/sdi-heterozygous seedless individuals (Fig. 4), which rules out the presence of cis-acting misexpression mutations in the sdi+ haplotype of this gene. In the absence of allelic imbalance, mitotic recombination between the two alleles of 'Sultanina'

could not recover VviAGL11 expression as it was suggested previously for the origin of 'Sultanina Monococco' somatic variants [START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF].

Finally, after RNA-seq screening of coding sequences within the sdi mapping interval (Table 2), the SNV causing the Arg197Leu substitution in VvAGL11

was the only predicted deleterious variant in which a full linkage with seedlessness phenotype was confirmed in a collection of grapevine cultivars (Fig. 5A). Although this missense variation was previously discarded according to its homozygous presence in the seeded cultivar 'Asyl Kara' [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF], in our study this cultivar was homozygous for the Arg codon similar to all other 106 seeded cultivars analyzed (Supplemental Table 6 and Supplemental Table 7). Remarkably, as consistent genetic proof, this polymorphism is the only candidate mutation that is not detected in 'Sultanina Monococco' seeded somatic variants of the original seedless cultivar 'Sultanina' (Fig. 6). Given that unlikely coincidence in near-isogenic somatic variants, it can be assumed that decades ago were proposed for the main origin of seedlessness in grapevine (Bouquet and Danglot, 1996;Lahogue et al., 1998).

Molecular determination of stenospermocarpy by the sdi missense mutation

In agreement with the absence of the VviAGL11 missense mutation in seeded somatic variants of 'Sultanina' (Fig. 6C), AGL11 homologs participate in the development of maternal seed tissues that is triggered after fertilization in other plant species (Colombo et al., 1997;[START_REF] Mizzotti | SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat[END_REF]Malabarba et al., 2017). Resembling the phenotype of grape stenospermocarpy (Pearson, 1932;Barritt, 1970;[START_REF] Pratt | Reproductive Anatomy in Cultivated Grapes -A Review[END_REF]Striem et al., 1992;Malabarba et al., 2017), cosuppression of FLORAL BINDING PROTEIN7 (FBP7) and FBP11, two AGL11 homologs present in petunia (Petunia hybrida), caused the development of shrunken seeds that lack seed coat sclerification with a consequent degeneration of the endosperm, while the embryo frequently remained viable (Colombo et al., 1997). As reported for FBP7 and FBP11 transcripts in wildtype petunia or AGL11/STK protein in Arabidopsis (Colombo et al., 1997;[START_REF] Mizzotti | SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat[END_REF], VviAGL11 is specifically transcribed in the developing seed coat in 'Pinot Noir' seeded grape cultivar (Malabarba et al., 2017). These coincidences suggest that AGL11 function is impaired in grapevines carrying the Arg197Leu substitution.

Resembling the Arg197Leu substitution identified in grapevine (Table 2, Fig. 5 and Fig. 6), variation of endocarp lignification in different accessions of oil palm (Elaeis guineensis) is associated with five independent missense substitutions in the MADS-box domain of SHELL, an oil palm homolog of AGL11 [START_REF] Singh | The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK[END_REF][START_REF] Ooi | Non-tenera Contamination and the Economic Impact of SHELL Genetic Testing in the Malaysian Independent Oil Palm Industry[END_REF]. The Arg197Leu substitution in VviAGL11 is located This domain participates in binding activity of MIKC tetramers to DNA and it may also determine protein-protein interaction specificity (Honma and Goto, 2001;[START_REF] Melzer | The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in 'floral quartet'-like complexes in vitro[END_REF]van Dijk et al., 2010). According to this function, and considering also that VviAGL11-SEPALLATA heterodimers interact with additional VviAGL11 units [START_REF] Mellway | Interaction analysis of grapevine MIKC(c)-type MADS transcription factors and heterologous expression of putative veraison regulators in tomato[END_REF], the Arg197Leu substitution might cause dominant effects by disrupting the action of VviAGL11containing multiprotein complexes, even in the presence of the seeded allele in heterozygous individuals as postulated for mutant alleles of SHELL [START_REF] Singh | The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK[END_REF]Singh et al., 2015). This mechanism would be compatible with the incomplete dominance of the sdi mutation that can be inferred in view of the more extreme seedless phenotype that is often displayed by sdi+/sdi+ homozygous individuals [START_REF] Mejia | Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[END_REF][START_REF] Ocarez | Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[END_REF].

In spite of genetic proofs leading to the missense substitution in VviAGL11

(Table 2, Fig. 5 and Fig. 6) and all other indirect evidence mentioned above, a heterologous assay in Arabidopsis was inconclusive about the role in seedlessness determination of the VviAGL11 protein encoded by the sdi+ haplotype (Malabarba et al., 2017). However, species-specific functions of AGL11 homologs are conceivable since, contrasting with co-suppressed petunia lines and sdi stenospermocarpy, the Arabidopsis stk loss-of-function mutant over-accumulates polyphenols in the seed coat in the absence of endosperm degeneration or embryo arrest [START_REF] Mizzotti | SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat[END_REF]. In fact, while 28 DEGs between sdi+ and sdi-RG×CS offspring were the closest grapevine homologs to any out of the 248 de-regulated genes in the stk mutant [START_REF] Mizzotti | SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat[END_REF], 71% of these DEGs showed inverted expression response tissues and fertilization products [START_REF] Mizzotti | The MADS box genes SEEDSTICK and ARABIDOPSIS Bsister play a maternal role in fertilization and seed development[END_REF]Figueiredo et al., 2016).

