**5.5 Identification of the genetic traits of aromatic character of cabernet sauvignon**

Methoxypyrazines are a family of volatile compounds found in many fruits and vegetables and especially in grapes, providing herbaceous flavors (green capsicum aroma) to the wines of some varieties such as Cabernet Sauvignon or sauvignon blanc. While several methoxypyrazine biosynthetic pathways have been proposed, none of the metabolic steps have been genetically confirmed. Dunlevy et al. [24] used a F2 population derived from a F1 microvine obtained by crossing the Cabernet Sauvignon and a picovine. The Cabernet Sauvignon variety is capable of producing the molecule 3-isobutyl-2-methoxypyrazine (IBMP), the major compound associated with capsicum flavors, while the microvine that derives from Pinot Meunier produces very little amount of this compound. In F1 offspring, all individuals produced IBMP, suggesting a homozygote dominant genotypic status for this trait in Cabernet Sauvignon. The phenotyping of the F2 individuals identified 43 lines able to accumulate IBMP, while 21 individuals lacked this compound confirming the dominant homozygous genotype for Cabernet Sauvignon and the homozygous recessive genotype for picovine progenitor.

After genotyping and phenotyping, the entire F2 progeny, a 2.3 Mb locus determining IBMP accumulation in grape berries, was found on chromosome n°3. Of the 261 genes identified in the corresponding QTL, two candidate methyltransferase genes have been identified, *VvOMT3* and *VvOMT4*. Screening a collection of 91 grapevine genotypes differentially accumulating IBMP into the grapes indicated *VvOMT3* as the most likely candidate to explain the genetic determinism of the green capsicum trait in grapevine fruits. Moreover, the data suggested that the low level of methoxypyrazines found in most cultivated grape varieties resulted from human selection for mutations in methyltransferase. The markers identifying this locus are valuable tools for the selection of grape varieties that are aromatically typified by IBMP and recalling Cabernet wines.

## **5.6 Effect of application of exogenous stimulants of fruit metabolism**

The microvine plant model which displays unique reproductive organ behavior offers new experimental options for grapevine fruit physiological studies, not only because of the size of the plants which facilitate experimental handling in greenhouse or growth cabinet but also because it is possible to study several developmental stages at once. Taking advantage of the biological properties of the microvine, two studies were recently performed to study the impact of exogenous compound application to the ML1 microvine grapes on the aroma accumulation during ripening. The first study was about the impact of vine-shoot aqueous extracts, which have been proposed as bio-stimulants to be sprayed to the canopy to modify wine aromatic profile. Sanchez-Gomez et al. [44] experimented the effect of vine-shoot extract foliar application on the composition of the grapes at 21 stages of development. The application was carried out from BBCH53 (detached inflorescences) to BBCH85 (berry softening) to reveal stage-specific responses of the accumulation of

*Advances in Grape and Wine Biotechnology*

respiration and/or through H<sup>+</sup>

relatively low experimental flow.

growth and cellular respiration rate, malic acid respiration, as a supplemental energy source in the fruit, is not compulsory. In cool climate, the allocation of carbon to the fruit can support glycolysis, malate synthesis, and sugar accumulation into the vacuole. Conversely, under hot climate, cytoplasmic sugars could be limiting when the cell starts to accumulate sugar in the vacuole at the onset of ripening. Thus, the malate would be drained from the vacuole to supply energy through

/sugar exchange at the tonoplast.

