In Greco Bianco, both morphological and molecular markers were used to identify distinct clonal groups (Villano et al., 2023). Even within this minor variety, an “exploitable variation” in vigour and yield per vine was detected. Certain clones allocated a larger proportion of biomass to vegetative growth, whereas others prioritised reproductive output. This shows that the vigour–yield balance is not fixed at the varietal level, but is a clone-specific trait that can be matched to site potential, training system and management objectives. Similarly, clonal evaluations of Tempranillo in the Rioja region reported wide ranges in production parameters (Portu et al., 2024). These studies highlighted a key agronomic trade-off: the relationship between yield and quality is often non-linear. Some clones maintained high yields without the pronounced decline in polyphenolic parameters typically associated with over-cropping, indicating that the genotypic capacity to ripen fruit under load differs between clones. Altogether, these results confirm that clonal material does not form a homogeneous block, but rather a set of agronomically distinct options within the same variety.

A specific, highly heritable component of yield is fruit set, defined as the proportion of flowers that develop into berries. In Malbec, clonal trials have quantified natural genetic variation in fruit-set rates (Calderón et al., 2025). These trials identified clones with consistently high coulure (flower drop) and others with high fruit retention. This variation is particularly relevant in the context of climate change, where adverse weather during flowering (heat waves, cold spells, or rain events) is expected to become more frequent. Clones with naturally higher fruit-set potential provide a buffer against “sink-limited” years, contributing to more stable yields across vintages. This supports the view that yield stability depends not only on climate and management practices but also on intrinsic genetic traits that vary at the clonal level.

In Merlot selections from California’s Central San Joaquin Valley, Fidelibus et al. (2007) reported clonal differences in the rate of soluble-solids accumulation and acidity degradation (Fidelibus et al., 2007). At a common harvest date, distinct clones showed significant variation in soluble solids and pH. This phenological spread can be exploited to stagger harvest operations, distribute winery loads in time or select “low-sugar accumulation” clones as a strategy to mitigate the impact of warming on potential alcohol. Similar observations were reported for Cabernet franc in van Leeuwen et al. (2013).

In aromatic varieties, variation in secondary metabolites is the defining quality parameter. Clonal differences in terpene profiles have been documented both at the molecular level, through variants in the DXS gene that determine Muscat flavour, including in the Chardonnay musqué clone (Emanuelli et al., 2010; Duchêne et al., 2009), and at the oenological level, with clone-specific aroma profiles persisting from berry to sparkling wine (Prévide Bernardo et al., 2022). These examples also illustrate the difficulty of drawing a sharp boundary between varietal and clonal variation, since the same biosynthetic genes can harbour both inter-varietal polymorphisms and intra-varietal somatic mutations. Beyond aromatics, clonal variation in phenolic composition represents an equally relevant quality parameter. In Tempranillo, clonal evaluations revealed a wide range in colour intensity and anthocyanin content, with some clones showing stable expression across years and sites and others showing strong environmental dependence (Portu et al., 2024). In Malbec, a large‑scale analysis of 131 clones grown in a single vineyard demonstrated substantial clonal variation in anthocyanin profiles, largely driven by differences in malvidin derivatives, and linked these differences to differential expression of genes involved in hydroxylation (F3′5′H), methylation (OMT1) and transport (AM2) of anthocyanins (Muñoz et al., 2014). These patterns reveal substantial mechanistic diversity in anthocyanin biosynthesis among clones. Combined with evidence of genotype-by-environment interactions documented in multi-site studies (Pagliarani et al., 2019; Robinson et al., 2025), they support the hypothesis that some clones may perform differently under contrasting environmental conditions, making them particularly relevant for strategies aimed at colour stability and phenolic balance under climate-change scenarios.

Water scarcity is emerging as one of the major challenges for viticulture in many traditional growing regions. Recent physiological studies have started to unravel the mechanisms underlying intravarietal variability in water-use efficiency (WUE) (Tortosa et al., 2016). In Grenache, for example, clones with contrasting WUE strategies were identified, and these differences were linked to intrinsic variations in stomatal conductance ( g _s) and mesophyll conductance ( g _m) (Buesa et al., 2022). These results show that “water-saver” clones already exist within widely planted varieties and can be targeted to improve drought tolerance without changing cultivar. A comparative analysis of inter- and intra-varietal diversity concluded that, for WUE, the intra-varietal fraction of variance can equal the inter-varietal fraction (Medrano et al., 2015; Tortosa et al., 2016). This finding has profound implications: instead of replacing a variety such as Tempranillo with a more drought-tolerant cultivar (thus altering the regional identity), growers could replant with drought-tolerant clones of Tempranillo, preserving typicity while adapting to warmer and drier conditions.

