OENO One A molecular marker set combining a retrotransposon insertion and SSR polymorphisms is useful for assessing diversity in Vitis Pelsy Frédérique Bevilacqua Lucie Merdinoglu  SophieBlancandDidier Strasbourg Universitéde INRAE UMR-A SVQV 1 3 1 1 Colmar F-68000 France Abstract

Molecular markers, based on DNA polymorphisms, are useful tools for identifying individuals, establishing phylogenetic relationships, managing collections of genetic material or assisting breeding. In the present study, we developed a marker set to differentiate Vitis species, grapevine varieties or clones belonging to the same variety. This novel marker set combines, in four PCR amplifications, the presence/absence of a remarkable retrotransposon, Tvv1-Δ3460, inserted at its single locus and the SSR polymorphism present within its two LTRs. By studying a collection of Vitaceae accessions, we showed the prevalence of two allelic forms of Tvv1-Δ3460 - one of which was partially truncated - in Vitis species. Out of the twenty-five studied Vitis species, the insertion of a Tvv1-Δ3460 element was detected in twenty, including Vitis vinifera. The homozygous vs heterozygous state of the element insertion was determined by amplifying the empty site. Additionally, each Tvv1-Δ3460 LTRs included a microsatellite sequence useful for designing markers based on LTR length. The LTR-SSR markers distinguished most of the fifty-two cultivars and revealed polymorphism within five of the seven varieties studied.

Keywords: molecular markers, grapevine, retrotransposon, SSR

 Introduction

Molecular markers are useful tools for identifying individuals, establishing phylogenetic relationships, managing collections of genetic material or assisting breeding. They are based on various types of DNA polymorphisms found in genomes (Gupta et al., 1999; Schulman, 2006). Microsatellite markers, whose polymorphism takes advantage of the variable number of simple sequence repeats (SSR) at a given locus, is undoubtedly the most extended molecular marker system for grapevine. SSR markers are highly transferable, co-dominant, and very useful for identifying grapevine cultivars (Merdinoglu et al., 2005; This et al., 2004) and for studying Vitis phylogeny (Di Gaspero et al., 2000). However, most available SSR markers fail to distinguish clones that derive from repeated vegetative propagation cycles from a unique single individual. Nevertheless, a standard set of five SSR markers (VMC3a9, VMC5g7, VVS2, VVMD30 and VVMD32) which can reveal clonal polymorphism has been proposed (Pelsy et al., 2010). Somatic variations giving rise to clone diversity within grapevine varieties have also been investigated by genomic approaches; 15 distinguishable Chardonnay clones have been identified by 1620 SNPs and InDels, which can be exploited to define markers for clone-specific genotyping (Roach et al., 2018).

Mobile elements are abundant, rapidly evolving and widespread in the genomes of plants. They actively contribute to molecular polymorphism and, therefore, form the basis of other molecular marker systems. They take advantage of the activity or the structural variations of transposable elements. Sequence-specific amplification polymorphisms (SSAP) markers, which reveal the pattern of insertion of elements belonging to the same family (Waugh et al., 1997), have been developed to analyse genetic diversity and relatedness in the genus Vitis (Moisy et al., 2008b). Retrotransposon polymorphism fingerprinting (RUP), which amplifies the highly variable untranslated leader (UTL) region of the Tvv1, has revealed a unique pattern in each of 94 Vitaceae accessions and is conserved between clones (Pelsy, 2007). Finally, inter retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) have been shown to discriminate the white table grape cultivar Italia from its coloured variants (Rubi, Benitaka, Brasil and Black Star) derived from clonal propagation of somatic mutations (Strioto et al., 2019).

In the grapevine genome, several retrotransposons have been found inside genes, such as Vine1 in Adhr (Verriès et al., 2000), or close to genes, such as Gret1 in the promotor of VVMybA1 (Kobayashi et al., 2004). Others have been characterised by a computerized sequence similarity search and grouped into families sharing an amino acid identity of ≥ 90 % (Moisy et al., 2008b), among which the Tvv1 family, which is a Ty1-copia like LTR-retrotransposon, has been extensively studied (Moisy et al., 2008a). Tvv1 full-length copies of around 5 kb in size share an internal region with a single highly conserved open reading frame and, upstream, an untranslated leader (UTL) region which is highly variable in size. This internal region is flanked by LTRs between 149 and 157 bp long (Pelsy and Merdinoglu, 2002). The Tvv1 family also comprises unique copies of remarkable Tvv1 elements that have suffered large deletions and are fixed at single loci. Among them, Tvv1-Δ3460, which has undergone a major 3,460 bp long deletion in the coding sequence compared to the full-length copies, is located on chromosome 8 of the grapevine genome. In Pinot noir cv, Tvv1-Δ3460 is 2,074 pb long, but it slightly differs in size in the genomes of other varieties hosting this element, mainly because of the number of TA motifs in a microsatellite stretch located in both LTR sequences (Moisy et al., 2008a).

Molecular markers based on the insertion of retrotransposons can be used for identifying grapevine species and cultivars (D'Onofrio et al, 2010), but they generally fail to reveal clonal polymorphism (Pelsy, 2007). Nevertheless, transposable elements are responsible for the main proportion of somatic mutations affecting four Pinot Noir clones (Carrier et al., 2012) and have been found to cause diversification of 15 Zinfandel clones (Vondras et al., 2019). In the present study, we developed a marker set based on the combination of the presence/absence of the remarkable retrotransposon Tvv1-Δ3460 at its insertion site and the SSR polymorphism within its LTRs. By studying a collection of Vitaceae accessions, we showed the relevance of Tvv1-Δ3460-based markers to distinguish in a simple way most of the Vitis accessions, and to reveal clonal polymorphism within different varieties.

