**4.4 Seedlessness**

Grapevine seedlessness is one of the best examples of cultivar innovation resulting from original somatic mutation in table grapes. Somatic variants defective in seed development appeared spontaneously along the history of grapevine cultivation and they have been propagated vegetatively [6]. Seedless variants in grapevine are classified into two major classes: (i) parthenocarpy, when fruits are set and develop without fertilization resulting in small berries free of seeds [75] and (ii) stenospermocarpy, when fertilization and embryo formation is not altered but later seed development is aborted [76]. Parthenocarpic varieties have been widely used for the production of Corinto seedless raisins, but, as their sterility makes sexual transmission of the causal mutation impossible, the use of this trait remains limited to those genotypes in which parthenocarpy appeared spontaneously. Recent work in Corinto Bianco, a parthenocarpic variant derived from Pedro Ximenes cultivar [75], has pointed out to meiotic alterations precluding the development of viable gametes as the origin of the mutant phenotype [29]. On the other hand, an ancient somatic mutation producing a stable stenospermocarpy phenotype likely emerged in a white-berried oriental cultivar known as 'Kishmish,' also known as 'Sultanina' or 'Thompson Seedless' [77]. Since the mutation responsible for stenospermocarpy has a lower impact than parthenocarpy in berry size and does not lead to sterility (pollen is fertile and embryos can also be rescued

**39**

**Figure 4.**

*Red globe and crimson seedless fruits.*

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

breeding [13, 78, 79] (**Figure 4**).

from seed traces), it has become the major source of seedlessness in table grape

The stenospermocarpy phenotype has been associated with abnormal development of the inner ovule integument [80], which ends in impaired development and lack of lignification of maternal seed coat tissues [81]. Genetic analyses of seedlessness trait in several F1 progenies derived from at least one stenospermocarpic progenitor identified segregations that could be explained by the presence of a dominant locus named Seed Development Inhibitor (*SDI*) interacting with several recessive loci [82, 83]. Later, quantitative genetic analyses identified the *SDI* locus as a major QTL on linkage group 18, explaining up to 70% of the phenotypic variance for different seed variables [84–87]. Based on co-localization of this QTL with a grapevine homolog of the Arabidopsis MADS-box transcription factor gene *AGAMOUS-LIKE11* (*AGL11*), responsible for ovule morphogenesis and seed coat differentiation [88], *VviAGL11* was considered the best candidate gene for the *SDI* locus [86, 87]. More recently, using an independent positional study combined with targeted sequencing in a large collection of seeded and stenospermocarpic grapevine cultivars, a single nucleotide missense mutation in *VviAGL11* was identified as the causal origin of the dominant seedless phenotype [26]. This mutation causes the substitution of a conserved arginine 197 into leucine (**Figure 1**), which could disrupt the function of multimeric complexes containing *VviAGL11* proteins in a dominant manner. Interestingly, amino acid sequence variants of oil palm *AGL11* homologs have also been selected in this crop to reduce the level of seed coat lignification [89]. Apart from the relevant application of the identification of the causal point mutation in *VviAGL11* to develop efficient marker-assisted selection strategies for seedless grape breeding, this information paves the way to the development of targeted genome editing for the genetic improvement of seedless table grapes. Stenospermocarpic seedlessness could also be useful in black-berried wine grapes as a way to avoid the negative effects of unripe seeds in the sensory quality of red wines [90]. Ripening imbalance between pulp and seeds can become a problem under climate change conditions [3], what could be addressed with the use of