Apparently, this impaired flux in developing seeds of stenospermocarpic grapes leads to SA-dependent autoimmune responses (Fig. 3), and to endosperm degeneration and embryo development arrest [START_REF] Pratt | Reproductive Anatomy in Cultivated Grapes -A Review[END_REF]), which at the transcriptome level associate with the repression of secondary metabolism and MADS-box genes and the activation of photosynthesis-related genes (Fig. 3, Supplemental Diverse approaches converge on the SNV causing the Arg197Leu substitution in VviAGL11 as the seedlessness responsible mutation underlying the grapevine SDI locus. While molecular and expression assays rejected the hypothesis of cis-acting misexpression mutations, the deleterious effect predicted for this amino acid change, the full association between the variant allele and stenospermocarpy in a collection of grapevine cultivars, and the concurrent post-zygotic variation of both the missense polymorphism and seedlessness phenotype in somatic variants of the 'Sultanina' cultivar consistently point to this SNV as the causal mutation. This finding, together with previous discoveries in oil palm [START_REF] Singh | The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK[END_REF], show that variation in the amino acid sequence of AGL11 homologs has been selected in parallel during the domestication of distant monocot and dicot crops to control the proportion between pericarp and seed tissues of fruits as well as their level of sclerification.

While seedlessness is the most relevant fruit feature for the greatly active table grape breeding field, our discovery enables the development of the most efficient marker-assisted selection to track the major locus controlling this trait.

The simultaneous tracking of the sdi mutation and of minor SDI-independent loci controlling the degree of lignification and the size and number of seed traces will optimize the generation of new seedless cultivars in future table grape breeding programs. Remarkably, knowledge of the sdi mutation can also be exploited to introduce stenospermocarpy into other genetic backgrounds through controlled genome editing. Paulsen rootstock conducted under parral trellis, ferti-irrigated and cultivated under the same management practices.

Materials and Methods

For the study of sequence variation at selected loci, 124 grapevine cultivars and two RS×MO F 1 individuals were used (Supplemental Table 6 and Supplemental

Seedlessness trait assessment

For RG×CS F 1 progeny and progenitors, seed content was evaluated at harvest during three years (2007)(2008)(2009). For each individual and year, 20 fruits randomly collected from all the clusters produced by the plant were analyzed to estimate seed or seed trace number per berry and dry weight of seed content per berry (SDW). SDW was measured after 48 h of incubation of seeds and seed traces in an 80ºC oven.

The seedlessness phenotype was also de visu registered as a qualitative trait for every RG×CS F 1 individual in the same three seasons. For recombinants in the SDI mapping interval, de visu determination of seedlessness was conducted in 2012 and 2013 as well. To this aim, seeded and seedless individuals were classified according to the presence of hard seeds with totally sclerified integuments or soft seed traces with unsclerified or partially sclerified integuments, respectively, as previously described (Costantini et al., 2008).

Similar de visu classification of the seedlessness trait was carried out at least et al., 2002;Cabezas et al., 2006;Costantini et al., 2008), 178 of which showed segregation types suitable for genetic mapping. Primers were modified at the 3' (van Ooijen et al., 2002). Two parametric methods, Interval Mapping (IM) (Lander and Botstein, 1989;van Ooijen, 1992) through 1000 permutations for each trait (Churchill and Doerge, 1994). QTL

position was estimated from the location of the maximum LOD value and a minimum confidence interval (minimum overlapping interval for 1 LOD intervals independently detected for each year of analysis on each QTL and map).

Fine mapping of the SDI QTL

RG×CS F 1 individuals were screened for meiotic recombination around the SDI QTL according to the genotype at the microsatellite locus VVIN16 [START_REF] Merdinoglu | Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR[END_REF], and CAPS-28.53. This CAPS was designed for a BstBI (New England Biolabs, Inc., MA, USA) restriction enzyme target specific of the sdi+ haplotype in this progeny (Supplemental Table 2). Primer pairs used to obtain this and other target amplicons are described in Supplemental Fast DNA Polymerase (KAPA Biosystems, Cape Town, South Africa) was used for amplification from ~50 ng of gDNA. Amplification products were purified with ExoSAP-IT (USB Products Affymetrix, Cleveland, OH, USA) following the manufacturer's instructions and then sequenced by Sanger capillary electrophoresis using the same primers as in PCR. For every marker, the allele present in the sdi+ haplotype was inferred according to the genotype observed in cross progenitors, F 1 individuals with extreme seed/seedless phenotype and the two RS×MO sdi homozygous F 1 individuals (Supplemental Table 2).

Transcriptome analysis RNA extraction. Total RNA was extracted from frozen tissue powder using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich) with an added on-column DNase digestion step with the RNase-Free DNase Set (Qiagen).