Materials and methods 1. Plant material and DNA extraction

The plant material consisted in Vitaceae accessions divided into three groups. The Vitaceae group comprised Ampelopsis (5 accessions),Parthenocissus (1 accession), and Vitis genera (Muscadinia rotundifolia (4 accessions), Asian species (6 accessions), North American species (16 accessions))and inter-specific hybrids (2 accessions). The V. vinifera group contained cultivated grapevine varieties (52 accessions) and wild vines (6 accessions). Due to the dioicity of wild vines, a male and a female were chosen for each origin of sampling: Ste Croix en Plaine (53) and Mandeure (C25) in France, and Martigny (50) in Switzerland. All accessions are kept in the ampelographic collection of INRAE-Colmar (France). In addition, the segregation of the LTR-SSR alleles was assessed in a progeny of Riesling x Gewurztraminer comprising 11 randomly chosen individuals. The third group comprised seven wine grape varieties represented by a random collection of certified clones conserved in the French national repository (ENTAV, Le Grau du Roi, France), and by accessions recovered from field selections and kept in different germplasm repositories at INRAE, the French National Research Institute for Agriculture, Food and Environment : Cabernet franc (17 clones) , Cabernet-Sauvignon (37 clones), Chenin blanc (19 clones), Grolleau (48 clones), Pinot noir (23 clones), Riesling (27 clones), and Savagnin (45 clones including 22 Savagnin blanc, 19 Gewurztraminer, 4 Savagnin rose).

Total DNA was purified from young expanded leaves from individual plants using Dneasy TM Plant Mini-Kit (Qiagen, Hilden, Germany) as described by the supplier.

2. PCR conditions and fragment analysis

According to Moisy et al. (2008a), the internal PCR products A-750 and B-1500 characterised the Tvv1-Δ3460 copy (Figure1). Due to the presence of full-length copies of Tvv1 elsewhere in the genome, both amplifications also produce 4,3Kb and 4Kb long fragments respectively (Moisy et al., 2008a). When present at its insertion site, Tvv1-Δ3460 was revealed by a long-range PCR product 2500 bp long, F-2500, using Pvv78x primers designed in the host flanking regions of the insertion. These primers also amplify the empty site, E-450, as well as a potential full-length Tvv1 element that can be inserted at the site (Moisy et al., 2008a). To take advantage of the polymorphism of the microsatellite stretch located in both LTR sequences of Tvv1-Δ3460, 5' and 3' LTR-SSR markers primers were designed using Primer3 software (Rozen & Skaletsky, 1998) and synthesised by MWG Biotech AG (Ebersberg, Germany). One primer of each pair was HEX and 6-FAM fluorophore-labelled (PE Applied Biosystems, Warrington, UK) respectively to amplify the 5' and 3' LTR-SSR markers in multiplex (Table 1). All primer locations are given in Figure 1 and all new sequences in Table 1. PCR amplifications were carried out according to Hocquigny et al. (2004). The programme consisted of the following steps: 5 min at 94 °C, followed by 30 cycles of 30 s at 92 °C, 30 s at 52 °C, 30 s at 72 °C, and a final extension step of 7 min at 72 °C. PCR fragments were resolved on an automated 310C ABI PRISM DNA sequencer (PE Applied Biosystems, Foster City, CA), and sized with an ROX labeled-internal standard (50-654 bp) (PE Applied Biosystems, Foster City, CA). Microsatellite alleles were scored using GenScan (version 3.1) and Genotyper (version 2.5.2) software (PE Applied Biosystems, Foster City, CA). All polymorphisms were confirmed by at least two analysis.

Figure 1. Schematic representation of the three alleles of the locus.

Arrows boxes represent LTRs including the SSR stretch, the black box the UTL and the triple line ORF regions. The 5 bp-sequence duplicated to flank the retrotransposon is represented by a triangle. Dotted lines represent the 5' deletion of 5'ΔTvv1-Δ3460 and of its flanking region. Black arrows represent primers used in the study, whose numbers are shown in Table 1. Primers 1 and 2 amplify 5'LTR-SSR and primers 3 and 4 amplify 3'LTR-SSR. Pvv78x primers, located in the host region flanking the insertion, amplify either Tvv1-Δ3460 (F-2500) or the empty site (E-450). Primer positions are given according to the LTRs. Primers that generate A-750 an B-1500 fragments are given in Moisy et al. (2008a).

Figure 1. Schematic representation of the three alleles of the locus.

Arrows boxes represent LTRs including the SSR stretch, the black box the UTL and the triple line ORF regions. The 5 bp-sequence duplicated to flank the retrotransposon is represented by a triangle. Dotted lines represent the 5' deletion of 5'ΔTvv1-Δ3460 and of its flanking region. Black arrows represent primers used in the study, whose numbers are shown in Table 1. Primers 1 and 2 amplify 5'LTR-SSR and primers 3 and 4 amplify 3'LTR-SSR. Pvv78x primers, located in the host region flanking the insertion, amplify either Tvv1-Δ3460 (F-2500) or the empty site (E-450). Primer positions are given according to the LTRs. Primers that generate A-750 an B-1500 fragments are given in Moisy et al. (2008a).