Adaptation to water deficit is not restricted to above-ground traits. Studies evaluating genetic diversity under osmotic stress have examined root architecture in both rootstocks and scion clones, relating clonal variation in root branching density and depth to the capacity for soil exploration and water uptake (Peiró et al., 2020). Genotypes with more aggressive and deeper root systems are better able to maintain turgor under osmotic stress, thereby sustaining physiological activity during prolonged dry periods. These “hidden” below-ground traits represent an important component of intra-varietal diversity and should be considered when defining drought-adapted clonal material.

Adjusting phenology is another key strategy to mitigate the effects of climate change (Wolkovich et al., 2017). Delayed phenology is an interesting lever for adaptation to climate change, because it shifts grape ripening later in the season when temperatures are lower (van Leeuwen et al., 2024). A study on natural genetic variation in phenology quantified the range of budburst, flowering, and veraison dates among clones within the same variety (Van Houten et al., 2020). Similar patterns emerged in the evaluation of 13 new putative clones of Graciano, where Portu et al. (2025) recorded broader ranges in the timing of four phenological stages (budburst, flowering, veraison, and harvest) compared with two registered clones of the cultivar, highlighting intra-varietal potential for delayed cycles. This intra-varietal plasticity extends to complex traits, as demonstrated by Robinson et al. (2025), who analysed 14 years of data across eight clonal populations (including Pinot noir and Riesling), revealing substantial phenotypic variation in agronomic ( e.g., yield), quality ( e.g., sugar), and disease-related traits, with moderate to high broad-sense heritability. Three phenological stages emerge as key areas where heterogeneity could have a high impact on productivity. Clones with a delayed budburst date could evade late frost in the spring. Moreover, vineyards with a spread in the flowering time could be more tolerant to events like Zonda wind (Norte, 2015) which during short periods of time produce a dramatic increase of the temperature and decrease of atmospheric humidity, both conditions known to severely affect the fruit set by desiccation of stigma (Greer & Weston, 2010) or, more generally, all sorts of adverse conditions during flowering. The identification of “late-ripening” clones in otherwise early-ripening cultivars provides a tool to delay harvest into cooler autumn periods, thereby preserving acidity and aromatic freshness. A delay of 7–10 days in veraison relative to the clonal population mean effectively shifts the ripening window away from the peak of heat stress, which is particularly relevant for regions such as Mendoza or South Australia. Robinson et al. (2025) further highlight strong genetic correlations, underscoring the need for multi-trait selection to balance trade-offs in clonal breeding. This underscores the role of GxE interactions when searching for clonal diversity benefits, as observed in Portu et al. (2025), where environmental variability influenced trait expression across clones.

SSRs target highly polymorphic repetitive regions and became the standard for variety identification because of their high reproducibility and discriminating power among cultivars. However, their mutation rate, though high in evolutionary terms, is often too slow or too stochastic to capture the somatic mutations that accumulate between clonal generations. In cv. Keshmeshi, for example, a combination of SSRs and AFLPs was used to assess diversity among selected clones (Doulati Baneh et al., 2009). Although some clonal variation was detected, the authors concluded that the resolution was limited and that the true variability of the population was likely underestimated. In other words, the markers could not show the fine-scale mutations responsible for many of the observed phenotypic differences.

This limitation was particularly evident in a study of a spontaneous mutant and selected clones of cv. Italia (Fanizza et al., 2003). Despite clear phenotypic divergence (the mutant showed conspicuous morphological differences), AFLP analysis revealed very few polymorphisms. This discrepancy highlighted a fundamental issue: the genetic changes driving somatic variation (often single-nucleotide variants or specific structural rearrangements) may occur outside the restriction sites or primer-binding regions sampled by AFLPs. Similarly, an assessment of the Muscat group, including Muscat of Alexandria clones, used SSRs and AFLPs to evaluate diversity (Regner et al., 2015). While the analyses confirmed varietal identity and separated some accessions, the ability to discriminate closely related clones from selection programs was low. Collectively, these early studies established two key points: clones are genetically distinct entities, but the “signal” of their divergence is often too faint to be detected by low-resolution marker systems.