Microarray analysis. Developing fruits at fruit set stage (~2 WAF, flowering time 20 May 2010) were collected on 3 June 2010, around midday, from four seeded (660-43, 660-50, 669-45, 671-19) and five seedless (660-40, 660-41, 660-57, 669-19, 671-34) RG×CS F 1 individuals and immediately frozen in liquid nitrogen. Individuals were selected according to contrasting genotypes in the VMC7F2 marker [START_REF] Pellerone | Grapevine microsatellite repeats: Isolation, characterisation and use for genotyping of grape germplasm from Southern Italy[END_REF], which co-localizes with the SDI QTL (Table 1), and to extreme phenotypes for SDW in the corresponding progeny distribution tail (Supplemental Table 1). Each individual was analyzed in separate as independent biological replicates. At least 12 developing fruits collected from ≥2 different clusters were ground for each RNA extraction. (Supplemental Table 1). Selected seeded individuals belonged to the higher tail of the distribution for SDW phenotype in the progeny, whereas seedless individuals without extremely low SDW were selected to ease the extraction of seed traces. Berries at pea size (10-12 mm) developmental stage were collected on 12 June 2012 (~4 WAF, flowering time 18 May 2012) around midday and immediately frozen in liquid nitrogen. Berries were allowed to briefly thaw in the laboratory, opened by cutting with a scalpel and then, seed content was rapidly recovered with tweezers and refrozen in nitrogen. Each individual was analyzed in separate as independent biological replicates. Seed content from 12-50 fruits collected from ≥2 different clusters was used for each RNA extraction. RNA-seq was performed in the Centre for Genomic Regulation (CRG) (Barcelona, Spain). The six corresponding cDNA libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Prep kit starting from 1 µg of total RNA as described previously [START_REF] Royo | Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco[END_REF]. A mean fragment size of 302 bp was obtained. Library sequencing was performed on an Illumina HiSeq (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/) was carried out using TopHat2 v.2.0.13 (Kim et al., 2013). TopHat2 was run under default parameters with the exception of gap length ≤8, mismatch ≤8 and edit distance ≤8 that were allowed in 125 nt reads. These values were adjusted to compensate for the relatively high genetic distance between the table grape genotypes studied here and the reference genome of the wine grape-related PN40024 reference genome. As post-filtering, only uniquely mapped single copy reads with quality ≥20, aligned in the same chromosome and paired in the expected orientation were kept for further analysis. After filtering, an average of more than 15 million reads per replicate was considered for subsequent analysis (Supplemental Table 10). The htseq-count tool (v.0.5.4p5) from HTSeq (Anders et al., 2015) was used to estimate unambiguous read count for each 12X V1 annotated transcript. Normalization following the trimmed mean of Mvalues (TMM) method [START_REF] Robinson | A scaling normalization method for differential expression analysis of RNA-seq data[END_REF], as well as seed trace versus seed DEGs search (adjusted Benjamini-Hochberg P-value ≤0.05 and ≥2-fold change) were performed in edgeR v.2.2.6 (Robinson et al., 2010).

Analysis

Finally, reads per kb of exon per million fragments mapped (RPKM) was calculated using edgeR and low expressed transcripts were filtered out if average RPKM was <1 in both seed traces and seeds.

Functional analysis of differentially expressed genes. Lists of DEGs identified from microarray and RNA-seq analyses were compared using Venny by RNA-seq (Supplemental Table 1).

Search of RNA-seq data for candidate sequence variation. To detect sequence variation specific to the sdi+ haplotype in expressed transcripts, RNAseq alignments used for differential expression analysis were also analyzed for the presence of SNPs and INDELs, basically following a similar pipeline to that described previously [START_REF] Royo | Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco[END_REF], but adjusted to boost variant calling in both low expressed and allelic imbalanced genes. For variant calling, three tools implemented in SAMtools package v.1.5 (Li et al., 2009;Li, 2011) whereas the effect of sdi candidate amino acid substitutions on protein function was predicted using PROVEAN (Choi et al., 2012).

Hormone analysis

To compare phytohormone levels between developing seeds and seed traces, five seedless (665-16, 669-60, 670-17, 675-17, 660-57) and five seeded (660-43, 660-50, 665-17, 671-19, 677-06) RG×CS F 1 individuals were analyzed (Supplemental Table 1). Additionally, two independent biological replicates of RG and CS, each from a different plant, were analyzed as well. Fruits at pea size stage (~4 WAF) were collected simultaneously to RNA-seq samples on 12

June 2012 and immediately frozen in liquid nitrogen. For each replicate, seed content from ≥20 fruits was extracted. SA and active GA (GA 1 and GA 4 ) levels Reactions were performed in a final volume of 20 µl with 5 µl of a 1:10 dilution of cDNA. Gene-specific primers (Supplemental Table 9) were designed using the Oligo Explorer 1.2 software (Gene Link) and the gene sequences from the grapevine 12X.0 genome assembly (Jaillon et al., 2007) as design templates.

For allele-specific RT-qPCR, the 3' end of forward and reverse primers coincided with SNP positions in a way that each primer was specific for one

Genotyping of candidate mutations in VviPPAT2

For validation of candidate SNVs detected from RNA-seq data, the gene VviPPAT2 (VIT_18s0041g01870) was partially sequenced in a collection of 93 grapevine accessions (Supplemental Table 6). Primer pairs PPAT2-R151C and PPAT2-Q195L were used for PCR and Sanger sequencing to this end (Supplemental Table 9).