Table 1. 5’LTR-SSR and 3’LTR-SSR primers.
titre du tableau
marker primer name nb location label sequence
5'LTR-SSR 5_3460_fw 1 5' Tvv1-D3460 host region CAGAGTCAAT TTCCTTCCCC AT
5_3460_rv 2 UTL Tvv1-3460 HEX CGTGACCCAA GAAGAAAAAG AA
3'LTR-SSR 3_3460_fw 3 Rnase Tvv1-3469 FAM AGAGCAACTT GGGGATATTT TT
3_3460_rv 4 3' Tvv1-D3460 host region AGTCATTTGG AACCAGTGGA TC

Results

The presence/absence of Tvv1-Δ3460 at its expected insertion site was revealed by three PCR amplifications. The A-750 and B-1500 fragments, which overlap the specific internal region of Tvv1-Δ3460, characterised this element (Moisy et al., 2008a). The Pvv78x primers designed in the host flanking regions of the insertion amplified a 2500 bp-long fragment, F-2500, when Tvv1-Δ3460 was present. Conversely, this pair of primers produced a 450 bp-long fragment, E-450, when the site was empty. In some accessions, A-750 was amplified but not B-1500 or E-450. This combination of PCR fragments revealed a new element whose 5'LTR was truncated, which was named 5'ΔTvv1-Δ3460. No amplification of this new element was possible using the Pvv78x primers leading to the conclusion that its 5'host flanking region must have been deleted as well. Finally, the insertion of a full-length Tvv1 copy, with an average size of 5 kb, was investigated at the given site by long-range PCR using Pvv78x primers. Among the accessions in the study, none revealed the expected 5,5 Kb-long fragment.

In addition, as can be seen in Table 1, two markers, 5’ and 3’ LTR-SSR, were developed to characterise Tvv1-Δ3460 LTRs. 5’LTR-SSR was amplified using primer 5_3460_fw, annealing upstream of Tvv1-Δ3460, and paired with primer 5_3460_rv designed in the UTL sequence of the element. Similarly, 3’LTR-SSR was amplified using primer 3_3460_fw, designed in the RNAse sequence of Tvv1-Δ3460, and paired with primer 3_3460_rv located downstream of the element.

1. Tvv1-Δ3460 in Vitaceae species

A collection of 34 Vitaceae species (Table 2) was investigated for the presence/absence of Tvv1-Δ3460. Ampelopsis species did not amplify A-750, B-1500 or E-450, which is most likely due to a lack of homology of the primers designed from the sequence of V. vinifera with the target sequence of Ampelopsis. The Parthenocissus, the 4 Muscadinia and 5 of 24 Vitis accessions amplified E-450 and not A-750 or B-1500, leading to the conclusion that Tvv1-Δ3460 is absent in these accessions, while the Pvv78x primers are effective at amplifying the empty site.

Of the remaining 19 Vitis, 14 accessions that amplified both A-750 and B-1500, but not E-450, kept Tvv1-Δ3460 in a homozygous state. Conversely, four accessions displayed A-750, B-1500 and E-450 indicating the presence of Tvv1-Δ3460 combined with its empty site. Finally, Vitis rupestris du Lot amplified A-750, but not B-1500 or E-450. This new pattern of amplification revealed the new element 5’ΔTvv1-Δ3460. In this collection, 9 of the 18 North American Vitis or hybrids of North American accessions amplified the empty site of insertion of Tvv1-Δ3460, but did not amplify any of the 6 Asian Vitis.

The polymorphism of the LTRs of the inserted elements were investigated using 5’ and 3’ LTR-SSR markers (Table 1). In the collection of the 19 Vitaceae species hosting at least one copy of Tvv1-Δ3460, a total of 18 alleles of 240 to 290 bp in size and 16 alleles of 322 to 371 bp in size were scored for 5’and 3’LTR-SSR markers respectively. The two LTR-SSR markers defined a specific genotype for all of these Vitis species, except for V. riparia Millardet and V. riparia Muller, which shared the same genotype. The LTR-SSR genotype of V. rupestris du Lot is consistent with the association of two copies of 5’ΔTvv1-Δ3460, whose 3’LTR were different in size.