Carrier et al. (2012) provided a paradigm-shifting demonstration that TEs are a major cause of somatic polymorphism in V. vinifera. Their work showed that inserting retrotransposons such as Gret1 into regulatory regions can drastically alter phenotypes. A classic example is the modulation of berry colour: TE insertions in the VvMybA1 gene cluster can switch anthocyanin synthesis off (producing white grapes) or on (reverting to red or grey variants). This mechanism redefined clonal variation as not merely the slow accumulation of point mutations, but a landscape of potentially dramatic structural changes driven by mobile elements, often activated by stress. Consistently, the white variety of Tempranillo has been shown to carry a large number of genomic rearrangements compatible with such processes (Carbonell-Bejerano et al., 2017).

Beyond individual TE insertions, the grapevine genome is subject to larger-scale structural rearrangements. Pelsy (2010) described chromosome replacements and deletions as frequent drivers of clonal polymorphism, particularly for berry colour. Large deletions or the replacement of one chromosome arm by its homolog (loss of heterozygosity) in regions harbouring colour genes generate the Pinot blanc, gris, and noir variants from a common origin. Population genetics and comparative genomics further reveal their role in clonal divergence, with haplotype-phased assemblies showing hemizygosity in approximately one in seven genes, strong purifying selection against inversions and translocations, and dramatic gene gain and loss in domesticated lineages, including transitions toward asexual reproduction genes and ~8 % accession-specific families, highlighting somatic variation as a driver of intravarietal diversity (Zhou et al., 2019; Long et al., 2024). These “macroscopic” genomic events help to explain why some clones are phenotypically unstable and why AFLPs (which sample the genome randomly) often fail to detect the causal structural changes. A further dimension of clonal structural diversity is periclinal chimerism, in which somatic mutations affect a single meristematic cell layer and generate stable mosaics in which distinct genotypes coexist across tissues of the same plant. This mechanism has been documented in grapevine at the berry colour locus and beyond: chromosome-level replacements confined to one cell layer explain several pale-berry bud sports of Cabernet-Sauvignon and Pinot (Pelsy et al., 2015; Walker et al., 2006), and phased long-read assembly of Merlot has identified 51 to 53 periclinal chimeras per haplotype, several within coding regions (Sichel et al., 2023). For massal selection, chimerism represents an invisible layer of diversity that escapes whole-plant genotyping and can generate phenotypically distinct individuals through cell-layer sorting during vegetative propagation.

In a landmark study, Roach et al. (2018) used population-level sequencing of multiple Chardonnay clones to infer their evolutionary history. By identifying thousands of high-confidence single-nucleotide variants (SNVs), they constructed a phylogenetic tree of the variety that resolved distinct clonal lineages. The analyses also revealed evidence of ancestral inbreeding, providing insight into the historical origins of Chardonnay and its founder genotypes. Importantly, the clonal lineages inferred from genomic data were consistent with historical records of dissemination and with documented phenotypic differentiation among clones. This work demonstrated that clones do not form a flat, unstructured set of entities, but rather a stratified population with deep evolutionary branches.

A similar WGS-based strategy applied to Malbec revealed clonal lineages that are geographically structured (Calderón et al., 2021). Whole-genome resequencing combined with custom genotyping allowed the identification of specific markers that distinguish pre-phylloxera European material from South American massal selections. The results showed that the introduction bottleneck into the New World, followed by several generations of local massal selection, generated distinct genomic signatures. This provides a molecular basis for the concept of “Argentine Malbec” as not only phenotypically, but also genomically differentiated from its French progenitors. This evidence supports the concept that South American viticulture (particularly in Argentina and Chile) represents a unique global case study. Unlike European regions where post-phylloxera replanting was often driven by early clonal standardisation, these countries maintain tens of thousands of hectares of commercial vineyards established through massal propagation. Consequently, these regions act as large-scale in situ reservoirs of pre-clonal genetic diversity, preserving a complexity that has been eroded elsewhere. This vast genetic capital, validated by the genomic divergence found in Malbec (Calderón et al., 2021), offers an unparalleled resource for adaptation and selection.