Targeted re-sequencing of VviAGL11

The gene VviAGL11 (VIT_18s0041g01880) plus a 2-kb upstream sequence (9,849-bp in total corresponding to positions chr18:26,888,672-26,898,521 in the PN40024 12X.0 grapevine reference genome) were sequenced in a collection of 110 accessions (Supplemental seedless and seeded accessions (Supplemental Table 8). Microsatellite marker genotyping was performed as described elsewhere (Ibáñez et al., 2009). Parsed Citations

Accession numbers

  Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 9 explained between 71 and 83% of the total variance for SDW depending on the 192 www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 10 year and the linkage map. It co-localized with the SDI QTL detected previously in different genetic backgrounds related with stenospermocarpic seedlessness

F

  1 population was therefore screened for meiotic recombinants by studying the VVIN16 SSR marker and a newly developed Cleaved Amplified Polymorphic www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 11 Sequence (CAPS) marker (CAPS-28.53) mapping at either side of the VMC7F2 218 marker, respectively (Fig. 2). This interval comprises a 5.1 Mb physical distance 219 in the 12X.0 grapevine reference genome assembly (Fig. 2), which is equivalent 220 to 5.5 Mb in the recently published 12X.v2 assembly as one small scaffold has Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 12

  Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259

  Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 19

  . While both SNVs were validated by PCR in the CS progenitor, no genotype-phenotype association was confirmed. For the SNV at position chr18:26,859,228 (C:T), the T nucleotide www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 20 allele inferred the sdi+ haplotype of CS from the RNA-seq analysis was present in ten seeded cultivars ('Afus Ali', 'Alphonse Lavallee', 'Aramon', 'Aubun', 'Morio Muskat', 'Naparo', 'Planta Nova', 'Pedro Ximenes', 'Semillon' and 'Verdil'); and similarly, for the SNV at position chr18:26,871,891 (A:T), the T nucleotide allele of the sdi+ haplotype in CS was present in two seeded cultivars ('Cornichon

  These variants were considered to be linked in the sdi+ haplotype because the analysis included two sdi+/sdi+ homozygous seedless 'Ruby Seedless' × 'Moscatuel' (RS×MO) F 1 individuals obtained from a seedless by seedless www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 21 cross. From all these positions, only the genotype for the SNP at position chr18:26,889,437 (A:C, according to the sequence in the plus strand) was fully associated with the seedlessness phenotype. Remarkably, this SNP causes the Arg197Leu substitution with the predicted deleterious effect already detected in the RNA-seq variant calling (Table

  and Pabonwww.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 22 Mora et al. (

  somatic variations (either genetic or cellular) that altered the genotype of the missense SNV are the cause of the polymorphic seed development phenotype displayed by post-zygotic variants of 'Sultanina'. This causality proof, together with all other genetic and molecular evidence described above, led us to postulate that VviAGL11 is the major dominant regulator gene and the SNV resulting in the Arg197Leu substitution is the monogenic sdi mutation that two www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 29

  Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 30 in the C domain characteristic of MIKC-type MADS-domain proteins (Fig. 5B).

  end with a fluorochrome (NED, 6-FAM, VIC or PET) by Applied Biosystems. PCR amplifications were performed with Ecogen Taq polymerase. PCR products were analyzed with ABI Prism 3730 (Applied Biosystems) at the Unidad de Genómica-Campus Moncloa del Parque Científico de Madrid. Detection of amplified fragments and allele assignment was carried out using www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 37 GeneMapper™ v3.7 (Applied Biosystems). Additionally, F 1 individuals and progenitors were genotyped for 335 SNP markers described in Lijavetzky et al. (2007) and Cabezas et al. (2011), 112 of which showed segregation types suitable for genetic mapping and were analyzed as described previously (Cabezas et al., 2011). Genetic map construction. Genetic maps were built according to a 2-way pseudo-testcross strategy (Grattapaglia and Sederoff, 1994) using JoinMap 3.0 (van Ooijen and Voorrips, 2001). DH (Doubled Haploid) and CP (Cross Pollinators) models were assumed for the construction of parental and consensus maps, respectively. The global segregation of each marker was tested for the expected Mendelian segregation ratio using a χ 2 goodness-of-fit test (P<0.01). Markers were grouped in the same LG according to the threshold LOD≥5.0 and ordered by paired markers with LOD≥3.0 and recombination frequency (REC) ≤0.35. These parameters were reduced to LOD≥1.0 and REC≤0.45 when required to adapt linkage groups to current information on the physical genome map. Mapping distances were calculated (in cM) using the Kosambi function (Kosambi, 1944). QTL detection. QTL analyses were carried out on RG×CS mapping progeny independently for each of the three SDW evaluations (seasons 2007, 2008 and 2009) using the framework maps of each progenitor and the consensus framework map for the cross. QTL mapping was performed using MapQTL 4.0

  of RNA integrity as well as synthesis, labeling, and hybridization of cDNA to NimbleGen microarray 090818 Vitus exp HX12 (NimbleGen-Roche) www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 40 and robust multi-array average (RMA) normalization were performed as indicated elsewhere (Carbonell-Bejerano et al., 2014). Linear models for microarray data (limma) were run in Babelomics (Medina et al., 2010) to search for DEGs between seedless and seeded individuals. DEGs were identified considering a Benjamini-Hochberg adjusted P-value ≤0.05 and ≥2-fold change as significance thresholds. RNA-seq and differential gene expression analysis. RNA was obtained from seed traces of three seedless (669-60, 670-17, 675-17) and seeds from three seeded (665-17, 671-19, 677-06) RG×CS F 1 individuals. These individuals were selected according to contrasting genotypes for the SDI QTL marker VMC7F2

  Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 41 2000 using v4 chemistry (flow cells and sequencing reagents). Paired-end strand-specific reads of 125 nt were produced. Gapped alignment of reads

(

  http://bioinfogp.cnb.csic.es/tools/venny/). Gene lists were further analyzed for www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 42 functional enrichment compared to the whole set of transcripts predicted in the 12X V1 annotation of the grapevine reference genome following a grapevinespecific functional classification (Grimplet et al., 2012). The analysis was carried out in FatiGO as described elsewhere (Carbonell-Bejerano et al., 2014). Search of RNA-seq data for candidate allelic imbalance. Aligned reads within the sdi fine-mapped interval were visually inspected for allele-specific expression with the Integrative Genomics Viewer (IGV) software (Thorvaldsdottir et al., 2013). For VviAGL11, the allelic ratio in each sample was estimated by comparing the frequency of each allele at heterozygous SNP positions in filtered Binary Alignment Map (BAM) files. A mean value was estimated for all the SNPs detected along the gene according to the parameters indicated in the following paragraph. The sdi+ haplotype was inferred from the comparison of two seedless RG×CS F 1 individuals that shared the same allele inherited from RG for the SDI locus with the three seeded individuals analyzed

  were measured at the Plant Hormone Quantification service of the Institute for Plant Molecular and Cell Biology (IBMCP), Valencia, Spain. In summary, 200 mg fresh weight of seeds or seed traces was extracted for each replicate as indicated above for RNA-seq analysis. Frozen ground tissue was then analyzed www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 44 using an ultra performance liquid chromatography -tandem mass spectrometer (UHPLC-MS) system (Q-Exactive, ThermoFisher Scientific). The statistical treatment of results was carried out using SPSS software (v.24.0 for Windows; IBM Corp., Somers, NY, USA). Reverse transcription quantitative PCR (RT-qPCR) RNA was obtained from either berry skin, flesh, pericarp, seeds or seed traces. Pea size fruits collected from the ITUM experimental field on 26 June 2011 were used for three RG×CS F 1 individuals (660-41, 660-43, 660-57) and RG and CS progenitor accessions. 'Sultanina' samples from ESP217-5186 accession were collected at fruit set (6 June 2011) in the ICVV collection. At least 8 fruits were used for each RNA extraction. For reverse transcription from total RNA (1 µg), SuperScript™ III First Strand (Invitrogen, USA) and oligo (dT) were used following manufacturer's instructions. Transcript levels were determined by RT-qPCR using a 7500 Real-Time PCR System (Applied Biosystems) and SYBR Green PCR Master Mix (Applied Biosystems).

Figure 1 .

 1 Figure 1. Stenospermocarpic phenotype and segregation in a RG×CS F 1 population. A, Berries of 'Red Globe' (RG) and 'Crimson Seedless' (CS) cross progenitors. Representative berries, seeds (RG) and stenospermocarpic seed traces (CS) at maturity are shown. B, Histogram showing the distribution of seed dry weight per berry (SDW) trait in RG×CS F 1 progeny. SDW values

Figure 2 .

 2 Figure 2. Fine mapping of the sdi mutation in two seedless × seeded F 1 populations. Scheme of crossover mapping for recombinants around the SDI locus in A, 'Red Globe' × 'Crimson Seedless' (RG×CS) and B, 'Imperial Napoleon' × 'Crimson Seedless' (IN×CS) F 1 mapping populations. Each class of recombinant F 1 individuals is depicted in different lines, where red, blue and grey colors denote chromosome fragments corresponding to the sdi+ seedless, sdi-seeded, or undetermined haplotypes, respectively. F 1 individuals considered and their phenotype (seed, seeded; trace, seedless) are indicated for each class in the left and right sides of the corresponding line, respectively.Markers studied in each F 1 mapping population, the sdi fine-mapped interval and the genes included on it according to distances in the PN40024 12X.0 reference genome and 12X V1 gene annotations are represented as well.

Figure 3 .

 3 Figure 3. Transcriptome and phytohormone level comparisons between seedless (sdi+) and seeded (sdi-) RG×CS F 1 individuals. A, Venn diagram comparison of differentially expressed genes (DEGs) identified in 2 weeks after flowering (WAF) fruits (NimbleGen microarray) and 4 WAF aborting/developing seeds (RNA-seq). The number of up-and down-regulated DEGs in seedless (sdi+) versus seeded (sdi-) individuals in each experiment is shown. B, Summary of functional categories over-represented in up-and down-regulated DEGs in each experiment. This panel corresponds to a summary of the full list

Figure 4 .

 4 Figure 4. VviAGL11 transcript levels. A, Representation of RNA-seq reads aligned in a region of VviAGL11 with a high density of heterozygous single nucleotide polymorphisms (SNPs). Image obtained from IGV viewer. Read depth is shown separately for each studied seedless (sdi+) and seeded (sdi-) RG×CS F 1 individual. The frequency of each allele at single nucleotide variant (SNV) positions compared to the reference genome is denoted in colors. The read depth range depicted for each individual is indicated as well. A scheme of the portion of VviAGL11 12X V1 gene model annotated in this region of the PN40024 12X.0 reference genome is shown in blue at the bottom of the image with coding sequence exons (cds) numbered from the start codon. B, Average VviAGL11 allelic frequency in seedless RG×CS F 1 individuals estimated from RNA-seq data. C, Average VviAGL11 allelic frequency in seeded RG×CS F 1 individuals estimated from RNA-seq. In B and C, frequencies were normalized to the sdi-allele inherited from RG, for which expression levels are depicted in blue. Only positions with a heterozygous genotype in the three replicates (six and three SNPs in seedless and seeded individuals, respectively) were considered to estimate average frequencies and SD (represented by error bars) in the three seeded or seedless replicates. D, VviAGL11 expression estimated

Figure 5 .