Table 2. Distribution of Tvv1-Δ3460, 5’ΔTvv1-Δ3460 and the empty site within the Vitaceae panel and genotypes at 5’LTR-SSR and 3’LTR-SSR.
titre du tableau
Accession name Geographic origin B-1500 A-750 E-450 F-2500 5’LTR-SSR 3’LTR-SSR
1500 bp 750 bp 450 bp 2500 bp allele 1 allele 2 allele 1 allele 2
Ampelopsis aconitifolia Asia - - - - -
Ampelopsis cordata North America - - - - -
Ampelopsis heterophylla Asia - - - - -
Ampelopsis japonica Asia - - - - -
Ampelopsis pedonculata Asia - - - - -
Parthenocissus quinquefolia North America - - + - - -
Muscadinia rotundifolia Carlos North America - - + - - -
Muscadinia rotundifolia Dulcet North America - - + - - -
Muscadinia rotundifolia Régale North America - - + - - -
Muscadinia rotundifolia YxC North America - - + - - -
Vitis aestivalis North America - - + - - -
Vitis amurensis Asia + + - 276 285 322 324
Vitis arizonica North America + + - 271 283 343 355
Vitis armata Asia + + - 271 278 323
Vitis berlandieri Colombard hybrid + + + 240 324
Vitis berlandieri Planchon North America + + + + 277 322
Vitis candicans North America - - + - - -
Vitis cinerea North America - - + - - -
Vitis cordifolia 9 couderc hybrid - - + - - -
Vitis Davidii Asia + + - 279 283 322 324
Vitis doaniana North America + + + 290 327
Vitis ishikari Asia + + - 268 282 322
Vitis labrusca Concorde North America + + - 277 332 349
Vitis labrusca Isabelle North America + + - 276 330 334
Vitis linsecumii North America - - + - - -
Vitis monticola Large Bell North America + + + 269 328
Vitis reticulata Asia + + - + 269 322 361
Vitis riparia Gloire de M North America + + - 252 331
Vitis riparia Millardet North America + + - 273 331 355
Vitis riparia Muller North America + + - 273 331 355
Vitis rubra North America + + - + 264 274 333 337
Vitis rupestris du Lot North America - + - - - 327 371
Vitis titania Asia + + - 252 280 328 333
Vitis vulpina North America + + - + 275 331

The sign ‘+’ indicates amplification of the fragment and ‘-‘ no amplification. Tvv1-Δ3460 is characterised by the amplification of A-750, B-1500 and F-2500, but not of E-450. 5’ΔTvv1-Δ3460 is characterised by the amplification of A-750, but not of B-1500, E-450 or F-2500. The empty site is characterised by the amplification of E-450, but not of A-750, B-1500 or F-2500.

2. Tvv1-Δ3460 in Vitis vinifera

The presence/absence of Tvv1-Δ3460 was also evaluated in a collection of V. vinifera accessions comprising 52 cultivated varieties and 6 wild vines. On the one hand, all the accessions amplified A-750, while none amplified E-450, indicating the prevalence of Tvv1-Δ3460insertionin the V. vinifera species. On the other hand, 55 of the 58 accessions amplified B-1500. The three varieties Mourvèdre, Roussanne and Semillon that did not amplify B-1500 presumably hosted only 5’ΔTvv1-Δ3460 (Table 3).

Table 3. Distribution of Tvv1-Δ3460 and 5’ΔTvv1-Δ3460 within the Vitis vinifera panel.
titre du tableau
Accession name B-1500 A-750 E-450 5’LTR-SSR 3’LTR-SSR
1500 bp 750 bp 450 bp Allele 1 Allele 2 Allele 3 Allele 1 Allele 2 Allele 3
Aligoté B + + - 275 284 332 351
Aubin vert B + + - 284 348 351
Auxerrois + + - 284 348 351
Bachet Noir N + + - 278 330 348
Beaunoir + + - 275 278 330 332
Cabernet franc N + + - 277 330 342
Cabernet-Sauvignon N + + - 277 330 348
Carignan N + + - 276 284 346 351
Chardonnay B + + - 275 284 332 351
Chenin B + + - 276 348 352
Cinsaut N + + - 276 330 348
Clairette B + + - 276 278 330 348
Colombard B606 + + - 276 277 279 330 353
Corbeau N + + - 277 330 348
Côt N596 + + - 275 332 348
Folle Blanche B + + - 279 330 340
Franc noir de la Haute Saone + + - 284 348 351
Gamay Blanc Gloriod B + + - 284 332 351
Gamay N + + - 275 284 332 351
Gewurztraminer R 643 + + - 277 330 348
Gouais B + + - 278 284 330 351
Grenache N + + - 275 330 340
Grolleau B + + - 285 349 351
Knipperlé 61D + + - 275 284 332 351
Marsanne B + + - 277 348
Mauzac B + + - 277 342 348
Melon B + + - 284 348 351
Merlot N + + - 277 330 348
Mourvèdre - + - - 340 342
Muscat d'Alexandrie B + + - 239 240 276 324 330
Muscat d'Alsace R + + - 240 296 324 340
Muscat cendré 336 B + + - 275 332 348
Muscat de Hambourg N + + - 276 330 336
Muscat de Saumur B + + - 240 324 348
Muscat Ottonel B + + - 240 324 348
Muscat Reine des Vignes B + + - 276 330 348
Muscat petits grains + + - 240 296 324 340
Persan + + - 275 332 348
Peurion N + + - 240 297 324 340
Pinot N162 + + - 275 332 348
Riesling B 49 + + - 285 351
Romorantin B929 + + - 275 278 330 332
Roublot + + - 275 278 330 332
Roussanne B - + - - 340 342
Sacy B + + - 278 330 348
Sauvignon B + + - 274 339 348
Sémillon B - + - - 342 332
Sylvaner 50 + + - 276 284 330 351
Syrah n + + - 274 332 348
Tannat + + - 274 278 330 332
Ugni Blanc B + + - 240 276 324 330
Viogner B + + - 278 330 342
VSil50K + + - 275 330 332
VSil50l + + - 275 277 328 330 348
VSil.53I + + - 269 275 277 330 348
VSil.53J + + - 275 277 330 342
VSil.C25S2B + + - 275 332
VSil.C1S6 + + - 274 284 332 351

The sign ‘+’ indicates amplification of the fragment and ‘-‘ no amplification. Tvv1-Δ3460 is characterized by A-750: +, B-1500: +, E-450: - and 5’ΔTvv1-Δ3460 by A-750: +, B-1500: -, E-450 :-. The genotypes at 5’LTR-SSR and 3’LTR-SSR are given.