**5.4 Identification of QTLs of development stable under fluctuating environments**

Air temperature elevation combined with the shift of all phenological stages to warmer period is causing critical changes on vine yield and grape composition. Plant breeding has the potential to offer new cultivars with stable yield and quality under warmer conditions, but this requires the identification of stable genetic traits. The investigation about the stability of developmental QTLs with regard to abiotic factors is complicated with the non-dwarf varieties, because of its biological properties (long juvenile period, big size of the plants). Most of previous studies were carried out outdoors, in uncontrolled environmental conditions and with a

Houel et al. [25] reported the first experiment performed with microvines, to identify QTLs of vegetative and reproductive development, testing their stability under fluctuating environments. A F1 mapping population consisting of 129 microvines derived from the PV00C001V0008 x ugni blanc fleshless berry mutant was genotyped using an Illumina® 18 K SNPs chip (single-nucleotide polymorphism). Forty-three vegetative and reproductive traits were phenotyped over four vegetative cycles in the field, and a subset of 22 characters were measured over two climatic chamber culture cycles under two contrasting temperature regimes. Ten stable QTLs were identified for the development and composition of the berry and the leaf area on the parental genetic maps. A new major QTL accounting for up to 44% of variance of the berry weight was identified on the chromosome 7 in the ugni blanc parent. This QTL co-locates with QTLs of number of seeds per berry (accounting for up to 76% of the total variance), QTLs of fruit acidity before maturation (up to 35% of explained variance), and yield components such as the number of clusters and berries per cluster (up to 25% explained variance). In addition, a minor leaf surface QTL was found on the chromosome 4 in the same parent. This study which combined the use of microvine population to boost and facilitate the phenotyping with high-throughput genotyping technologies was innovative in grapevine genetics and also for perennial fruit crops. It allowed the identification of 10 stable QTLs, including the first QTLs of *V. vinifera* berry acidity detected in an

This progeny was also included in a study addressing the diversity for fruit volume, main sugar, and organic acid amounts in *V. vinifera* [43]. A panel of 33 genotypes, including 12 grapevine varieties and 21 microvine offspring, were characterized. Fruit phenotyping focused on two critical stages of fruit development: the end of green growth phase when organic acidity reaches a maximum and the physiological ripe stage when sugar unloading and water uptake stop. To determine the date of sampling for each critical stage, fruit texture and growth were carefully monitored. Analyses at both stages revealed large phenotypic variation for malic and tartaric acids as well as for sugars and berry size. At ripe stage, fruit fresh weight ranged from 1.04 to 5.25 g and sugar concentration from 751 to

. The content in organic acids varied both in quantity (from 80 to

) and in composition, with malic to tartaric acid ratio ranging from 0.13 to 3.62. At the inter-genotypic level, data showed no link between berry growth

**18**

intraspecific cross.

1353 mmol.L<sup>−</sup><sup>1</sup>

361 meq.L<sup>−</sup><sup>1</sup>

glycosylated aroma precursors at BBCH89 (ripe stage). Fifty grape sampling time points spreading to 86 days were established and normalized using the cumulative growing degree days parameter. The results confirmed that vine-shoot extract treatment had a positive impact on the accumulation of glycosylated compounds [45], especially aglycones such as alcohols, terpenes, and C13-norisoprenoids, with a higher impact when the treatment was applied at grape ripening stage.

The same approach was carried out to characterize the behavior of glycosylated aroma precursors in microvine fruits after foliar application of guaiacol. Previous outdoor experiments have showed that spraying guaiacol on vines could modify the contents of aroma compounds in grape and grape-derived wines. It was shown that such treatments could increase guaiacol glycoconjugates in leaves, shoots, and fruits of Monastrell variety, where there was a release of aglycone compounds during wine processing. However, the effect of such application and its timing on glycosylated aroma precursor pool remained unstudied. Sanchez-Gomez [46] studied the effect of guaiacol sprays when applied at several fruit developmental stages on glycosylated compound accumulation. The applications were carried out from phenological stage BBCH71 (fruit set) to BBCH85 (berry softening), to reveal stagespecific responses of the accumulation of glycosylated aroma precursors at BBCH89 (ripe stage). Data confirmed that guaiacol is an elicitor of the accumulation of glycosylated aromatic compounds in the microvine fruit, with a higher efficiency of application during ripening stages of the fruits. Geraniol, a terpene compound, exhibited the higher increase increment with up to 50-fold high concentration after guaiacol spraying than in the control.