 5 Figure 5. Sequence variation in AGL11 and association with seedlessness. A, Nucleotide sequence variation in the sdi+ haplotype of VviAGL11 and association of the sdi-linked variants with seedlessness in a collection of grapevine cultivars. VviAGL11 (exons + introns) plus the 2-kb upstream sequence were targeted for Illumina paired-end sequencing in 105 seeded and 5 seedless accessions. The position of single nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs) identified in the sdi haplotype compared to the PN40024 reference genome is depicted on a scheme of the gene (encoded in the minus strand) with coding sequences (CDS) numbered from the start codon of the gene. The frequency of the variant allele in stenospermocarpic seedless and seeded accessions is indicated for specific candidate polymorphisms selected from the full list of polymorphisms available in Supplemental Table 7. Genotype-phenotype correlation of 100% was only identified for the SNP causing the Arg197Leu substitution in the sdi allele, which is highlighted in red. B, VviAGL11 protein domain model and

Figure 7 .

 7 Figure 7. Model of grape seedlessness determined by the sdi locus. The left panel represents a heterozygous sdi+/sdi-individual in which the sdi Arg197Leu substitution prevents the activation of gene expression by multiprotein complexes containing the mutant VviAGL11 protein (depicted in red). This lack of gene expression activation precludes seed coat differentiation, which triggers salicylic acid (SA) production along with the over-expression of WRKY, Homeobox-domain (HD), and NAC transcription factors, and finally leads to endosperm degeneration and embryo developmental arrest in seed traces. The right panel depicts the activation of seed morphogenesis that takes place in seeded sdi-/sdi-individuals. Complexes containing the wild-type VviAGL11 protein (depicted in blue) directly or indirectly activate the expression of genes involved in seed coat sclerification, which enables endosperm maintenance and embryo development.

  . Stenospermocarpy is widely used in the production of seedless table grape cultivars because berry

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Table 2

 2

	) and nine recombinant F 1 individuals

further restrict the interval length because material for these recombinants is no longer available. According to grapevine 12X V1 gene annotations www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 13

Table 4

 4

		). WRKY, Homeobox and NAC
	Downloaded from	on August 6, 2018 -Published by	www.plantphysiol.org
	Copyright © 2018 American Society of Plant Biologists. All rights reserved.

272 as these TF families were over-represented in seed trace-upregulated genes as 273 well. Likely connected with lack of seed differentiation, genes upregulated in 274 seed traces were also over-represented in photosynthesis-related categories.

		WAF) using NimbleGen microarrays. Although we used whole setting fruits
		because the extraction of seed content was difficult at this stage, this strategy
		eased the analysis of a higher number of replicates. In this case, only 335
		DEGs were detected (adjusted p-value ≤0.05 in limma and ≥2-fold change).
	275
	276	As expected, genes downregulated in seed traces were over-represented in
	277	functional categories related with seed morphogenesis such as
	278	'phenylpropanoid metabolism' (Fig. 3B and Supplemental Table 4). Collectively,
	279	these results are in line with general trends described by Wang et al. (2016)
	280	following a similar approach.
	281	To assess for transcriptome differences at earlier stages, a similar comparison
		between sdi+ and sdi-RG×CS F 1 individuals was carried out at fruit set stage (2
		15
		Comment citer ce document :
	Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez,
	Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The
	Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box
	GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259

Table 4

 4

	). Immune-like

Table 3

 3

	). When seed tissues

17 repression in sdi+ individuals). Regardless, any potential allelic imbalance in

VviAGL11 transcript levels was assessed from the RNA-seq dataset.

  -to-typeness with the genetic profile indicated for 'Asyl Kara' in the Vitis International Variety Catalogue (VIVC) database (Supplemental Table8and www.vivc.de). 'Asyl Kara' was also homozygous for the seeded allele (T:T) in position chr18:26,889,399 (Supplemental Table

	. 'Asyl Kara' was independently genotyped several
	times for this position, confirming the C:C genotype. The accession of 'Asyl
	Kara' used at the Instituto de Ciencias de la Vid y del Vino (ICVV) collection

comes from the Vassal-Montpellier collection and, following microsatellite marker genotyping, we confirmed in the same DNA used for VviAGL11 www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 23 sequencing its true

Table 1

 1

	485
	486	other available accessions of 'Sultanina Monococco'. Genotyping of 13
	487	microsatellite markers confirmed the identity of the four studied 'Sultanina
		Monococco' accessions as post-zygotic variants of 'Sultanina' (Supplemental

). Seeds produced by 'Sultanina Monococco' were generally filled and www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 24 most of them sank in water (Fig. 6B). Moreover, 59.9% germination success 482 under regular soil conditions was obtained for 'Sultanina Monococco' 483 (2085Mpt1 accession) self-cross filled seeds, confirming that they are 484 functional. A similar seeded variant phenotype was visually confirmed in two 488 Table 8). 489 www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 25

Table 3 ,

 3 Supplemental Table 5 and SupplementalTable 7). Specifically for VviAGL11 we show that rather than misexpression, which was related by other www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 27

Table 3 ,

 3 Supplemental Table4). These molecular responses are

	very similar to those reported during SA-dependent seed abortion in
	incompatible Arabidopsis inter-specific hybrids that, like sdi-determined
	stenospermocarpy, proceed with abnormal seed coat development (Burkart-
	Waco et al., 2013).