Carménère exemplifies the accumulation of somatic variation in a narrow-founder cultivar maintained primarily through massal propagation. Introduced from France in the mid-19th century and preserved in Chile in the absence of phylloxera, the variety experienced a severe genetic bottleneck followed by over 150 years of vegetative multiplication. Whole-genome resequencing of 12 Chilean clones (36 biological replicates) using a new chromosome-scale Carménère reference genome identified more than 9000 clone-private SNVs and small indels per clone (Garcia et al., 2026). Although most variants mapped to repetitive regions, a substantial subset affected coding sequences, with genes involved in plant–pathogen interactions, transport and secondary metabolism being most frequently impacted. These results demonstrate that historic massal populations can preserve significant intravarietal diversity even after strong founder effects.

To bridge the gap between cost and resolution, Urra et al. (2023) developed a proof-of-concept panel based on high-throughput amplicon sequencing targeting 46 InDel markers, demonstrating that much higher clonal resolution than SSRs is achievable at manageable cost. The design principles and discrimination statistics of this approach are discussed in detail in Section 7.3.1.

GBS has emerged as a powerful tool for characterising large clonal collections and guiding conservation strategies. Applied to Primitivo, it generated approximately 38,000 SNPs across 35 clones, enabling discrimination of genetically unique individuals and identification of groups associated with oenological traits (Procino et al., 2025). The methodological details and broader applications of GBS-based population typing are covered in further detail in Section 7.2.1. The molecular characterisation of grapevine clones has evolved from a state of near "blindness" to high-definition genomic resolution. We now know that the grapevine genome is fluid, shaped by mobile elements and structural rearrangements that generate continuous somatic polymorphism. The transition from SSRs and AFLPs to WGS and high-throughput genotyping has confirmed that intravarietal diversity is not a conceptual artefact but a measurable genomic reality. These tools now make it possible to distinguish clonal lineages, trace the historical trajectories of varieties, and, crucially, design data-driven strategies for the conservation of massal populations.

The main mechanism explored in grapevine is variation in cytosine methylation across the genome, particularly in promoter regions and regulatory elements. Increased methylation in a promoter typically represses transcription, effectively silencing the associated trait; conversely, demethylation can restore gene expression. This mechanism was first demonstrated for skin colour, where hyper-methylation of the VvMybA1 promoter results in green berries (Azuma et al., 2009). Evidence for sequence-independent epigenetic divergence among clones comes from studies tracking methylation changes under environmental pressure in vegetatively propagated material (Varela et al., 2020; Varela et al., 2024; Baránek et al., 2015). This mechanism is not limited to individual sports or clones; it appears to have played a broader role throughout domestication. Rodriguez-Izquierdo et al. (2024) compared methylation patterns between 18 wild and cultivated grapevine accessions using reduced-representation bisulfite sequencing. They found significantly higher global methylation levels in cultivated varieties, identifying 9.955 differentially methylated cytosines (DMCs), of which 78 % were hypermethylated in cultivars. Core methylated genes (consistently methylated in both wild and cultivated) were enriched in stress response and terpenoid/isoprenoid metabolic pathways, while differentially methylated genes were associated with peroxisome protein targeting, ethylene regulation, histone modifications, and defence responses. These findings suggest that DNA methylation contributed significantly to the fixation of adaptive traits during domestication, potentially acting as a molecular substrate for the emergence of intra-varietal diversity observed today. Epigenetic responses to specific stresses further illustrate this dynamic. De Giovanni et al. (2015) surveyed methylation in NCED genes (key in ABA biosynthesis and drought response), identifying symmetric and asymmetric CpG islands that modulate expression under normal vs drought conditions. This suggests NCED as candidates for ‘epigenetic memory’, where stress-induced methylation patterns could persist, influencing phenotypic plasticity and adaptation in clones. The dynamics of these changes highlight both reversibility and persistence: Baránek et al. (2015) showed that in vitro stress induces methylation alterations, with 40 % reverting within a year (transient acclimation) but 60 % persisting as mitotically heritable epimutations. Sequencing variable regions linked 19.5 % to known genes ( e.g., Gret1 retrotransposon, auxin-responsive factor) and 51.2 % to hypothetical stress-sensitive proteins, suggesting epigenetic drift contributes to clonal divergence under environmental pressure.

