Vitis vinifera

TO TINKER OR NOT TO TINKER
WITH GRAPE VARIETIES:
IS THAT THE QUESTION?
Michele Morgante
Agriculture in the past
and viticulture in the present
Publio Virgilio Marone, Georgiche
Virgilio introduces Mecenate to apicolture. (Georgiche IV, 1-66)
PLANT BREEDING: TRAIT INTROGRESSION
THROUGH BACKCROSSING
PLANT BREEDING AND INDUCED
MUTAGENESIS
• Chemical mutagens
• Physical mutagens (radiations)
PLANT BREEDING AND MOLECULAR
BIOLOGY: TRANSGENIC PLANTS
PLANT BREEDING AND GENOMICS:
LINKING GENE AND PHENOTYPE VARIATION
Bioinformatics
mRNA profiling
Protein profiling
Gene inactivation &
activation
Gene sequences (ESTs)
Genome sequence
Gene
Phenotype
Role of alleles
and haplotypes
Genetic
variation
DNA genotyping (SNPs)
Genetic diversity
Phenotypic
variation
Linkage maps
Association mapping
Physical/gene maps
Marker assisted
Selection (MAS)
+
Improved use of
germplasm
resources
Metabolic profiling
Phenotypic profiling
3-5 years gain
in new tobacco
varieties to
market
PRESENT PLANT BREEDING IN GRAPE
• Grape varieties are 200 years old
• No contribution of modern plant breeding
• Clonal propagation immortalizes varieties
• Clonal selection chooses somatic mutants
• Limited variability exploited within each
variety
• Very sensitive to diseases
• High yield
FUTURE PLANT BREEDING IN GRAPE
• Grape varieties are highly heterozygous
• Great genetic diversity available
• Start crossing program and create new genetic
combinations
• Improve disease resistance and decrease fungicide
use
• Improve quality
• Decrease yield
• Molecular breeding (MAS)
• Transgenics
MOLECULAR BREEDING RESEARCH
1.Germplasm and crossing populations
2.Downy mildew resistance
3.Powdery mildew resistance
4.Selection for fruit quality
5.Fingerprinting of the Italian grape
germplasm
1. Germplasm and cross populations at IGA
• 204 grape accessions:
31 Vitis species, 53 interspecific hybrids &
Cultivars of Vitis vinifera (mainly local)
PN40024: the
sequenced grape
Rooted cuttings for inoculation tests
• 2.5 ha vineyards for breeding:
40 cross populations: ~7,000 seedlings
120 F1 per each cross + some extended
Seedlings, cuttings for disease tests, clonal
replicates grafted on rootstocks
Greenhouses for seed germination
Seedling populations in the field
Clonal replicates: selection for fruit quality
2. Breeding for downy mildew resistance
Damage on foliage
Damage on bunches
Damage on flowers/fruitlets
Yield loss and deterioration of fruit quality
Sources of DM-R in the grape germplasm
North American grapes
Vitis sp. 2n=38
Muscadinia sp. 2n=40
European grape
Vitis vinifera 2n=38
Asian grapes
Vitis sp. 2n=38
Genetic mapping based on segregation of field phenotype
CROSS 1
Chardonnay x Bianca
46 individuals
452 loci, 3.9 cM gaps
CROSS 2
CROSS 3
Cabernet S x 20/3
Pinot noir x 99-1-48
46 individuals
46 individuals
381 loci, 6.2 cM gaps
292 loci, 7.9 cM gaps
Comparison of the major resistance locus identified in each parent
3. Breeding for powdery mildew resistance
North American grapes
Vitis sp. 2n=38
Muscadinia sp. 2n=40
European grape
Vitis vinifera 2n=38
Sources of PM-R in the grape germplasm
North American grapes
Vitis sp. 2n=38
Muscadinia sp. 2n=40
European grape
Vitis vinifera 2n=38
Vitis vinifera cultivar
‘Kishmish vatkana’
presumed site
of origin
current site of cultivation
Vitis vinifera ‘Kishmish vatkana’
•Morphological features of pure viniferas
•Seedless (stenospernocarpy)
•Leaves & berries asymptomatic of PM
infections in field and greenhouse
•Monogenic segregation based on leaf
scores of visible masses of conidiophores
4. Selection for berry quality & enological attitude
stage
trait
May-June
Downy mildew
resistance
July-August
Agronomic traits
type of selection
+
resistant
observations
susceptible
+
Fungal sporulation on leaves
Vigour Class 1 extreme weakness
Vigour: 5 classes canopy
> 2 clusters/shoot
No. Of shoots/bud
No. Clusters/shoot (fertility)
AugustSeptember
+
Yield and
fruit quality
Bunch diseases and disorders
Sugar accumulation (°Brix weekly)
Clusters features (size, shape, loose/tight)