*Advances in Grape and Wine Biotechnology*

**38**

**4.4 Seedlessness**

*both meristem layers yields white berries.*

**Figure 3.**

Grapevine seedlessness is one of the best examples of cultivar innovation resulting from original somatic mutation in table grapes. Somatic variants defective in seed development appeared spontaneously along the history of grapevine cultivation and they have been propagated vegetatively [6]. Seedless variants in grapevine are classified into two major classes: (i) parthenocarpy, when fruits are set and develop without fertilization resulting in small berries free of seeds [75] and (ii) stenospermocarpy, when fertilization and embryo formation is not altered but later seed development is aborted [76]. Parthenocarpic varieties have been widely used for the production of Corinto seedless raisins, but, as their sterility makes sexual transmission of the causal mutation impossible, the use of this trait remains limited to those genotypes in which parthenocarpy appeared spontaneously. Recent work in Corinto Bianco, a parthenocarpic variant derived from Pedro Ximenes cultivar [75], has pointed out to meiotic alterations precluding the development of viable gametes as the origin of the mutant phenotype [29]. On the other hand, an ancient somatic mutation producing a stable stenospermocarpy phenotype likely emerged in a white-berried oriental cultivar known as 'Kishmish,' also known as 'Sultanina' or 'Thompson Seedless' [77]. Since the mutation responsible for stenospermocarpy has a lower impact than parthenocarpy in berry size and does not lead to sterility (pollen is fertile and embryos can also be rescued

*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*  from seed traces), it has become the major source of seedlessness in table grape breeding [13, 78, 79] (**Figure 4**).

The stenospermocarpy phenotype has been associated with abnormal development of the inner ovule integument [80], which ends in impaired development and lack of lignification of maternal seed coat tissues [81]. Genetic analyses of seedlessness trait in several F1 progenies derived from at least one stenospermocarpic progenitor identified segregations that could be explained by the presence of a dominant locus named Seed Development Inhibitor (*SDI*) interacting with several recessive loci [82, 83]. Later, quantitative genetic analyses identified the *SDI* locus as a major QTL on linkage group 18, explaining up to 70% of the phenotypic variance for different seed variables [84–87]. Based on co-localization of this QTL with a grapevine homolog of the Arabidopsis MADS-box transcription factor gene *AGAMOUS-LIKE11* (*AGL11*), responsible for ovule morphogenesis and seed coat differentiation [88], *VviAGL11* was considered the best candidate gene for the *SDI* locus [86, 87]. More recently, using an independent positional study combined with targeted sequencing in a large collection of seeded and stenospermocarpic grapevine cultivars, a single nucleotide missense mutation in *VviAGL11* was identified as the causal origin of the dominant seedless phenotype [26]. This mutation causes the substitution of a conserved arginine 197 into leucine (**Figure 1**), which could disrupt the function of multimeric complexes containing *VviAGL11* proteins in a dominant manner. Interestingly, amino acid sequence variants of oil palm *AGL11* homologs have also been selected in this crop to reduce the level of seed coat lignification [89]. Apart from the relevant application of the identification of the causal point mutation in *VviAGL11* to develop efficient marker-assisted selection strategies for seedless grape breeding, this information paves the way to the development of targeted genome editing for the genetic improvement of seedless table grapes. Stenospermocarpic seedlessness could also be useful in black-berried wine grapes as a way to avoid the negative effects of unripe seeds in the sensory quality of red wines [90]. Ripening imbalance between pulp and seeds can become a problem under climate change conditions [3], what could be addressed with the use of

**Figure 4.** *Red globe and crimson seedless fruits.*

seedless wine varieties. Finally, editing of *AGL11* homologs could also be useful to generate seedlessness in other fruit crops.

## **5. Final considerations on the use of somatic variation**

The application of NGS to the study of somatic variation in grapevine is increasing our knowledge on the nucleotide sequence variation underlying phenotype variation. By direct comparison of somatic variants, this technology has the potential to identify causal candidates at the gene and gene variant levels. Regardless, genetic and molecular approaches are still required to confirm the role of those candidates. So far, NGS approaches have been used to unravel widely used classical phenotypes as those described along the chapter. When combined with genome edition technologies, they constitute new tools for the genetic improvement and adaptation of traditional elite grapevine wine cultivars.

The first conclusion that comes out from the review of currently available information in grapevine is that due to the essential heterozygous condition of emergent somatic mutations, only dominant mutations can generate somatic variant phenotypes. More frequently, these dominant mutations involve gains of function resulting from either SNV that generate nonneutral amino acid substitutions [26, 46, 47] or gene overexpression and misexpression caused by transposon insertions [42, 43] or recombinations [41, 69]. Loss of function mutations has also been described but so far only in the case of SV that unmasks the effect of recessive null alleles present at the color locus in cultivars that are heterozygous for functional and null alleles of the responsible *MYBA* genes [15]. Another interesting conclusion relates to the particular relevance that chimeric expression of the mutations can have in the generation of specific cultivars such Meunier or the gray-berried variants. These examples show once more how the same mutation can lead to different phenotypes depending on the meristem cell layers affected.