In the V. vinifera collection, the amplification of 5’ and 3’ LTR-SSR markers each provided 13 alleles of 239 to 297 pb and 324 to 353 bp in size respectively. These two markers made it possible to characterise 38 genotypes which differed from those previously characterised in the collection of 34 Vitaceae species. Twenty-nine genotypes were displayed by one variety only, 5 genotypes by 2 varieties, 1 genotype by 3 varieties and 4 genotypes by 4 varieties. Varieties sharing the same genotype were known to be related such as the progeny of Pinot noir × Gouais blanc cross. Out of the 58 Vitis vinifera, 22 showed two alleles at each locus, indicating the presence of two full copies of Tvv1-Δ3460 with polymorphic LTRs. Thirty-one accessions, among them Pinot noir and Gewurztraminer, displayed one allele for 5’LTR-SSR and 2 alleles for 3’LTR-SSR. These varieties associated either two copies of Tvv1-Δ3460 with 5’LTRs of the same length and polymorphic 3’LTRs, or one copy each of Tvv1-Δ3460 and 5’ΔTvv1-Δ3460. To clarify the 5’ LTR-SSR genotype of Pinot noir (allele 275), 13 varieties known to be Pinot noir × Gouais blanc progeny (Bowers etal. , 1999) were considered. All of these varieties displayed one allele of Gouais blanc [278:284], either alone or associated with allele 275 of Pinot noir. This result suggests the segregation of a null allele of Pinot noir resulting from the lack of 5’LTR of 5’ΔTvv1-Δ3460. Thus, Pinot noir is heterozygous [275:-], because of the association of Tvv1-Δ3460 and 5’ΔTvv1-Δ3460, as well as Auxerrois or Melon [284:-].

The 5’ LTR-SSR marker null allele resulting from the 5’ΔTvv1-Δ3460 insertion is quite common and carries out segregation within many V. vinifera varieties. For example, the genotype of Cabernet-Sauvignon [277], whose parents are Cabernet franc [277] and Sauvignon B [274], must be heterozygous for the null allele [274: -] inherited from Sauvignon B [274:-].

Finally, two varieties, Marsanne and Riesling, and a V. viniferassp. silvestris accession, VSil.C25S2B, amplified only one allele at each locus. As none of these accessions amplified the empty site of insertion, their genotypes may result from the association of either two copies of Tvv1-Δ3460 with LTRs of the same length, or of Tvv1-Δ3460 and 5’Δ- Tvv1-Δ3460, which have 3’LTR of the same length. To clarify the genotypes of Riesling (alleles 285 at 5’LTR-SSR and 351 at 3’LTR-SSR) and Gewurztraminer (alleles 277 at 5’LTR-SSR and 330:348 at 3’LTR-SSR), the segregation of these alleles was analysed in 11 individuals of the progeny of a Riesling x Gewurztraminer cross. At 5’LTR-SSR locus, one descendant amplified both 277 and 285, three only 277, four only 285 and 3 did not amplify the marker. This result indicated the hemizygous genotypes of Riesling [285:-] and Gewurztraminer [277:-] at 5’LTR-SSR. Conversely, all progeny displayed allele 351 of Riesling at 3’LTR-SSR, in association with either allele 330 (7 progeny) or allele 348 (7 progeny) of Gewurztraminer. The genotype of Riesling for the 3’LTR-SSR marker is therefore homozygous [351:351], while that ofGewurztraminer is heterozygote [330:348]. This pattern of amplification leads to the conclusion that the two varieties hosted Tvv1-Δ3460 and 5’ΔTvv1-Δ3460, but Riesling has elements which display 3’LTR of identical size, while those of the Gewurztraminer elements are different.

3. LTR-SSR polymorphism within seven French wine grape variety collections

The capacity of the two Tvv1-Δ3460 LTR-SSR markers to reveal intra-varietal polymorphism was evaluated in seven clone collections of wine grape varieties: Cabernet franc, Cabernet-Sauvignon, Chenin blanc, Grolleau, Pinot noir, Riesling, Savagnin. These collections comprised a total of 216 accessions of certified clones and introductions which had been preserved in French repositories. These collections of clones were chosen for comparison since they had previously been evaluated with 12 SSR markers for the collections of Grolleau, Cabernet franc, Chenin blanc, and with 30 markers for those of Cabernet-Sauvignon, Pinot noir, Riesling and Savagnin (Pelsy et al., 2010).

In this study, the reference genotype was defined as that the genotype shared by the majority of the accessions of a collection (Table 4). All varieties amplified one allele at locus 5’LTR-SSR and six amplified two alleles at locus 3’LTR-SSR, except Riesling. Two variants were detected at locus 5’LTR-SSR: one Savagnin that displayed a 275 bp-long allele, instead of the 277 bp-long reference allele, and one Cabernet franc that showed the heterozygous genotype [277:285], instead of the reference genotype, which was most probably homozygous [277:277]. With the appearance of 10 new alleles, the 3’LTR-SSR locus was far more susceptible to polymorphism than 5’LTR-SSR (2 new alleles). One to 5 variant genotypes were observed in 5 of the varieties, mainly as a result of the addition of a new allele to the reference genotype, leading to triple-allele genotypes characterising periclinal chimeras.

