**4.2 Muscat flavor**

*Advances in Grape and Wine Biotechnology*

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

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

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

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.

**4.1 Meunier phenotype**

extreme dwarf phenotype [47].

of periclinal chimeras in grapevine.

**34**

states.

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, Chardonnay, or Chasselas [46, 56].

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 plants [60].

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

#### **Figure 1.**

*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 development and lignification of the seed coat.*

domain of DXS protein (**Figure 1**). A serine substitution by a proline in position 272 in Chardonnay Muscat, an arginine substitution by cysteine in position 306 in Gewurztraminer, and a deletion involving five amino acids in position 285–289 on Chasselas Musqué [46]. Altogether, the correlation of independent nonlethal spontaneous Muscat mutations in this conserved DXS domain with the presence of Muscat aroma in all studied cases suggests its relevance in protein function. In fact, the Muscat amino acid substitution influences the enzyme kinetics by increasing its catalytic efficiency and it is also able to dramatically increase monoterpene levels in transgenic tobacco plants [60].

The closely related genetic relationships among Muscat varieties could be interpreted as resulting from the original selection of a somatic variant in which this characteristic aroma emerged and was propagated vegetatively. Occasional hybridization of this variant with other cultivars grown in ancient times as well as more recent directed hybridizations would have generated the currently available plethora of Muscat varieties (**Figure 2**). The identification of independent nonneutral amino acid substitutions or amino acid deletions in the same protein region clearly identifies *VviDXS* as a target gene to improve Muscat aroma through breeding (**Figure 1**). Specific nonneutral amino acid substitutions are not easily obtained from mutagenesis programs. However, the current catalog of known sequence variants in *VviDXS* provides several specific amino acid changes that could be recreated through genome sequence editing to introduce the Muscat flavor trait in any desired cultivar.

#### **4.3 Berry color**

Berry color is a very relevant trait determining consumer preferences in table grapes as well as the type of wines that can be elaborated from wine grape cultivars. In this way, red and *rosé* wines are made from black-berried cultivars, while white-berried cultivars are used for making white wines. In grapevine, berry color results from the biosynthesis and vacuolar accumulation of anthocyanins in berry skin cells during the ripening process from *veraison* stage. Variation for berry color is determined by a major locus on linkage group 2 [61, 62]. This berry color locus co-localizes with a cluster of tandemly repeated *VviMybA* genes [63]. Among them, *VviMybA1* and *VviMybA2* are expressed in the berry skin of blackberried cultivars from *veraison* [64]. The function of these transcription factors is required to trigger the expression of target genes such us UDP-glucose:flavonoid 3-O-glucosyltransferase (*UFGT*), encoding the limiting enzyme activity for the anthocyanin biosynthetic pathway [64, 65]. Original wild grapevines producing black berries and berry color diversity could have emerged as a result of somatic variation and be selected as a domestication trait in cultivated out-crossed forms [66]. Black-berried cultivars carry at least one functional copy of both *VviMybA1* and *VviMybA2* linked in a functional allele of the color locus. White-berried cultivars do not synthesize anthocyanins in the berry skin and they lack functional copies of these *MYBA* genes at the color locus. Most white-berried cultivars are homozygous for the canonical null allele of the locus in which *Gret1* retrotransposon insertion in the promoter of *VviMybA1* along with a small INDEL causing a frameshift in *VviMybA2*, respectively, causes loss-of-function in the two genes [41, 64]. Most of the diversity in berry color observed among grapevine varieties has been related to nucleotide sequence variation in this locus [67].

