Allelic diversity of domestication genes associated with anthocyanincontent and profile in wild grapevine (Vitis vinifera ssp sylvestris)

Rosa Arroyo-García

The grapevine, Vitis vinifera, ssp. sativa was likely domesticated from V. vinifera ssp. sylvestris The sylvestris subpecies has an Euroasiatic distribution The domestication process likely involved:

Selection of female and hermaphrodite plants Selection of plants producing larger, sweeter and colorful fruit Establishment of cutting propagation

Zohary, 1995

Grapevine domestication

Phenotypic variation between wild and cultivated grapevine

Morphological differences• Leaf morphology • Flower type • Fruit size • Seed size

wildcultivated

Leaves

Fruit

Seeds

Differences at biotic and abiotic stresses

Flowers

Habitats of wild grape populations in the Iberian Peninsula

•Sandly soils

•River forest

• River banks

• Coastal sheers and beaches

S1 S2 S3S4

S5S6

S7

S8S9

S10S11

S12

S13

237 samples

61 locations

13 river bank populations

PopulationsGuadiana S8Guadalhorce S12Guadalete S11Guadalquivir S9Palmones S13Marisma S10Ebro S1Deva S2Playa S3Cadagua S4Nervion S5Cares S6Ason S7

V. vinifera ssp sylvestris in the Iberian Peninsula

Are they a mixture of wild forms, cultivated forms and rootstocks?

Genetic and Morphological Characterization

1010Putative hybridsPutative hybrids

Paretage analyses with cultivated grapevine and rootstocks

219 genotypes

Genetic Characterization

Morphological Characterization

192 genotypes219 genotypes

Mol Ecol (2012). 21(4)800-16

Population Structure in wild grapevine

Two major genetic groups of wild grapevine: Northern and Southern

Two major genetic groups of wild grapevine: Northern and Southern

Morphological characterization

34 Phenotypic traits evaluated in 192 accessions

21 OIV traits evaluated 3 consecutive years: 051, 053, 067, 068, 070, 81-1, 81-2, 082, 83-1, 83-2, 202, 203, 206, 208, 220, 223, 225, 230, 236, 241, 503

9 Agronomic valuesProduction per plants (grape weight) Sugar contentTotal acidityAlcohol probable degreeCluster weightBerries per cluster (3 clusters)Must yield (%)Berry weightFlower sex

2 Phenological traitsBud breaking timeVeraison time

2 Enological traitscontent and profile of anthocyanins

IMIDRA, El EncínMadrid

Trait: Berry Color

The grape ancestor has black skinned berries.

Berry color in cultivated grapevines display wide range of variation

The color of berry skin is determinated by the quantive and composition of anthocyanins.

Anthocyanin content/ profile in wild vines has not been reported.

The grape ancestor has black skinned berries.

Berry color in cultivated grapevines display wide range of variation

The color of berry skin is determinated by the quantive and composition of anthocyanins.

Anthocyanin content/ profile in wild vines has not been reported.

V. vinifera ssp sylvestris126 distinct female genotypes

Total Anthocyanin mg/kg grapes

Wild accessions 400-3795 Cultivated García-Beneytez (2003) 204-2068

Cultivated García-Rodríguez (2006) 431-1513

Levels of anthocyanins are slighly high but similar to those presented by winemaking grapes.

Trait:Anthocyanin content

The distribution of the anthocyanin content is different betwen Northen and Southern

populations.

Anthocyanin Biosynthesis

One locus constituted for a cluster of MYB-type transcriptional factor genes

VvMybA1 and VvMybA2 were shown to be functionallyinvolved in berry pigmentation.