Thus, clonal polymorphism was revealed in 5 of the 7 clone collections. Four variants were characterised out of the 17 clones of Cabernet franc, 3 out of the 19 clones of Chenin, 2 out of the 23 clones of Pinot noir, 3 out of the 27 clones of Riesling and 10 out of the 45 clones of Savagnin, most of the latter being Savagnin blanc. One clone of Cabernet franc was polymorphic at both loci. Altogether, it was possible to unambiguously distinguish 11 clones out of the 216 studied by a unique genotype, while 2 to 5 variants shared the same genotype.

Conversely, no variants were detected among the 37 and 48 clones of Cabernet-Sauvignon and Grolleau respectively.

Table 4. 5’LTR-SSR and 3’LTR-SSR genotypes of clones belonging to seven wine grape varieties.
titre du tableau
Variety total clone nb certified clone nb 5’LTR-SSR 3’LTR-SSR
Reference genotypes Variant genotypes Variant clone nb Reference genotypes Variant genotypes Variant clone nb
Cabernet franc 17 2 277 277-285 1 330-342 328-342 1
330-332-342 1
330-350 1
Cabernet-Sauvignon 37 22 277 - 330-348 -
Chenin 19 0 276 - 348-352 348-350-352 3
Grolleau 48 285 - 349-351 -
Pinot noir 23 23 275 - 332-348 332-348-350 1
332-334-348 1
Riesling 27 5 285 - 351 339-351 1
351-353 2
Savagnins 45 7 277 275 1 330-348 330-344-348 1

New alleles are indicated in bold.

Discussion

Having effective and easy-to-use markers capable of quickly identifying Vitis species, grapevine varieties and clones belonging to the same variety is still a challenge today. In this study, we describe a novel set of molecular markers based on a combination of the variation in the presence of the remarkable retrotransposon Tvv1-Δ3460 at its insertion locus and the SSR polymorphism of its LTRs to assess grapevine diversity. We used it to explore species, varietal and intra-varietal diversity. In 4 PCR amplifications (B-1500, E-450 and 5’ and 3’ LTR-SSR in multiplex), it was possible to characterise all the species and varieties. In addition, the 3’LTR-SSR marker was shown to be highly relevant in revealing clonal polymorphism.

1. Presence vs absence of Tvv1-Δ3460 is informative

The Tvv1-Δ3460 locus showed different alleles in the Vitaceae species revealed by the long-range PCR using Pvv78x primers. Ampelopsis accessions did not amplify Tvv1-Δ3460 or its empty site, which is likely because the primers designed from a V. vinifera sequence were not homologous enough for their target sites in Ampelopsis, due to the phylogenetic distance between both genera. The Parthenocissus, the four Muscadinia and five of the American Vitis or hybrid accessions (V.aestivalis , V.candidans , V. cinerea, V.cordifolia Couderc and V.linsecumii ) only amplified the empty site of the locus (Figure 2). This result agrees with previous studies which showed the divergence between the genera Ampelopsis and Vitis, but brought Parthenocissus quinquefolia as close as Muscadinia to Vitis (Pelsy, 2007).

Four American Vitis or hybrid accessions (V.berlandieri Colombard, V. berlandieri Planchon, V. doaniana, V. monticola Large Bell) amplified both the full and the empty site of Tvv1-Δ3460 (Figure 2). All the remaining accessions, including the Asian accessions and the wild or cultivated V. vinifera, displayed a Tvv1-Δ3460 element, but never the empty site. These results indicate that the Tvv1-Δ3460 insertion is specific to the Vitis species. Either the allele with the full site or the one with the empty site of Tvv1-Δ3460 were present homozygous or heterozygous in the American Vitis or hybrids accessions. Conversely, all the Asian accessions and V. vinifera varieties hosted a Tvv1-Δ3460 insertion at the locus. This insertion was most probably dispersed through natural intermixing, due to the close proximity of Asian Vitis with the European species. However, the empty site which is remained in half of American Vitis confirms the disjunction between the Old and New World (Zecca et al., 2012).

The formation of deleted elements may occur either during the retrotransposition process prior to integration, or by illegitimate recombination within an integrated full-length element. Since no full copy of Tvv1 was amplified via long-range PCR amplification using the Pvv78x pair, the large internal deletion characteristic of Tvv1-Δ3460 most likely occurred during the retrotransposition process before integration.

Finally, 5’ΔTvv1-Δ3460, a new element that was subject to a deletion of the 5’ region of Tvv1-Δ3460 and of its 5’host region, was identified in V. rupestris du Lot and in many V. vinifera varieties. Nevertheless, the 4 PCR does not allow us to conclude that the V. rupestris du Lot and V. vinifera varieties share the very same 5’ΔTvv1-Δ3460 element, or that independent deletions lead to different 5’ΔTvv1-Δ3460 elements.

In some varieties, two copies of Tvv1-Δ3460 or of 5’ΔTvv1-Δ3460 can be combined, such as in Sylvaner or Semillon respectively, but with 3’LTRs of different lengths. Other varieties, such as Pinot noir, Riesling and Gewurztraminer, combine Tvv1-Δ3460 and 5’ΔTvv1-Δ3460. The elements of Pinot noir and Gewurztraminer have 3’LTRs of different lengths, while those of Riesling have 3’LTR of the same length (Figure 2).

