**4. Nucleotide sequence variation underlying grapevine somatic variation**

The availability of grapevine reference genomes and the advent of NGS technologies have paved the way for the identification of the nucleotide diversity underlying variation for relevant phenotypic traits in grapevine. Somatic variants are excellent tools for this goal, since they allow studying the mutation effect in a common genetic background when comparing somatic variants to the direct ancestor of the same cultivar. This facilitates the identification of the causal genes and gene variants. In fact, in the last years, the molecular and genetic basis of an increasing number of phenotypic traits has been elucidated using somatic variants as experimental systems.

We consider transcriptome RNA-seq comparisons as an excellent diagnosis tool for the screening of candidate genes because this technology has the potential to trace mutations that alter either gene expression or coding sequences. In our hands, the process starts with a careful phenotypic analysis comparing the progenitor normal plant and the somatic variants. Concurrently, we develop self-cross derived progenies of both genotypes for segregation analyses. The main objective of the phenotypic analysis is to understand the developmental origin of the emerged trait. In this manner, we can identify a target organ, tissue, and developmental stage in which the mutation is initially expressed and take samples of it from each variant to conduct a transcriptome comparison. The interpretation of gene biological function from the developmental and phenotypic variation can frequently be misleading since, as mentioned before, many of these mutations have dominant gain-offunction effects.

Under these premises, transcriptome comparison, both at gene expression and sequence levels, combined with the results of segregation analyses of mutant phenotype in self-cross populations of each variant can provide a preliminary identification of putative candidate genes. These candidates will have to be confirmed by directly comparing their sequences in normal and somatic variants of the same cultivar. Both in transcriptome and sequence analyses, it is important to consider the possible chimeric state of causal mutations in the somatic variants.

When the described approaches lead to the identification of sequence variation susceptible of generating the mutant phenotypic effect, it is still required to confirm that this sequence variation is the cause of the phenotype. When the responsible mutation is present in the L2 layer and can be transmitted through gametes, co-segregation of the mutant phenotype with the candidate sequence variants would support a causality relationship although it is not a definitive proof. Genetic transformation to restore normal or variant phenotypes can be a difficult and time-consuming alternative in grapevine. Other possibilities like allele-specific expression analyses or sequence characterization of a large number of variants or cultivars displaying the same phenotype have been used in different cases to proof that a candidate gene variant is in fact responsible for a relevant phenotypic effect [26, 42, 43, 46].

In the next section, we review several examples of studies taking advantage of somatic variants to understand the molecular genetics of four relevant grape traits.

## **4.1 Meunier phenotype**

The Meunier phenotype accounts for the tomentose (hairy) phenotype of shoot tips and leaves in cultivar Pinot Meunier derived from Pinot noir. Plants derived by somatic embryogenesis from different L1 and L2 cell layers of Pinot Meunier showed different phenotypes, demonstrating that Pinot Meunier is a periclinal chimera carrying a mutant L1 line responsible for the Meunier phenotype. In addition, those plants regenerated from L1 somatic embryos displayed a new dwarf phenotype with short internodes [20]. Further characterization of those dwarf plants showed that they produced inflorescences and bunches in all nodes along the length of the shoots [47]. Grapevine nodes develop either inflorescences or tendrils that share a common ontogenetic origin from uncommitted primordia in grapevine [48]. Application of gibberellins (GAs) and GA biosynthesis inhibitors has been shown to modify tendril and inflorescence development in grapevine [49]. This phenotype suggested that the Pinot Meunier was associated with an altered response to gibberellins, what was confirmed by the high levels of active gibberellins detected in the dwarf plants paralleled by their insensitivity to the application of these hormones [47]. It is also similar to the phenotype of *gai* mutants of *Arabidopsis*, carrying mutations in GAI, a negative regulator of GA response [50]. In fact, dwarf plants derived from Pinot Meunier L1 were shown to carry a point mutation in a *GAI* homologous gene converting a leucine residue into a histidine within its conserved DELLA domain (**Figure 1**), the GA-sensitive domain unique to all members of this family of regulatory proteins [47]. The final proof confirming the role of this mutation in the origin of the dwarf phenotype came from the genetic analyses performed on self- and out-crosses of the mutant dwarf plants regenerated from the L1 of Pinot Meunier. The results showed that the mutated allele behaved in a semi-dominant manner, with homozygous mutant plants displaying a more extreme dwarf phenotype [47].