The concept of terroir has traditionally been framed in terms of soil properties, climate, and management practices (Seguin, 1969; van Leeuwen & Seguin, 2006; Muñoz et al., 2026; Muñoz et al., 2021). Recent work adds a molecular dimension to this framework, proposing that the environment “programmes” the genome through stable, site-specific methylation patterns (Dal Santo et al., 2016).

● Global methylation patterns: Studies of global DNA methylation have shown that methylation landscapes differ significantly between vineyards and regions, generating distinct epigenetic fingerprints (Xie et al., 2017). These patterns are robust enough to cluster vines according to their geographical origin, suggesting that local environments induce stable changes in gene-expression potential that contribute to terroir-specific phenotypes.

● Clone-dependent responses in Malbec: Work on Malbec in Mendoza has provided some of the most detailed evidence of this interaction (Varela et al., 2020). It was shown that vineyard environments influence both phenotypic traits and methylation profiles in a clone-dependent way: some clones exhibit high epigenetic plasticity and respond strongly to environmental change, whereas others show relatively rigid methylation patterns.

● Plasticity versus stability over time: A three-year study following transplanted Malbec material revealed that certain methylation marks adjust rapidly after relocation (acclimation), while others remain stable and retain a “memory” of the original environment (Varela et al., 2024). This raises important questions for clonal certification: a given clone produced in a low-altitude nursery and the same genetic clone propagated for decades at high altitude may differ substantially at the epigenomic level, even if their DNA sequence is identical (Varela et al., 2024, Varela et al., 2020).

● Clone-specific responses to repeated drought stress: In two cultivars (Debina and Dichali), repeated drought cycles induced differential morpho-physiological, genetic and epigenetic responses, with ‘Dichali’ showing higher tolerance (Maniatis et al., 2024). Grafted plants on ‘Richter 110’ rootstock exhibited greater resilience than self-rooted ones, suggesting beneficial scion–rootstock interactions. Gene expression revealed up-regulation of most stress-related genes in ‘Dichali’, including a key module involving miR159 up-regulation and strong down-regulation of the transcriptional co-repressor VvTOPLESS-4-like. MSAP analysis further identified genotype-specific DNA methylation patterns under stress. Although conducted in pots under controlled conditions, these results illustrate how autochthonous germplasm can harbour clone-dependent epigenetic and genetic plasticity that enhances adaptation to recurring abiotic stress in Mediterranean environments.

These observations support a model in which epigenetic mechanisms act as a molecular substrate for terroir, modulating clonal phenotypes in response to environmental history. This view is reinforced by evidence that grapevine can form stable, mitotically heritable epigenetic memories of (a)biotic stresses, providing a mechanism for long-term acclimation in clonally propagated perennials (Berger et al., 2025).

Historically, clonal selection was closely tied to sanitary selection: the elimination of viruses to restore vigour and yield (Golino et al., 2017b; Walter & Martelli, 1998). A pioneering comparison between healthy and leafroll-affected planting stocks showed that high viral loads cause severe agronomic penalties, including reduced vigour, lower yields, and delayed sugar accumulation, thereby justifying the implementation of clean-stock programmes (Lider et al., 1975). However, this binary view (infected versus clean) oversimplifies the biological reality. In practice, vines are rarely virus-free; instead, they harbour complex viromes comprising multiple latent or mildly pathogenic viruses, such as members of the Betaflexiviridae ( e.g., grapevine rupestris stem pitting-associated virus), that may not cause obvious symptoms but subtly alter physiological processes (Meng & Rowhani, 2017). Moreover, grapevines can also host mycoviruses through their associated fungal communities, further complicating the holobiont structure (Debat et al., 2025).

The interaction between a given clone and its viral community is dynamic and can generate long-term phenotypic trajectories. A 17-year longitudinal study on Pinot noir compared vines infected with different sources of grapevine leafroll-associated viruses and documented distinct, strain-specific effects on ripening and yield (Reynard et al., 2022). Rather than being simply “less productive”, infected vines followed alternative phenotypic trajectories, with altered phenology, delayed maturation and modified sugar–acid balances that persisted over nearly two decades. More broadly, several studies have shown that viral infections can mimic genetic or epigenetic effects on traits such as vigour, yield, sugar accumulation, and colour development. A mild leafroll infection, for example, may reduce sugar accumulation and delay harvest, a combination of traits that could be misinterpreted in a blind trial as a “late-ripening clone” rather than as a clone–virus interaction (Dayer et al., 2017).