Conclusions

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  F 1 mapping populations, (RG×CS, n = 292 and IN×CS, n = 299) were generated from controlled crosses carried out in 2003 and 2008, respectively. For these crosses, emasculated flowers of RG and IN seeded cultivars were pollinated with pollen collected from CS that in both populations was the donor of the sdi mutation. One plant for each F 1 individual and three plants for each progenitor were grown in the same experimental field belonging to the Sociedad Murciana de Investigación y Tecnología de Uva de Mesa (ITUM) located in Blanca, Murcia, Spain. Plants were grafted onto 1103

	Plant material

www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 34 Two table grape (Vitis vinifera)

Table 7

 7

	Spain. These plants were grafted in 2010 onto 110 Richter rootstock, and their
	true-to-typeness was verified by genotyping SNP markers and comparing the
	results with the ICVV-SNP database (Cabezas et al. 2011). RG and CS
	samples were obtained from ITUM, whereas RS×MO F 1 individuals and the MO
	cultivar were obtained and cultivated as described elsewhere (Carreño et al.,
	2015). Material for 'Chasselas Doré' was obtained from plants, grafted onto 110
	Richter in 2003, in the 'Vitis Germplasm Bank' (ESP-080) of the Instituto

). Samples of 120 cultivars were obtained from the Grapevine Germplasm Collection of the Instituto de Ciencias de la Vid y del Vino (ICVV, ESP-217). At ICVV, ten plants per cultivar were maintained under the same agronomical conditions in 'Finca La Grajera' located in Logroño, La Rioja, Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA) located in 'Finca El Encín' in Alcalá de Henares (Madrid, Spain). In addition to www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259

Table 9

 9

	. PCR

amplification and Sanger capillary electrophoresis sequencing. In most cases, primers were designed to align in coding sequences according to grapevine www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 39 12X V1 gene annotations (http://genomes.cribi.unipd.it/). For PCR, KAPA2G

  were used to compare each sample to the PN40024 reference genome: samtools mpileup for genotype probability estimation, bcftools for variant calling, and finally, strand bias and baseQ bias filters were applied using varfilter. Subsequently, using ad www.plantphysiol.org on August 6, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 43hoc Bash shell and Perl scripts[START_REF] Royo | Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco[END_REF], we selected polymorphisms within the SDI fine-mapping interval specific to the three seedless individuals according to the following filters: average depth of contrasting alleles (variant or

	reference) per F 1 individual ≥5 counts, frequency of variant allele in sdi+
	individuals ≥20%, frequency of variant allele in seeded individuals <2.5% and
	frequency of spurious alleles (alleles other than the reference and the first
	variant) <2.5% in both samples. In this case, reads corresponding to each
	transcription strand direction were independently computed. The effect of
	detected candidate sdi mutations considering grapevine 12X V1 gene
	annotations was estimated using SnpEff v.2.0.3 (Cingolani et al., 2012),

  American Society of Plant Biologists. All rights reserved. Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259

	(VIT_16s0098g01190),	ELONGATION	FACTOR	1-α	(EF1-α	,
	VIT_06s0004g03220)	and		GLYCERALDEHYDE-3-PHOSPHATE
	DEHYDROGENASE (GPDH (VIT_17s0000g10430) using the ∆∆Ct method. For
	allele-specific RT-qPCR, two independent biological replicates from different
	stenospermocarpic 'Sultanina' plants were analyzed and gDNA from the same
	accession was used for allelic ratio normalization assuming a balanced ratio in
	the genome. Graphical representations of relative transcript levels with standard
	deviations were performed using the Microsoft Excel software. The statistical
	treatment of results was carried out using SPSS software (v.24.0 for Windows;
	IBM Corp., Somers, NY, USA).
	allele. No-template controls were included for each primer pair, and each PCR
	was performed in triplicate. Amplification data were analyzed using the 7500
	SDS software 1.3 (Applied Biosystems). Relative transcript levels were
	calculated	after	normalization	to	the	grapevine	UBIQUITIN
		on August 6, 2018 -Published by Copyright © 2018 45 Downloaded from	www.plantphysiol.org

Table 7

 7 American Society of Plant Biologists. All rights reserved.Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P.

	) by BGI company

(Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 Validation by PCR and amplicon sequencing of the sdi candidate mutation identified in VviAGL11 was carried out using the primer pair CAPS-26.88 from Supplemental Table 9 on gDNA of 15 additional seedless cultivars as well as in the seeded cultivar 'Asyl Kara' and in two 'Sultanina Monococco' accessions (Supplemental Table 6). Genotyping of 13 microsatellite markers in the same DNA sample used to genotype sdi candidate variants by PCR and Sanger sequencing was carried out to verify the identity of 'Asyl Kara' and 'Sultanina'

Table 1 . Quantitative trait loci (QTLs) identified for seed dry weight per 1079 berry (SDW) in the 'Red Globe' (RG) × 'Crimson Seedless' (CS) F 1 mapping

 1

	Microarray hybridization and RNA-seq data have been deposited in the NCBI
	GEO database under GSE106668 and GSE107014 accession numbers,
	respectively. BAM files for read alignments of VviAGL11-targeted NGS data
	were deposited under BioProject ID PRJNA418130 (SRP124845) of the NCBI
	Sequence Read Archive.