Similar hairy phenotypes have also been found in other cultivars given the names of some derived varieties like Garnacha "peluda" (hairy in Spanish), a name that refers to the tomentose phenotype. However, whether this phenotype has the same genetic and molecular basis as the Meunier phenotype has not been investigated. The Meunier phenotype constitutes a great example of how a relevant agricultural trait can be generated by a mutation in chimeric state in a somatic variant, a feature that is lost when the mutation is present in all plant cell layers. Because bibliographic references to Pinot Meunier or Schwarzriesling in Germany date back at least to the seventeenth century [51], this case itself proves the stability of periclinal chimeras in grapevine.

Understanding the molecular basis of this phenotype opens the possibility to recreate with genome editing tools such as CRISPR/Cas9 [52] the causative single point mutation or other point mutations known to have similar effects on the DELLA domain of the GAI regulatory proteins. However, the replication of the Meunier phenotype in the same or other cultivar backgrounds will be difficult because it will require the mutation to be stable only in the L1 cell layer, something that could require more sophisticated cell culture and plant regeneration techniques. This exemplifies the specificity of phenotypes resulting from chimeric states.

To end, it is important to mention that the capacity of these dwarf plants to flower rapidly from the initiation of the first tendril makes them useful model systems for genetic studies in grapevine [53]. In this case, genome editing to recreate mutations in the DELLA gene and regenerate whole mutant plants to obtain dwarf models in cultivars other than Pinot would be more feasible.

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**Figure 1.**

*development and lignification of the seed coat.*

*Somatic Variation and Cultivar Innovation in Grapevine DOI: http://dx.doi.org/10.5772/intechopen.86443*

The Muscat flavor in grapevine describes an intense floral aroma present in the berries of some specific cultivars and their derived wines. It is linked to the high accumulation of monoterpenoids such as linalool, geraniol, nerol, citronellol, and α-terpineol, all having a low olfactory perception threshold [54]. This aroma has been strongly appreciated since ancient times and a family of closely related Muscat varieties was spread from the Eastern Mediterranean area by Greeks and Romans and can still be found with different names in many locations of the world [55]. The Muscat flavor has also been found in somatic variants of cultivars like Savagnin,

Genetic analyses of Muscat aroma in grapevine have been performed in biparental progenies involving Muscat varieties [57, 58] and in self-cross derived populations of Muscat Ottonel and Gewurztraminer (a Muscat flavor somatic variant of Savagnin) [59]. Muscat aroma segregated as a dominant trait and at least one common major QTL responsible for Muscat aroma was detected in all progenies, located in linkage group 5. A positional candidate gene, 1-deoxy-D-xylulose-5-phosphate synthase (*VviDXS*), was proposed to account for the terpenoid overproduction phenotype [58, 59]. This gene encodes the first enzyme of the plastidial methylerythritol phosphate (MEP) pathway, which functions upstream in monoterpene and diterpene biosynthesis. Several investigations have shown that this enzymatic reaction is a biosynthetic step of the pathway that limits terpenoid biosynthesis in

Based on those hypotheses, Emanuelli et al. [46] re-sequenced the *VviDXS* grapevine gene in a collection of 148 grape varieties, including Muscat-aromatic as well as other aromatic and neutral accessions. Among the SNP significantly associated with the presence of Muscat aroma, they identified the putative causal SNP responsible for the Muscat phenotype. This SNP is present in all Muscat varieties and generates a predicted nonneutral substitution of a lysine by an asparagine in residue 284 of *VviDXS*. Interestingly, Muscat-like aromatic somatic variants also displayed unique nonsynonymous mutations in close positions of the same

*VviGAI, VviDXS, and VviAGL11 proteins and mutations responsible for Meunier, Muscat, and Seedlessness traits. Protein domains are represented according to Pfam database. L38H mutation in the DELLA domain results in the lack of GA-response in pinot Meunier. K284N mutation is present in all Muscat varieties. Three additional independent mutations were identified in the same DXS domain in Muscat-like aroma somatic variants: S272P in chardonnay Musqué, deletion (Del) of five amino acids 285–289 in Chasselas Musqué, and R306C in Gewürztraminer. R197L mutation in AGL11 located in the C-terminus of the protein alters* 

**4.2 Muscat flavor**

plants [60].

Chardonnay, or Chasselas [46, 56].