Dominant gain-of-function mutations identified in grapevine somatic variants exemplify how new gene functions can be created by mutations changing expression to different cell types, developmental stage, or transcription levels, o by the alteration of a key amino acid in functional protein domains. While the effects of loss of function mutations are generally easy to predict when the function of the affected genes is known, gain of function is much more unpredictable and represents a source of innovation that can create new possibilities for genetic improvement. Their dominant nature makes them especially useful not only for the improvement of traditional cultivars but also to breed new cultivars. Systematic screening of the large clonal germplasm hosted in old vineyards and collections of ancient accessions of traditional cultivars can unveil very relevant information and variant traits to be exploited in conventional, genomics-assisted, or genetic engineering-mediated breeding.

## **Acknowledgements**

Current research activities in our group are funded by Project BIO2017-86375-R.

**41**

**Author details**

and José Miguel Martínez Zapater\*

Government of La Rioja), Logroño, Spain

provided the original work is properly cited.

\*Address all correspondence to: zapater@icvv.es

Pablo Carbonell-Bejerano, Carolina Royo, Nuria Mauri, Javier Ibáñez

Instituto de Ciencias de la Vid y del Vino (CSIC, University of La Rioja,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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

### **Conflict of interest**

The authors declare no conflict of interest.

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

*Advances in Grape and Wine Biotechnology*

generate seedlessness in other fruit crops.

**5. Final considerations on the use of somatic variation**

adaptation of traditional elite grapevine wine cultivars.

the meristem cell layers affected.

engineering-mediated breeding.

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

seedless wine varieties. Finally, editing of *AGL11* homologs could also be useful to

The application of NGS to the study of somatic variation in grapevine is increas-

The first conclusion that comes out from the review of currently available information in grapevine is that due to the essential heterozygous condition of emergent somatic mutations, only dominant mutations can generate somatic variant phenotypes. More frequently, these dominant mutations involve gains of function resulting from either SNV that generate nonneutral amino acid substitutions [26, 46, 47] or gene overexpression and misexpression caused by transposon insertions [42, 43] or recombinations [41, 69]. Loss of function mutations has also been described but so far only in the case of SV that unmasks the effect of recessive null alleles present at the color locus in cultivars that are heterozygous for functional and null alleles of the responsible *MYBA* genes [15]. Another interesting conclusion relates to the particular relevance that chimeric expression of the mutations can have in the generation of specific cultivars such Meunier or the gray-berried variants. These examples show once more how the same mutation can lead to different phenotypes depending on

Dominant gain-of-function mutations identified in grapevine somatic variants exemplify how new gene functions can be created by mutations changing expression to different cell types, developmental stage, or transcription levels, o by the alteration of a key amino acid in functional protein domains. While the effects of loss of function mutations are generally easy to predict when the function of the affected genes is known, gain of function is much more unpredictable and represents a source of innovation that can create new possibilities for genetic improvement. Their dominant nature makes them especially useful not only for the improvement of traditional cultivars but also to breed new cultivars. Systematic screening of the large clonal germplasm hosted in old vineyards and collections of ancient accessions of traditional cultivars can unveil very relevant information and variant traits to be exploited in conventional, genomics-assisted, or genetic

Current research activities in our group are funded by Project BIO2017-86375-R.

ing our knowledge on the nucleotide sequence variation underlying phenotype variation. By direct comparison of somatic variants, this technology has the potential to identify causal candidates at the gene and gene variant levels. Regardless, genetic and molecular approaches are still required to confirm the role of those candidates. So far, NGS approaches have been used to unravel widely used classical phenotypes as those described along the chapter. When combined with genome edition technologies, they constitute new tools for the genetic improvement and

**40**