2. 5’ and 3’ LTR-SSR: highly informative markers which can be used to identify Vitis accessions

In the Vitis species which hosted at least one copy of Tvv1-Δ3460, the 5’ and 3’ LTR-SSR markers are well-conserved, defining a specific genotype for all of these Vitis species. Moreover, when considering the 58 V. vinifera accessions (cultivated grapevine varieties and wild vines), the same group studied with 14 SSR markers showed that the number of alleles detected per locus ranged from 1 for VMC1e11a to 14 for VVS2, with an average of 8.6 (Pelsy, 2007). 5’ and 3’ LTR-SSR each amplified 13 alleles; they are therefore among the most informative markers for identifying V. vinifera accessions.

 3. LTR-SSR polymorphism within seven French wine grape variety collections

Not all microsatellite markers can reveal clonal polymorphism within clone collections, and therefore a standard set of five microsatellite markers (VMC3a9,VMC5g7, VVS2, VVMD30, and VVMD32) has been proposed (Pelsy et al., 2010).

It was possible to develop 5’ and 3’ LTR-SSR markers due to the systematic presence of an insertion - either of Tvv1-Δ3460 or 5’ΔTvv1-Δ3460 - at the locus of different grape varieties, and to a TA microsatellite stretch in the LTR sequences of these Tvv1 elements. Using these markers, polymorphism was assessed in the clone collections of seven varieties each comprising 17 to 48 clones.

The 3’ LTR-SSR marker revealed heterozygous genotypes for all varieties, except Marsanne and Riesling. The analysis of a Riesling progeny showed that this variety is homozygous for this marker [351-351]. This assertion is confirmed by the identification of two variant clones of Riesling, which showed two new genotypes at this locus, [339:351] and [351:353], and two new alleles, 339 and 353, which derived from the reference allele 351. However, it was not possible to determine from these data whether Marsanne hosts Tvv1-Δ3460 with identical 5’ and 3’LTRs or Tvv1-Δ3460 in association with 5’ΔTvv1-Δ3460, both with an identical 3’LTR.

Variant genotypes were revealed by 3’LTR-SSR in five of the seven clone collections. Thus, this marker is more effective than all the previously identified SSR markers: indeed, VMC3a9, VMC5g7 and VVS2 revealed variants in four of these collections of clones, while VVMD30 and VVMD32 in three and two collections respectively.

The 5 'LTR-SSR marker, meanwhile, often only amplified a fragment in the studied varieties, as was the case for Pinot noir, Riesling and Gewurztraminer, which are all hemizygous due to the combination of Tvv1-Δ3460 and 5’ΔTvv1-Δ3460, and 5’ΔTvv1-Δ3460 resulting in a null allele with 5’ LTR-SSR. Therefore, the 5’ LTR-SSR marker is less effective at revealing clonal polymorphism than the 3’ LTR-SSR marker.

In a previous study, the same clones were studied with SSRs to identify a standard set of five markers (VMC3a9, VMC5g7, VVS2, VVMD30 and VVMD32) which revealed clonal polymorphism within different varieties (Pelsy et al., 2010). The addition of the LTR-SSR markers improved the identification of the clones in 4 of the 7 varieties by distinguishing new clones. Therefore, while the standard set markers made it possible to assign a unique genotype to 1 to 5 clones in the collections, the additional LTR-SSR markers allowed 2 to 4 new clones with a unique genotype to be identified, thus increasing the number of distinguishable clones; for example, for Savagnins, the number of clones displaying a unique genotype increased from 5 to 9 (Table 5).

Table 5. number of distinguishable clones using one of the SSR sets and adding LTR-SSR markers.
titre du tableau
Variety total clone nb distinguishable clones nb
SSR set 1 SSR set 2 SSR set 3 LTR-SSR total
Cabernet franc 17 3 2 5
Chenin 19 3 0 3
Pinot noir 23 5 2 7
Riesling 27 1 2 3
Savagnins 45 5 4 9

SSR set 1 comprises VMC3a9, VMC5g7, VVS2, VVMD30, and VVMD32; set 2 comprises VMC3a9, VMC5g7, VVS2 and VVMD30 and set 3 comprises VMC3a9, VMC5g7, VVS2 and VVMD32.

Genomic analysis provides access to a large number of SNPs and InDels that can be exploited to define markers capable of characterising interclonal diversity in different grapevine varieties. Nevertheless, these approaches are limited by sequencing technology or the lack of a reference genome for the studied varieties (Roach et al., 2018). Furthermore, SSR markers, which are highly transferable between different varieties and Vitis species, are simple tools which can be used to identify variant clones in a wide range of varieties. Thus, to increase the capacity for revealing clonal polymorphism with SSR markers, we recommend that 3 ′ LTR-SSR be added to the standard set of previously characterised VMC3a9, VMC5g7, VVS2, VVMD30 and VVMD32 (Pelsy et al., 2010).

Conclusion

Deleted retroelements are unique, very stable and can be considered as Mendelian loci. They can be identified within different families of retrotransposon by PCR amplification with primers derived from the LTRs or the conserved PBS or PPT sequences. Using available grapevine genome sequences, their flanking sequences can then be identified to design primers that will yield easy-to-use co-dominant markers. Their specific dispersion in the Vitaceae make these markers valuable tools for studying their diversity.

 Acknowledgements

We would like to thank Lionel Ley and the members of the experimental unit of INRAE-Colmar for their very valuable technical support in the production of plants both in the greenhouse and in the vineyard. We would also like to thank Cyndy Ponama, Gisèle Butterlin, Roxanne Hardy and Angélique Ardiller for the laboratory experiments.