Candidate Genes

Fournier-Level et al., 2009

White allele

Red allele

Allelic diversity in VvMybA1

Gret1

a d3

VvmybA1

TS

VvmybA13´LTR5´LTR gag-pol

VvmybA1a

F1 R2

VvmybA1

TS

VvmybA1

VvmybA1c

VvmybA1

TS

VvmybA1E1 E2 E3P

White alelle

Wild type alelle

192 distint female genotypes from Spain

Structure of theVvmybA1 alelles

New allele with insertion in the promotor region of VvmybA1

Genotypes

The most frequent genotype are homozygous for the wild type alelle VvmybA1c

Alleles A B C D E F G HVvmybA1+44+111 + + +VvmybA1+111 +VvmybA1+44 + + +VvmybA1c + + +VvmybA1a + + +

% of Population 69,96 0,45 0,45 0,45 3,59 24,22 0,45 0,45

Allelic frequencies of VvmybA1

The new allele VvmybA1 +111 is not present in the cultivated grapevine

Relationship between genotypes and anthocyanin content at VvmybA1

Homozygous for the red wild alelle showed higher anthocyanin content than

heterozygous

G

VvmybA1 VvmybA2

G

Accession T7b/d3 a/d3Anthocyanin concentration (mg/grape kg )

BU 1.1' + + 460CA 9.7 + + 504CO 7.7 + + 558CO 6.8 + + 680CA 12.3 + + 830BI 1.3' + + 956CO 4.7 + + 982CO 1.5 + + 999J 2.4 + + 1007

Relationship between genotypes and anthocyanin content at VvmybA2

G

Gret 1

VvmybA1 VvmybA2

Genotype

?

Accesions with low anthocyanin concentration

156 163 223 277 295 305 312 378 382 391 418 425Bu 1.1 C A T G A A T T G A A ABu 1.1 T G T G A A T G A A G ACa 9.7 C G G A A A T G A A G ACa 9.7 T G T G A A T G A A G A

Ca 12.3 C A T G A A T T G A A ACa 12.3 C G T G A A T G A A G ABi 1.3 C G T A G G T G A G G ABi 1.3 C G T A A G T G A G G GWhite C G T A A G T T A G G G

Red C G T A A G T G A G G G

Change Cis by Gly

Relationship between genotypes and anthocyanin content at VvmybA2

New allele with a change of Cys by Gly in the coding region of VvmybA2

Step 1: Primer design

Step 2: Primer extension

Step 3: Detection

SNaPShot (Applied Biosystems)

• 32 cv: T (66%)• 16 cv: T/G (34%)

• 32 syl: T (80%)• 8 syl: T/G (20%)

G

SNaPShot (Applied Biosystems)

Allelic diversity of VvmybA2

Georgian samples

• 16cv: T (80%)• 4cv:T/G (20%)

• 171 syl: T (95%)• 9 syl: T/G (5%)

Spanish samples

Levels of anthocyanins in wild accessions are quite variable, but similar to those presented by winemaking grapes.

We have found a new alelles in VvmybA1 and VvmybA2 TF genes.

The alellic diversity in natural populations is higher in the center of diversity of the specie.

The anthocyanin content could be affected by additive or synergistic effects of these alelles.

Conclusions

Trait: Anthocyanin Profile

Castellani et al., 2007

Cyanidin

CyGl

PnGl

Delphinidin

DpGl

PtGl

MvGl

Glycosylation

Methylation of the OH groups in the

B-ring.

Acilation of the glucose

Acylated derivaded

Trait:Anthocyanin Profile

Trisubstituted Disubstituted

Glucose Glucose Glucose

Glucose Glucose

Cyanidin Peonidin Delphinidin

Petunidin Malvidin

Malvidin-acetil-glucoside

Malvidin-p-cumaril-glucoside

-Group A: without acylated anthocyanins

-Group B: anthocyanin monoglucosides type cianidin with acylated derivated

-Group C: anthocyanin monoglucosides type delphinin with acylated derivated (p-coumaraylated).