These observations lead to a critical hypothesis regarding historical clonal characterisation. Many “clones” described in the 20th century were selected solely on phenotypic grounds ( e.g., looser clusters, reduced vigour, delayed ripening) and with the sanitary selection tools available at the time of selection (ELISA or indexing), without systematic screening for latent or mild viral infections. The availability of transcriptomic tools has provided a new perspective in the sanitary status assessment of grapevines, enabling the identification of previously unknown viruses. In such cases, the recorded “clonal differences” may in fact reflect, at least in part, differences in virome composition rather than fixed genetic or epigenetic divergence. To test this hypothesis, we performed a meta-analysis of public transcriptomic datasets associated with phenotypic clonal evaluations. Specifically, we re-examined RNA-Seq data from 'Tempranillo' clones previously characterised for bunch compactness (Grimplet et al., 2019; BioProject PRJNA448744). Using a de novo assembly approach to reconstruct non-grapevine transcripts, we identified the presence of seven viral species not originally detailed in the physiological study, including Grapevine virus A (GVA) and GLRaV-2 in specific clones. Notably, the viral composition varied among clones that were being compared for morphological traits. This finding underscores a critical caveat: many clones in genomic repositories or historical trials may actually represent distinct holobionts, where phenotypic differences are driven or amplified by differential viral loads. If a “clone” is defined by a specific phenotype, but that phenotype is conferred by a viral endophyte, the removal of the virus during heat therapy would effectively “erase” the clone. This implies that a significant portion of what is categorised as “intravarietal diversity” may actually represent viral diversity superimposed on a uniform genetic background.

Recognising viruses and epigenetic variability as integral components of the extended genotype reframes the definition of a clone. A clone can no longer be considered a purely genetic entity; instead, its phenotype emerges from the combined effects of its DNA sequence, its environmentally programmed epigenome and its interacting virome. This tripartite complexity may help explain why massal selection, which conserves the full diversity of genomes, epigenomes and viromes within a population, is frequently associated with greater vineyard resilience, adaptability and wine complexity compared to strictly monoclonal plantings. However, this potential advantage remains largely observational and requires further experimental validation.

A foundational contribution introduced GBS, in which restriction enzymes digest genomic DNA and only the ends of the resulting fragments are sequenced (Elshire et al., 2011). This approach bypasses large fractions of repetitive DNA, including many TEs, and preferentially samples gene-rich regions. In terms of scale and cost, GBS enables the simultaneous discovery and genotyping of thousands of single-nucleotide polymorphisms (SNPs) across hundreds of individuals in a single sequencing run. In viticulture, Procino et al. (2025) demonstrated the power of this method in a study of Primitivo clones. Using GBS, they generated approximately 38,000 SNPs across 35 clones and used this high-density dataset to cluster clones into genetic groups that correlated with oenological traits. Likewise, the application of GBS and ddRAD in a collection of 138 clones from six Southern Italian varieties identified polymorphic groups and functional divergence in stress pathways, supporting the utility of these methods for characterising intra-varietal diversity in massal populations (Villano et al., 2023). This work shows that GBS is not merely a research tool, but a practical method for scanning germplasm collections to identify redundancy and uniqueness, and to rationalise conservation and breeding efforts.

Whereas GBS is a discovery-oriented technology, Kompetitive Allele-Specific PCR (KASP) is a high-throughput screening tool. Xi et al. (2023) demonstrated its efficacy in grapevine by developing KASP assays for a locus controlling stenospermocarpy (seedlessness), showing that this platform can robustly genotype key alleles at low cost. KASP relies on fluorescence-based detection to identify which allele is present at a given SNP or small InDel. In Rubus, Clare et al. (2023) showed that a minimal panel of 24–48 SNPs converted into KASP assays could distinguish virtually all commercial propagated lineages. While the genetic distances between Rubus lineages are broader than those typically separating grapevine clones within a variety, the methodological principle suggests that once variety- or clone-specific somatic mutations (SNVs) are identified through WGS, they can be converted into inexpensive KASP markers. Nurseries can then screen thousands of mother plants at low cost per sample, ensuring true-to-type propagation of prestigious clones or specific massal lines at an industrial scale. In this context, KASP becomes the operational tool that transforms genomic discoveries into routine diagnostics.