Bowers J. E. , Boursiquot J. M. , This P. , Chu K. , Johansson H. , Meredith C. P. , 1999. Historical genetics : the parentage of Chardonnay, Gamay, and other wine grapes of northeastern France. Science, 285, 1562-1565. https://doi.org/10.1126/science.285.5433.1562. Carrier G. , Le Cunff L. , Dereeper A. , Legrand D. , Sabot F. , Bouchez O. , Audeguin L. , Boursiquot J.-M.& This , P. , 2012. Transposable Elements Are a Major Cause of Somatic Polymorphism in Vitis vinifera L. PLoS ONE 7(3): e32973. https://doi.org/10.1371/journal.pone.0032973. Di Gaspero G. , Peterlunger E. , Testolin R. Edwards , K. J. & Cipriani , G. , 2000. Conservation of microsatellite loci within the genus Vitis. Theoretical and Applied Genetics 101, 301–308. https://doi.org/10.1007/s001220051483. Gupta P. K. , Varshney R. K. , Sharma P. C. , Ramesh B. , 1999. Molecular markers and their applications in wheat breeding. Plant Breeding, 118(5), 369-390. https://doi.org/10.1046/j.1439-0523.1999.00401.x. Hocquigny S. , Pelsy F. , Dumas V. , Kindt S. , Héloir M. C. , Merdinoglu D. , 2004. Diversification within grapevine cultivars goes through chimeric states. Genome, 47(3), 579-589. https://doi.org/10.1139/g04-006. Kobayashi S. , Goto-Yamamoto N. , Hirochika H. , 2004. Retrotransposon-induced mutations in grape skin color. Science, 304, 982. https://doi.org/10.1126/science.1095011. Merdinoglu D. , Butterlin G. , Bevilacqua L. , Chiquet V. , Adam-Blondon A.-F. , Decroocq S. , 2005. Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Molecular Breeding, 15, 349-366. https://doi.org/10.1007/s11032-004-7651-0. Moisy C. , Blanc S. , Merdinoglu D. , Pelsy F. , 2008a. Structural variability of Tvv1 grapevine retrotransposons can be caused by illegitimate recombination. Theoretical and Applied Genetics, 116(5), 671-682. https://doi.org/10.1007/s00122-007-0700-4. Moisy C. , Garrison K. , Meredith C. P. , Pelsy F. , 2008b. Characterization of ten novel Ty1 copia-like retrotransposon families of the grapevine genome. BMC Genomics, 9, 469. https://doi.org/10.1186/1471-2164-9-469. Pelsy F. , 2007. Untranslated leader region polymorphism of Tvv1 , a retrotransposon family, is a novel marker useful for analyzing genetic diversity and relatedness in the genus Vitis. Theoretical and Applied Genetics, 116(1), 15-27. https://doi.org/10.1007/s00122-007-0643-9. Pelsy F. , Hocquigny S. , Moncada X. , Barbeau G. , Forget D. , Hinrichsen P. , Merdinoglu D. , 2010. An extensive study of the genetic diversity within seven French wine grape variety collections. Theoretical and Applied Genetics, 120(6), 1219-1231. https://doi.org/10.1007/s00122-009-1250-8. Pelsy F. , Merdinoglu D. , 2002. Complete sequence of Tvv1, a family of Ty1 copia-like retrotransposons of Vitis vinifera L., reconstituted by chromosome walking. Theoretical and Applied Genetics, 105(4), 614-621. https://doi.org/10.1007/s00122-002-0969-2. Roach M. J. , Johnson D. L. , Bohlmann J. , van Vuuren H. J. J. , Jones S. J. M. , Pretorius I. S. , . . . Borneman A. R. , 2018. Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay. PLOS Genetics, 14(11), e1007807. https://doi.org/10.1371/journal.pgen.1007807. Rozen S. , Skaletsky H. J. , 1998. Primer3. Retrieved from http://www-genome.wi.mit.edu/genome_software/other/primer3.html. Schulman A. , 2006. Molecular markers to assess genetic diversity. Euphytica, 158, 313–321. https://doi.org/10.1007/s10681-006-9282-5. This P. , Jung A. , Boccacci P. , Borrego J. , Botta R. , Costantini L. , . . . Maul E. , 2004. Development of a standard set of microsatellite reference alleles for identification of grape cultivars. Theoretical and Applied Genetics, 109(7), 1448-1458. https://doi.org/10.1007/s00122-004-1760-3. Verriès C. , Bès C. , This P. , Tesnière C. , 2000. Cloning and characterization of Vine-1, a LTR- retrotransposon-like element in Vitis vinifera L., and other Vitis species. Genome, 43(2), 366-376. https://doi.org/10.1139/g99-139. Vondras A. M. , Minio A. , Blanco-Ulate B. , Figueroa-Balderas R. , Penn M. A. , Zhou Y. F. , . . . Cantu D. , 2019. The genomic diversification of grapevine clones. BMC Genomics, 20(1), 19. https://doi.org/10.1186/s12864-019-6211-2. Zecca G. , Abbott R. , Sun W.-B. , Spada A. , Sala F. , Grassi F. , 2012. The timing and mode of evolution of wild grapes (Vitis). Mol Phylogenet Evol, 62, 736 - 747. https://doi.org/10.1016/j.ympev.2011.11.015.