**2. Grapevine somatic variation**

Despite vegetative propagation is used in grapevine to multiply plants that are identical to the original type, spontaneous phenotypic variation occasionally appears on some shoots (known as bud sports) as a result of somatic mutations [16]. From bud sports, the new variant phenotype can be established as a whole plant and, eventually, as a new variety, using the same propagation strategy. Bud sports can display any type of phenotypic variation at any organ, leaf, stem, bunch, berries, seeds, etc. Variation can affect reproductive traits that determine yield and quality such as fertility, cluster compactness, berry color, or flavor. Somatic variation can also affect vegetative traits including plant vigor, leaf morphology, or even disease susceptibility. There are some cultivars like Pinot Noir, Sultanina, or Italia [14, 16] for which a large number of somatic variants have been identified for multiple traits. The number of sports appearing in a given variety is expected to increase proportionally with its age and vineyard surface. In addition,

the possibility that some genotypes are more prone to generate somatic variants has not been proven but cannot be discarded.

Spontaneous somatic variation results from the combination of mutations and cellular events. Initially, mutations take place in single meristematic cells associated with the DNA replication and cell division processes. Somatic mutations accumulate at a very low frequency. However, since current plants of traditional grapevine cultivars result from millions of mitotic divisions since the germination of the original seed, they accumulate a relatively large number of mutations in their genomes (see the next paragraph). For nuclear DNA, every somatic mutation can be considered to be heterozygous as they only affect one of the two existing genome copies per cell. These somatic mutations can range from single nucleotide substitutions to nucleotide insertions or deletions or even to large DNA sequence recombinations causing chromosomal reorganizations [16]. Other infrequent alterations include the change of ploidy level of the cell, reported in different varieties [17]. Somatic epimutations altering gene expression without affecting nucleotide sequence and causing new phenotypes have so far not been described in grapevine.

Most mutations do not have any effect on gene and cellular functions since only a small part of the genome sequence is involved in coding or regulatory functions [18]. Even mutations in coding sequences do not always generate amino acid changes in the encoded protein or if they do, still in many cases they behave as silent changes. Emergent somatic mutation will only affect one of the two copies of a given gene. This makes derived phenotypic effects to be mostly expected from dominant mutations either due to gains of function or haploinsufficiency. Independent recessive mutations causing loss of functional alleles in heterozygous loci carrying a null allele could also generate phenotypic effects although at low frequency. Importantly, deleterious mutations constraining essential cell functions will not accumulate because purifying somatic selection will prevent their propagation in the plant.

Cellular events associated with the stabilization of somatic mutations are conditioned by the tissue structure of plant meristems. The grapevine shoot apical meristem is organized in at least two cell layers, the outer L1 and the inner L2, from which all the cells of the plant derive [19]. These cell layers constitute almost closed compartments with very limited cell exchange between them. Cell division and differentiation in the L1 layer gives rise to all the epidermal cells of all the plant organs, while the L2 layer generates the cells that constitute all their internal tissues. The L2 cell layer is also responsible for gamete development within reproductive flower organs. Because mutations emerge spontaneously in either L1 or L2 layers, grapevine plants are genetic chimeras that carry slightly different genetic composition in L1- and L2-derived cell lines. In addition, vegetative multiplication from cuttings along centuries contributes to select and enrich part of the variation accumulating in the plant. At the same time, because of the lack of sexual reproduction, there is no purifying selection against mutations that could have deleterious effects on gametogenesis, fertilization, zygote formation, embryo development, seed germination, or juvenile growth.