Urra et al. (2023) presented a key advance with high-throughput amplicon sequencing. They designed a targeted panel of 46 InDel markers and, by amplifying and sequencing only these specific regions, achieved a discrimination rate of approximately 78 % among closely related clones. This strategy is particularly relevant for massal selection, because it enables the design of variety-specific panels ( e.g., “Malbec panel”, “Cabernet-Sauvignon panel”) composed of 50–100 hyper-variable loci. Using such a panel, a laboratory could process leaf samples from 1000 individual vines in a massal vineyard and assign each vine a molecular barcode, effectively mapping the genetic structure of the field. This level of resolution allows viticulturists to:

● quantify how many distinct genotypes are present;

● estimate their relative frequencies;

● identify rare or unique genotypes of high interest for conservation or selection.

In practical terms, Amplicon-seq panels provide a scalable, intermediate solution between low-resolution SSRs and full WGS, well-suited for characterising massal populations.

Consider a historic Malbec vineyard planted in 1920. Using GBS or a dense KASP/InDel panel, one could sample a statistically robust subset of the population (for instance, 10 % of the vines) and obtain the following information:

● Dominant genotype: Identification of the core genetic profile that defines the variety in that plot.

● Variant frequencies: Estimation of the proportion of plants carrying specific somatic variants, for example, alleles affecting anthocyanin pathways or traits associated with root architecture and vigour.

● Off-types: Detection of intruding varieties or clearly distinct clones that do not belong to the desired profile, which may have entered through accidental mixing or later re-plantings.

From these data, a viticulturist can derive a complexity index for the population and define reproducible profiles. Instead of offering a generic “Massal Selection Malbec”, a nursery could commercialise “Massal Selection Malbec – Profile A”, characterised by a specific ratio of genotypes ( e.g., 60 % Type A, 20 % Type B, 20 % Type C) known to produce a given organoleptic style (for example, enhanced floral notes associated with Type B). This approach transforms the amorphous concept of massal selection into a standardised yet inherently diverse product.

Work by groups such as Lijavetzky and collaborators has pioneered the use of high-density arrays and resequencing to discriminate clones within populations such as Malbec (Calderón et al., 2021). By identifying unique SNVs segregating among clonal lineages, it becomes possible to reconstruct the ancestry of massal populations. With this information, one can determine whether a given massal vineyard represents:

● a monoclonal expansion, where diversity is low and likely derived from a single historical introduction; or

● a polyclonal assembly, where diversity is high, indicating multiple introductions or substantial somatic diversification over time.

This distinction is crucial for conservation planning: polyclonal populations with rich clonal structure represent high-value resources, whereas monoclonal expansions, though perhaps interesting historically, contribute relatively little to the overall genetic pool.

Next-Generation Sequencing supports metagenomic approaches in which the total RNA (or DNA) of a vine is sequenced (Al-Rwahnih et al., 2025; Saldarelli et al., 2017). In RNA-seq experiments, reads derive not only from the plant transcriptome, but also from associated fungi, bacteria, and viruses (Debat et al., 2025). This makes it possible to reconstruct the complete virome of a massal population and to classify:

● Pathogenic agents: Acute viruses and viroids are associated with decline or severe quality losses.

● Latent/cryptic viruses: Ubiquitous and often asymptomatic agents such as Grapevine rupestris stem pitting-associated virus (GRSPaV) or Grapevine virus L (GVL), whose effects may be subtle or context-dependent (Cuello et al., 2026; Debat et al., 2019).

By combining host genotyping and virome analysis, one can generate a sanitary–genomic profile of each vine and of the population as a whole (Coetzee et al., 2010; Cuello et al., 2026; Vemulapati et al., 2025).

In the traditional clonal model, the presence of almost any virus justified the elimination of the plant. In a massal-genomic framework, a more nuanced strategy – surgical sanitation – becomes feasible. For example, in a massal population of 1000 vines, HTS might reveal that 50 vines harbour aggressive leafroll strains, while the remaining 950 carry only mild or latent viruses. Rather than discarding the entire population, one can remove only the 50 high-risk individuals, preserving the holobiont diversity (plant plus mostly benign virome) described previously. In this scenario, HTS data serve as a sanitary passport, certifying that the population is free of harmful agents without imposing virological sterility, which might itself alter subtle vine–virus–environment interactions relevant to terroir.