In addition, intracultivar variation for berry color, useful to select new derived varieties, has also been associated to variation in the berry color locus. In this way, spontaneous red-berried variants identified in white-berried table grape cultivars like Italia or in wine cultivars were shown to derive from recombination,

**37**

**Figure 2.**

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

locus, leaving only the null allele [15].

reverting the insertion of the *Gret1* retrotransposable element present in the promoter of *VviMYBA1* in white alleles, which at least partially recovers the expression of the gene [41, 68]. In other cases, red-berried variants emerged as new functional *MYBA* genes resulting from the recombination of nonfunctional homologous genes within the color locus [69]. On the other hand, black-berried cultivars heterozygous for the null allele occasionally display grape color variants with either red/gray or white berries depending on whether only the L2 or both L1 and L2 meristem cell layers (**Figure 3**), respectively, carry mutations at the color locus [21, 70–73]. Molecular characterization of red/gray and white berry somatic variants of Cabernet Sauvignon and Pinot Noir cultivars through Southern blots showed that the lack of berry skin anthocyanins was associated with deletion of the functional allele of the color locus [70, 74]. Later, the loss of heterozygosity along the color locus has been used to size the extent of deletions [21, 72, 73]. This heterozygosity loss has been directly related to spontaneous deletions involving the functional color locus allele and resulting in hemizygosity at the grape color

Altogether, these results demonstrate that intracultivar color variation appearing in either white or colored cultivars is mostly associated to structural variation at the color locus on linkage group 2, in combination with cellular events generating different chimeric situations and color patterns. Gain of color variants generally correspond to recombination events within the locus that generate gain-of-function mutations and dominant phenotypes. Loss of color variation seems to be restricted to black-berried cultivars heterozygous for a functional allele at the color locus and is associated with different deletions or complex chromosomal rearrangements eliminating this single functional copy [15]. Based on this information, bud irradiation with physical mutagens increasing the frequency of recombination and

deletion could be a strategy to generate new color variants in grapevine.

*Genetic relationships among Muscat varieties. Muscat à petits grains blancs is the progenitor of ancient variety Muscat of Alexandria and the putative ancestor of all the Muscat varieties. From them, additional Muscat varieties are derived by spontaneous or directed hybridizations (see [55] and* Vitis *International Variety* 

*Catalog (http://www.vivc.de)) as well as through somatic variation (\*).*

*Advances in Grape and Wine Biotechnology*

transgenic tobacco plants [60].

cultivar.

**4.3 Berry color**

domain of DXS protein (**Figure 1**). A serine substitution by a proline in position 272 in Chardonnay Muscat, an arginine substitution by cysteine in position 306 in Gewurztraminer, and a deletion involving five amino acids in position 285–289 on Chasselas Musqué [46]. Altogether, the correlation of independent nonlethal spontaneous Muscat mutations in this conserved DXS domain with the presence of Muscat aroma in all studied cases suggests its relevance in protein function. In fact, the Muscat amino acid substitution influences the enzyme kinetics by increasing its catalytic efficiency and it is also able to dramatically increase monoterpene levels in

The closely related genetic relationships among Muscat varieties could be interpreted as resulting from the original selection of a somatic variant in which this characteristic aroma emerged and was propagated vegetatively. Occasional hybridization of this variant with other cultivars grown in ancient times as well as more recent directed hybridizations would have generated the currently available plethora of Muscat varieties (**Figure 2**). The identification of independent nonneutral amino acid substitutions or amino acid deletions in the same protein region clearly identifies *VviDXS* as a target gene to improve Muscat aroma through breeding (**Figure 1**). Specific nonneutral amino acid substitutions are not easily obtained from mutagenesis programs. However, the current catalog of known sequence variants in *VviDXS* provides several specific amino acid changes that could be recreated through genome sequence editing to introduce the Muscat flavor trait in any desired