To manifest a mutant phenotype in a given plant organ, the mutation has to propagate through cell division from the original mutant meristematic cell. Initially, mutant daughter cells occupy a meristem cell layer (either the L1 or L2) or sectors of it, which subsequently gives rise to mutant organs by additional cell divisions. Once the mutant genotype is propagated in the L1, the L2, or both cell layers of a shoot apical meristem, the mutation could be transmitted by bud propagation. Periclinal chimeras with somatic mutations fixed in only one meristem cell layer are quite stable in grapevine, and indeed, some varieties like Pinot Meunier (L1 mutant) [20] or Pinot Gris (L2 mutant) [21] are chimeras that are stably maintained through

**31**

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

grape cultivars or as new derived cultivars [6, 14, 16].

**3. Genome sequence variation within cultivars**

vegetative multiplication as we explain in sections below. If the mutant daughter cells colonize both meristem cell layers by migration of mutant cells to the wild type layer, bud multiplication from such mutant buds will fix the mutation in all tissues of derived plants. This is the case of white-berried variants derived from originally black-berried varieties such as Pinot Blanc [21]. Since plants do not have a separated germline, somatic mutations present in the L2 can be transmitted through sexual reproduction as far as they are not lethal in the haploid phase. Somatic mutations generating new interesting phenotypes, stabilized in grapevine plants as periclinal chimeras, or extended to all cell layers, have been selected as new clones of wine

Sequencing and de novo assembly and annotation of the first grapevine genomes

[18, 22] provided a new body of knowledge and a new toolbox for the study of genome sequence diversity. Two different strategies were used for the first genome assemblies, a homozygous assembly based on PN40024, a partially inbred line derived from Pinot Noir, [18] or an assembly including both, consensus contigs of the two genome copies and independent contigs for each of the two haplotypes in more dissimilar genome regions of Pinot Noir (ENTAV 115) [22]. Both projects estimated a haploid genome size close to 500 Mb. More recently, long-read sequencing technologies such as PacBio are facilitating the release of haplotype-resolved assemblies, which are already available for the heterozygous grapevine cultivars Cabernet Sauvignon and Chardonnay [23, 24]. By the time being, the availability of reference genomes combined with the development of next-generation sequencing (NGS) technologies enable genome-wide analysis of the grapevine germplasm at affordable costs, which is extremely useful in genetic diversity studies as well as to search for mutations causing phenotypic variation [15, 24–26]. Although the use of these approaches to characterize somatic variation in grapevine is still scarce, an increasing number of publications are shedding light on the magnitude and type of

variation that accumulates at the genome level within given cultivars.

Somatic SNV (single nucleotide variants) and small insertions/deletions (INDEL) mutations are often the result of errors in DNA replication taking place during mitotic cell division. While the frequency of INDEL may exceed that of single base substitutions due for instance to low resolution of polymerases at homopolymeric or short repeats, INDEL are more difficult to detect using high-throughput sequencing methods due to the same reason. The first attempt to detect somatic polymorphisms at a genome-wide scale in grapevine used 454 GS-FLX sequencing technology to compare three Pinot Noir clones to the sequences in the genome assemblies of the Pinot-related accessions PN40024 and ENTAV-115 [27]. In this study, mean rates of 1.6 SNV, 5.1 INDEL, and 35.2 mobile element movements per Mb were described among clones. Short-read sequencing technologies led by Illumina provide a framework to accurately detect SNV and are also useful to detect small INDEL. In this manner, genome resequencing of three clones corresponding to different morphotypes of the ancient Italian wine cultivar Nebbiolo identified between 16 and 26 clone-specific SNV per Mb of genome [28]. However, these numbers might be over-estimated considering that the validation success was 61% for a quality-trimmed sub-selection of SNV [28]. More recently, the re-sequencing of 15 clones of Chardonnay compared to a de novo genome draft assembly for this cultivar identified a much more reduced number of SNV using a stringent k-merbased calling strategy variation [24]. The sum of SNV + INDEL ranged between 221 and 2 polymorphisms per clone (0.004–0.455 per Mb of genome), which

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

*Advances in Grape and Wine Biotechnology*

has not been proven but cannot be discarded.

the possibility that some genotypes are more prone to generate somatic variants

causing new phenotypes have so far not been described in grapevine.