Berry features (size, shape, colour)
Low yield
Bunch diseases and disorders (powdery
mildew, Botrytis, Acetobacter, berry and
stem shrivel)
Yield (Kg/vine)
No. of clusters/vine
Single cluster weight (5 clusters)
Berry weight (averaged over 3 samples of 15 berries)
harvest
Bunch & berry & picture pics
Sampling : - metabolite profiling (2 samples of 15 berries)
- sensorial test (1 samples of 20 berries)
- 1 Kg of grapes for nano-vinification
juice
Technologial
parameters
+
-
Sugar content (°Brix)
Titratable acidity
pH
wine
Flavour and
aroma
+
-
Analytical scores for macro-components of
wine quality: Alcool content, Residual sugars,
Titratable acidity, Volatile acidity, Malic acid,
Tartaric acid, Glycerol, K,
Metabolite profiles for micro-components of
flavour and aroma
Sensorial analysis: Wine testing
Propagation of selected lines
Image records of each selected line at harvest
Tools for assisted selection of fruit & wine quality
Cross Resistant-LowQuality x Susceptible-HighQuality
200x
200x+1
seed germination
200x+1
1° vegetative season
200x+1
Mature canes for grafting on commercial rootstocks
200x+2
1° vegetative season of grafted plants (rooting in nursery)
200x+3
2° vegetative season of grafted plants (vineyard)
200x+4
1° season of yield from vines (commercial vineyards)
DM-MAS
berry
Metabolite
profiling
SELECTION THROUGH THE SEASON
stage
trait
observations
May-June
Downy mildew
resistance
Fungal sporulation on leaves
July-August
Agronomic traits
Vigour: 5 classes canopy
Markers linked
to DM-R
No. Of shoots/bud
No. Clusters/shoot (fertility)
AugustSeptember
Yield and
fruit quality
Sugar accumulation (°Brix weekly)
Clusters features (size, shape, loose/tight)
Berry features (size, shape, colour)
Bunch diseases and disorders (powdery mildew, Botrytis, Acetobacter,
berry and stem shrivel)
Yield (Kg/vine)
No. of clusters/vine
Single cluster weight (5 clusters)
harvest
Berry weight (averaged over 3 samples of 15 berries)
berry
Bunch & berry & whole plant pics
Sampling : - metabolite profiling (2 samples of 15 berries
- sensorial test (1 samples of 20 berries)
Metabolite
profiles
- 1 Kg of grapes for nano-vinification
juice
Technologial
parameters
Sugar content (°Brix)
Titratable acidity
pH
wine
Flavour and
aroma
Analytical scores for macro-components of wine quality: Alcool content,
Residual sugars, Titratable acidity, Volatile acidity, Malic acid, Tartaric acid,
Glycerol, K,
Metabolite profiles for micro-components of flavour and aroma
Sensorial analysis: Wine testing
wine
Components
of quality
Flavour and
aroma-MAS
Metabolite profiling of grapes
•Mass spectral analysis of grape fruit volatiles (Waters GCT™ Premier system)
•GC/MS in electron ionisation (EI+) mode. ChromaLynx™ was used to identify
components present in the chromatograms and provide NIST library suggestions.
•Headspace volatiles were extracted by exposing a SPME fibre to the vial
headspace
Metabolite profiling of grapes
EI Chromatograms of Berry Skin
3
3562_12
TOF MS EI+
TIC
3.21e5
16.26
14.07
Bianca
23.80
%
24.93
30.81
33.65
29.48
26.11
34.05
28.68
16.48
13.56
19.25
24.36
33.11
20.09 20.96
45.83
38.08 38.82 39.47
27.79
1
12.00
3562_10
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
44.00
46.00
TOF MS EI+
TIC
5.67e5
16.29
24.93
%
14.07
29.77
Chardonnay
23.80
30.81
33.65
28.68
13.55
19.25 20.96 22.24
15.90
34.05
26.11
23.28
38.82
36.74 38.07
31.83
42.14
45.83
1
Time
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
44.00
46.00
PCA plot & separation
between groups of
samples
5. DNA Fingerprinting of the Italian National Catalogue
of grape cultivars
Markers:
Accessions:
38 tri-, tetra- and penta-nucleotide
SSR uniformly scattered across the
genome (1-3 per linkage group)
1152 grapes including:
• Vitis species
• Vitis hybrids
Selected from an initial panel of 94
SSR based upon:
• rootstocks
•low stuttering
• Vitis vinifera (national & regional cvs)
•highest power of discrimination
•regular allelic step
and validated on a panel of 48
cultivars
• Vitis vinifera (international cvs)