GENETIC and PLANT BREEDING of GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINAGRAPEVINE in ARGENTINA

1-1- Clonal selection: Clonal selection: Sebastián Gómez Talquenca (INTA)Sebastián Gómez Talquenca (INTA)

2- 2- Use of molecular marker to Use of molecular marker to characterize and assess geneticcharacterize and assess genetic diversity:diversity:

Liliana Martínez (UNCuyo)Liliana Martínez (UNCuyo)

3-3- Genetic transformation: Genetic transformation:Cecilia Agüero (UNCuyo)Cecilia Agüero (UNCuyo)

4- 4- Table grape breeding program:Table grape breeding program: Silvia Ulanovsky (INTA Rama Caída)Silvia Ulanovsky (INTA Rama Caída)

Sebastián Gómez TalquencaSebastián Gómez Talquenca

1- 1- Clonal selectionClonal selection

Sanitary selectionSanitary selection

Genetic selectionGenetic selection

Sanitary selectionSanitary selection

National regulatory framework National regulatory framework since 2002since 2002

Two levels of materialTwo levels of material– Certified Certified – StandardStandard

Certified stockCertified stock

ELISA test ELISA test IndexageIndexage

Wood graftingWood grafting– 3 years at field nursery3 years at field nursery– 2 years in tunneling nursery2 years in tunneling nursery

Green graftingGreen grafting

Certification supportCertification support

Development of new diagnostics Development of new diagnostics techniquestechniques

Identification and characterization Identification and characterization of local virus isolatesof local virus isolates

Clonal selectionClonal selectionDoneDone

19 Malbec clones with agronomical 19 Malbec clones with agronomical and enological evaluation in one and enological evaluation in one placeplace

Several head of clone selected forSeveral head of clone selected for– Syrah, Cabernet Sauvignon, Chardonnay, Syrah, Cabernet Sauvignon, Chardonnay,

Semillon, Sauvignon Blanc, Barbera, Semillon, Sauvignon Blanc, Barbera, Bonarda, Tempranillo, Torrontés RiojanoBonarda, Tempranillo, Torrontés Riojano

Quality vs. yield

Yield

Qualitative parameter

berry size

• ºBrix

• pHm

edia

media

• anthocianas(color)

18

7 15

19

16

12

8

17

6

20

14

109

13

114

1

52

3

0 50 100 150 200 250 300 350

A

B

C

D?

Clonal Selection of Malbec in MendozaClonal Selection of Malbec in MendozaClonal Selection of Malbec in MendozaClonal Selection of Malbec in Mendoza

Clones group A Clones group A

Clonal selection Clonal selection DoingDoing

Agronomical and enological evaluation of Agronomical and enological evaluation of Syrah (12 clones)Syrah (12 clones)

Preliminary agronomical and enological Preliminary agronomical and enological evaluation of Semillónevaluation of Semillón

Implantation of Bonarda, Tempranillo and Implantation of Bonarda, Tempranillo and Torrontés evaluation trials in one placeTorrontés evaluation trials in one place

Implantation of Malbec evaluation trials in Implantation of Malbec evaluation trials in multiple places (homologation of clones)multiple places (homologation of clones)

2.a- Identification of cultivar, clones and roostock grapevines by molecular markers: AFLP, SSR.

2.b- Assessment of genetic diversity by molecular markers

2.c- Caracterización molecular de aislamientos de Botryosphaeria spp.

Dra. Liliana MartínezDra. Liliana MartínezFacultad de Ciencias AgrariasFacultad de Ciencias AgrariasUniversidad Nacional de CuyoUniversidad Nacional de Cuyo

Mendoza, ArgentinaMendoza, Argentinalmartinez@fca.uncu.edu.arlmartinez@fca.uncu.edu.ar

RESULTS

Figure 1. Dendrograms of grape varieties using AFLP data. CrChic: Criolla Chica, CrGran: Criolla Grande, PGimen: Pedro Giménez, MoRos: Moscatel Rosado, MoAllo: Moscatel Amarillo, ToRioj: Torrontés Riojano, ToSan: Torrontés Sanjuanino, ToMen: Torrontés Mendocino, Chardon: Chardonnay, Tempra: Tempranillo. Clusters of European, “Criollas”, American accession, and Spanish and “Criolla” are indicated with letters “A”, “B”, “C” and “D”, respectively.

RESULTS

Moscatel de Alejandría x Criolla Chica

Torrontés RiojanoTorrontés SanjuaninoMoscatel Amarillo

“Polymorphism detection in ‘Malbec’ clones using microsatellites markers”

Martínez, L.; García Lampasona, S., Agüero, C. ; Cavagnaro,Pand Masuelli, R.

IX Lationamerican Congress of de Viticulture and Enology, Chile 2003.