a small part of the genome sequence is involved in coding or regulatory functions [18]. Even mutations in coding sequences do not always generate amino acid changes in the encoded protein or if they do, still in many cases they behave as silent changes. Emergent somatic mutation will only affect one of the two copies of a given gene. This makes derived phenotypic effects to be mostly expected from dominant mutations either due to gains of function or haploinsufficiency. Independent recessive mutations causing loss of functional alleles in heterozygous loci carrying a null allele could also generate phenotypic effects although at low frequency. Importantly, deleterious mutations constraining essential cell functions will not accumulate because purifying somatic selection will prevent their propaga-

Cellular events associated with the stabilization of somatic mutations are conditioned by the tissue structure of plant meristems. The grapevine shoot apical meristem is organized in at least two cell layers, the outer L1 and the inner L2, from which all the cells of the plant derive [19]. These cell layers constitute almost closed compartments with very limited cell exchange between them. Cell division and differentiation in the L1 layer gives rise to all the epidermal cells of all the plant organs, while the L2 layer generates the cells that constitute all their internal tissues. The L2 cell layer is also responsible for gamete development within reproductive flower organs. Because mutations emerge spontaneously in either L1 or L2 layers, grapevine plants are genetic chimeras that carry slightly different genetic composition in L1- and L2-derived cell lines. In addition, vegetative multiplication from cuttings along centuries contributes to select and enrich part of the variation accumulating in the plant. At the same time, because of the lack of sexual reproduction, there is no purifying selection against mutations that could have deleterious effects on gametogenesis, fertilization, zygote formation, embryo development, seed germi-

To manifest a mutant phenotype in a given plant organ, the mutation has to propagate through cell division from the original mutant meristematic cell. Initially, mutant daughter cells occupy a meristem cell layer (either the L1 or L2) or sectors of it, which subsequently gives rise to mutant organs by additional cell divisions. Once the mutant genotype is propagated in the L1, the L2, or both cell layers of a shoot apical meristem, the mutation could be transmitted by bud propagation. Periclinal chimeras with somatic mutations fixed in only one meristem cell layer are quite stable in grapevine, and indeed, some varieties like Pinot Meunier (L1 mutant) [20] or Pinot Gris (L2 mutant) [21] are chimeras that are stably maintained through

Spontaneous somatic variation results from the combination of mutations and cellular events. Initially, mutations take place in single meristematic cells associated with the DNA replication and cell division processes. Somatic mutations accumulate at a very low frequency. However, since current plants of traditional grapevine cultivars result from millions of mitotic divisions since the germination of the original seed, they accumulate a relatively large number of mutations in their genomes (see the next paragraph). For nuclear DNA, every somatic mutation can be considered to be heterozygous as they only affect one of the two existing genome copies per cell. These somatic mutations can range from single nucleotide substitutions to nucleotide insertions or deletions or even to large DNA sequence recombinations causing chromosomal reorganizations [16]. Other infrequent alterations include the change of ploidy level of the cell, reported in different varieties [17]. Somatic epimutations altering gene expression without affecting nucleotide sequence and

Most mutations do not have any effect on gene and cellular functions since only

**30**

tion in the plant.

nation, or juvenile growth.

vegetative multiplication as we explain in sections below. If the mutant daughter cells colonize both meristem cell layers by migration of mutant cells to the wild type layer, bud multiplication from such mutant buds will fix the mutation in all tissues of derived plants. This is the case of white-berried variants derived from originally black-berried varieties such as Pinot Blanc [21]. Since plants do not have a separated germline, somatic mutations present in the L2 can be transmitted through sexual reproduction as far as they are not lethal in the haploid phase. Somatic mutations generating new interesting phenotypes, stabilized in grapevine plants as periclinal chimeras, or extended to all cell layers, have been selected as new clones of wine grape cultivars or as new derived cultivars [6, 14, 16].