Berry color is a very relevant trait determining consumer preferences in table grapes as well as the type of wines that can be elaborated from wine grape cultivars. In this way, red and *rosé* wines are made from black-berried cultivars, while white-berried cultivars are used for making white wines. In grapevine, berry color results from the biosynthesis and vacuolar accumulation of anthocyanins in berry skin cells during the ripening process from *veraison* stage. Variation for berry color is determined by a major locus on linkage group 2 [61, 62]. This berry color locus co-localizes with a cluster of tandemly repeated *VviMybA* genes [63]. Among them, *VviMybA1* and *VviMybA2* are expressed in the berry skin of blackberried cultivars from *veraison* [64]. The function of these transcription factors is required to trigger the expression of target genes such us UDP-glucose:flavonoid 3-O-glucosyltransferase (*UFGT*), encoding the limiting enzyme activity for the anthocyanin biosynthetic pathway [64, 65]. Original wild grapevines producing black berries and berry color diversity could have emerged as a result of somatic variation and be selected as a domestication trait in cultivated out-crossed forms [66]. Black-berried cultivars carry at least one functional copy of both *VviMybA1* and *VviMybA2* linked in a functional allele of the color locus. White-berried cultivars do not synthesize anthocyanins in the berry skin and they lack functional copies of these *MYBA* genes at the color locus. Most white-berried cultivars are homozygous for the canonical null allele of the locus in which *Gret1* retrotransposon insertion in the promoter of *VviMybA1* along with a small INDEL causing a frameshift in *VviMybA2*, respectively, causes loss-of-function in the two genes [41, 64]. Most of the diversity in berry color observed among grapevine varieties has been

In addition, intracultivar variation for berry color, useful to select new derived

varieties, has also been associated to variation in the berry color locus. In this way, spontaneous red-berried variants identified in white-berried table grape cultivars like Italia or in wine cultivars were shown to derive from recombination,

related to nucleotide sequence variation in this locus [67].

**36**

reverting the insertion of the *Gret1* retrotransposable element present in the promoter of *VviMYBA1* in white alleles, which at least partially recovers the expression of the gene [41, 68]. In other cases, red-berried variants emerged as new functional *MYBA* genes resulting from the recombination of nonfunctional homologous genes within the color locus [69]. On the other hand, black-berried cultivars heterozygous for the null allele occasionally display grape color variants with either red/gray or white berries depending on whether only the L2 or both L1 and L2 meristem cell layers (**Figure 3**), respectively, carry mutations at the color locus [21, 70–73]. Molecular characterization of red/gray and white berry somatic variants of Cabernet Sauvignon and Pinot Noir cultivars through Southern blots showed that the lack of berry skin anthocyanins was associated with deletion of the functional allele of the color locus [70, 74]. Later, the loss of heterozygosity along the color locus has been used to size the extent of deletions [21, 72, 73]. This heterozygosity loss has been directly related to spontaneous deletions involving the functional color locus allele and resulting in hemizygosity at the grape color locus, leaving only the null allele [15].

Altogether, these results demonstrate that intracultivar color variation appearing in either white or colored cultivars is mostly associated to structural variation at the color locus on linkage group 2, in combination with cellular events generating different chimeric situations and color patterns. Gain of color variants generally correspond to recombination events within the locus that generate gain-of-function mutations and dominant phenotypes. Loss of color variation seems to be restricted to black-berried cultivars heterozygous for a functional allele at the color locus and is associated with different deletions or complex chromosomal rearrangements eliminating this single functional copy [15]. Based on this information, bud irradiation with physical mutagens increasing the frequency of recombination and deletion could be a strategy to generate new color variants in grapevine.

#### **Figure 2.**

*Genetic relationships among Muscat varieties. Muscat à petits grains blancs is the progenitor of ancient variety Muscat of Alexandria and the putative ancestor of all the Muscat varieties. From them, additional Muscat varieties are derived by spontaneous or directed hybridizations (see [55] and* Vitis *International Variety Catalog (http://www.vivc.de)) as well as through somatic variation (\*).*

**Figure 3.**

*Proposed genetic composition of shoot apical meristem (SAM) and berry color in Tempranillo somatic variants. L1 (outer) and L2 (inner) layers are represented in SAM, purple color indicates that cells in the layer carry a functional allele at the color locus and white color indicates the lack of functional color alleles in the cells. One functional allele in both meristematic layers is enough to develop black berries, while periclinal chimera with a mutant L2 cell layer in the SAM gives rise to gray color berries, the lack of functional alleles in both meristem layers yields white berries.*