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Table 2 . Nucleotide variation in the sdi+ haplotype generating protein sequence variation.

 2 Single nucleotide variants (SNVs) specific to the sdi+ haplotype located within the sdi fine mapping interval that were detected in the RNA-seq dataset (Supplemental Table5) with an impact on protein coding sequence are presented here. For each SNV, the position and gene annotation in the 12X V1 grapevine reference genome, the nucleotide change in the coding sequence of the sdi+ haplotype, the amino acid change, the PROVEAN score and the predicted effect of the change in the function of the protein are

	indicated. PROVEAN score ≤ -2.5 and > -2.5 are considered 'deleterious' and
	'neutral', respectively (Choi et al., 2012). Predicted deleterious changes are in
	bold.
	Genome position	Gene ID	Functional annotation	Coding sequence change	Amino acid change	Score	Predicted effect
	chr18:26,859,228 VIT_18s0041g01870	VviPPAT2	451 C>T	Arg151Cys	-6.44	Deleterious
	chr18:26,871,891 VIT_18s0041g01870	VviPPAT2	584 A>T	Gln195Leu	-4.27	Deleterious
	chr18:26,889,399 VIT_18s0041g01880	VviAGL11	628 A>G	Thr210Ala	-0.21	Neutral
	chr18:26,889,437 VIT_18s0041g01880	VviAGL11	590 G>T	Arg197Leu	-5.40	Deleterious
	chr18:26,914,516 VIT_18s0041g01890	Unknown protein	148 G>A	Asp50Asn	0.45	Neutral
	chr18:26,918,160 VIT_18s0041g01900	Translation elongation factor	997 A>T	Thr333Ser	1.01	Neutral

Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018). The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box GeneVviAGL11. Plant Physiology, 177 (3), 1234-1253. , DOI : 10.1104/pp.18.00259 52

Table 3 . Genotype of the two VviPPAT2 candidate single nucleotide variants (SNVs) in a collection of 93 accessions.

 3 The number of seedless and seeded accessions identified for each genotype are specified. For each position, the allele encoding candidate deleterious substitutions is denoted in bold. Accessions excluding these candidate SNVs are described in the footnote.

	The full list of genotypes for each accession can be found in Supplemental
	Table 6.
	Genotype	VviPPAT2 Arg151Cys	VviPPAT2 Gln195Leu	Seedless accessions	Seeded accessions
	1	T:T	T:T	1	0
	2	T:T	A:T	1	0
	3	C:T	A : T	1 4	1 a
	4	C:T	A : A	0	1 b
	5	C:C	A:A	0	28
	6	T:T	nd	0	1 c
	7	nd	T:T	2	0
	8	C:T	n d	0	7 d
	9	nd	A:T	2	1 e
	10	C:C	nd	0	1
	11 Footnote: a 'Verdil', b 'Pedro Ximenes', c 'Planta Nova', d 'Afus Ali', nd A:A 0 33
	'Alphonse Lavallee', 'Aramon', 'Aubun', 'Morio Muskat', 'Naparo', 'Semillon' and e 'Cornichon Blanc'. nd, no data was obtained.

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Acknowledgements

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We thank J.L. Pérez-Sotés and Servicio de Investigación Vitivinícola (SIVV, Gobierno de La Rioja) at ICVV collection and Rosa M. Arnau and María J.

Candel at IMIDA and ITUM for plant maintenance and technical support in phenotyping tasks. We are very grateful to Nachi Montemayor, Rufino Aguirrezabal, Miguel Angulo and Silvia Hernáiz for technical assistance. We also thank the Genomics service of the CNB-CSIC for running microarray hybridizations and Dr. Iván Carreño for sharing information regarding RS×MO F 1 progeny.

We thank J.L. Pérez-Sotés and Servicio de Investigación Vitivinícola (SIVV, Gobierno de La Rioja) at ICVV collection and Rosa M. Arnau and María J. Candel at IMIDA and ITUM for plant maintenance and technical support in phenotyping tasks. We are very grateful to Nachi Montemayor, Rufino Aguirrezabal, Miguel Angulo and Silvia Hernáiz for technical assistance. We also thank the Genomics service of the CNB-CSIC for running microarray hybridizations and Dr. Iván Carreño for sharing information regarding RS×MO F1 progeny.

This work was partially supported by project GRASP from European Union's Plant Genomics ERA-Net and projects BIO2011-026229 and BIO2014-59324-R from the Spanish Ministry of Economy (MINECO). This work also benefited from networking activities within the funded European Cooperation in Science and Technology (COST) Action FA1106 QualityFruit.

Comment citer ce document : Royo, C., Torres-Pérez, Mauri, Diestro, Cabezas, J. A., Marchal, C., Lacombe, T., Ibáñez, Tornel, M., Carreño, Martínez-Zapater, Carbonell-Bejerano, P. (Auteur de correspondance) (2018)