- Group D: anthocyanin monoglucosides type delphinin with acylated derivated (acetic acid)

Phenotype Characterization

126 distinct female genotypes

Revilla et al., 2010

Distribution of Anthocyanin profile correspond with geographic distribution

Phenotype Characterization

Acylated anthocyanin

Methylated anthocyanin

Family Anthocyanins South North% No Methylated DpGl 1,9-35,2 9,8-44,9

CyGl 0,3-38,2 1,5-42,3% Methyltated PtGl 2,2-21,1 8,8-21,7

PnGl 2,5-62,2 3,6-35,8MvGl 6,42-68,3 5,3-58,9

Family South North Total

Without acylated 4 19 23

with acylated 63 40 103

Candidate Gen: Vv 3´5´O- methyl-transferase 3.2Candidate Gen: Vv 3´5´O- methyl-transferase 3.2

Candidate genes: Methylation level of anthocyanin

- Gene expression in different berryenviromental stages

- Sequence gene candidate

Founier-Level , 2012

Candidate Gene: Expression of VvOMT3.2

Expression OMT 3.2Anthocyanin concentration

Percentage

% No Methylated anthocyanin

Percentage

% Methylated anthocyanin

% Methylated anthocyanin

% No Methylated anthocyanin

10 20 30 40 50 60....|....|....|....|....|....|....|....|....|....|....|....|....|.

SS3.5_Alelo1 MSSSSHRGILKTEALTKYILETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMSS3.5_Alelo2 MSSSSHRGILKTEALTKYLLETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMSS65 MSSSSHRGILKTEALTKYILETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMCO5.5 MSSSSHRGILKTEALTKYLLETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMCR1.5 MSSSSHRGILKTEALTKYLLETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMBrancellao MSSSSHRGILKTEALTKYLLDTSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMSANGUIVESE_Alelo1 MSSSSHRGILKTEALTKYLLETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIMSANGUIVESE_alelo2 MSSSSHRGILKTEALTKYLLETSAYPREHEQLKGLREATVEKHKYWSLMNVPVDEGLFISMLLKIM

100 110 120 130 140 150....|....|....|....|....|....|....|....|....|....|....|....|....|.

SS3.5_Alelo1 QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADGKEEGTLDFAMVDADKENSS3.5_Alelo2 QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADGKEEGTLDFAMVDADKENSS65 QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADGKEEGTLDFAMVDADKENCO5.5 QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADGKEEGTLDFAMVDADKENCR1.5 QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADGKEEGTLDFAMVDADKENBrancellao QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMTVLNDLIADGKEEGTLDFAMVDADKENSANGUIVESE_Alelo1 QDGKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADGKEEGTLDFAMVDADKENSANGUIVESE_alelo2 QDSKIIAVDPDKEAYQTGVPFIKKAGVEHKINFIQSDAMSVLNDLIADVSVLSVARATAAFNQTPL

190 200 210 220 230....|....|....|....|....|....|....|....|....|....|....|.

SS3.5_Alelo1 WFGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*SS3.5_Alelo2 WFGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*SS65 WFGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*CO5.5 WFGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*CR1.5 WFGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*Brancellao WFGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*SANGUIVESE_Alelo1 WSGSVARSEEEEMMDFERAGRVHLMKLNKFLASDPRVELSHLSIGDGVALCRRLY*SANGUIVESE_alelo2 --------------------------------------------------------

Candidate Gene: Sequence of OMT3.2

wild accessions N SNP Haplotypes Hd Pi valueshigh methylation 6 23 6 1 0,01low methylation 6 13 4 0,867 0,0072

The major components of the anthocyanin profile are anthocyaninmonoglucosides type delphinin and there are varaibles quantities of acylated anthocyanins.

In some accession anthocyanin monoglucosides type cianidin are predominate.

We have found accesions without acylated anthocyanins.

The two genetic groups (Northern-Southern) presented different characteristics related to their anthocyanin profile.

VvAOMT3.2 expression profile correlated with the level of methylated anthocyanin.

We have not found a novel VvAOMT variants associated with methylation level.

Conclusions

Alejandro Benito y Félix Cabello, IMIDRA Madrid, Spain

Ali Ergul, Ankara University, Turkey

Eugenio Revilla Autonoma University, Madrid, Spain

David CarrascoAlba Mª Vargas

INIA-CBGP

RTA2008-00036-01

Gabriella de Lorenzis; Osvaldo Failla Millan University, Italy

David Maghez, Georgia

COST-Action FA1003