RESULTADOSPolimorphism in clones 1 and 15Polimorphism in clones 1 and 15

Rest show the same allele patternRest show the same allele pattern

19 clones Malbec

6 loci: VVMD7, VVMD27, VVMD31, VVS2, ssrVrZAG62 y ssrVrZAG79

Diferentiation of Syrah clones

Few polimorphism

VrZAG 62 189 195 pb

VrZAG79

242 248 pb

SSAPSSAP

In Colaboration with University ofLjubljana, Slovenia

retrotransposonVine-1

Neg

ra C

orr

ien

t e T

acn

a

Neg

ra C

orr

ien

t e M

aje

s

Neg

ra C

orr

ien

t e N

CI

Italia M

oq

ueg

ua

Italia M

aje

s

Italia T

acn

a

Qu

eb

tan

ta

Cir

olla C

hic

aC

riolla G

ran

ce

Bu

red

eos

Bu

red

eos T

acn

aU

vin

aB

org

oñ

aM

ollar

ICA

Mollar

Maje

sTor r

on

tés S

an

juan

ino

Tor r

on

tés R

ioja

no

Tor r

on

tés M

en

docin

oM

oscate

l M

en

docin

oM

oscate

l R

osad

oP

ed

ro G

imén

ez

Malb

ec

Rosad

a V

itor

Moscate

l

Figure 1. Pattern alleles of Argentinean and Peruvian criollas varieties using VrZAG79 loci.

Comercial vineyards analysis with Comercial vineyards analysis with microsatellites markersmicrosatellites markers

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Figure 1. Allelic pattern of assayed varieties using primer VVMD31. Line 1: Zinfandel, 2: Chenin, 3: Uva Rara, 4: Croatina, 5: Corbeau, 6: Bonarda INTA, 7: Bonarda Piemontese, 8: Bonarda 65.3, 9: Bonarda 65.2, 10: Bonarda 65.1, 11: Bonarda 64.4, 12: Bonarda 64.2, 13: Bonarda 64.1, 14: Bonarda 6.2, 15: Bonarda 6.1, 16: Bonarda 4.2, 17: Bonarda 4.1, 18: Bonarda 3.2 and 19: Bonarda 3.1.

“Morphological and molecular characterization of Botryosphaeria

spp. isolated from different vineyard of Argentina

PhD thesis of Cecilia Cesari

ITS Sequences

•Hundreds and thousands copies in the genome• Appropiate length toSequence.•Highly variable regions among species

RibosomalsGenes

•Highly conserved genes : 18S, 5.8S y 28S

ITS(Internal Transcribed Spacer)

Projects Dra. Cecilia Agüero

Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins.

Salt tolerance analysis of Vitis vinifera cv. ‘Sultanina’ transformed with the vacuolar antiporter AtNHX1

Botrytis tolerance analysis of Vitis vinifera cv ‘Sultanina’ transformed with pear PGIP

3- Genetic transformation

‘Chardonnay’ Xylem Sap Proteins

•20

•25

•37

•60

•75

•100•150•250

Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins

Fig 1.

Chi1b

ATGAAGATATGGGGACTGCGTTTGTTCCCTTTAATGCTCTTAGCTATAGGTGGCGCCTTTGCACAAGAGCAATGTGGAAGGCAAGCCGGTGGAGCATTATGTTCAGGAGGGCTGTGTTGTAGCCAATATGGTTACTGTGGCAGCACTTCTGCCTACTGCTCCACTGGCTGTCAGAGCCAATGTCCTTCTGGTGGTTCCCCTTCTACTCCCTCCACTCCAACCCCAACTCCCAGTGGCGGCGGTGGGGATATTAGTTCTCTCATTAGCAAATCACTATTTGATGAAATGTTGAAGCACCGCAATGATGCTGCTTGCCCCGGCAAGGGCTTCTACACTTACGAAGCTTTCATTTCTGCTGTTAAGTCCTTTGGAGGTTTTGGAACGACTGGTGACACCAACACTCGGAAAAGAGAGATTGCTGCCTTTCTGGCTCAAACTTCACATGAAACCACAGGTGGTTGGGCATTGCTCCAGATGGACCATATGCATGGGGATATTGCTTCCTTCGGGAACAGGGCAACCCTGGAGACTACTGTGTTGCCAATCAACAATGGCCATGCGCTTCTGGTAAAAAATATTATGGCCGAGGTCCCATCCAAATTTCATACAACTACAACTACGGTCCAGCAGGAAAAGCCATA

MKIWGLRLFPLMLLAIGGAFAQEQCGRQAGGALCSGGLCCSQYGYCGSTSAYCSTGCQSQCPSGGSPSTPSTPTPTPSGGGGDISSLISKSLFDEMLKHRNDAACPGKGFYTYEAFISAVKSFGGFGTTGDTNTRKREIAAFLAQTSHETTGGWALLQMDHMHGDIASFGNRATLETTVLPINNGHALLVKNIMAEVPSKFHTTTTTVQQEKP

Jacobs,A.K., Dry,I.B. and Robinson,S.P. (1999) Induction of different pathogenesis-related cDNAs in grapevine infected with powdery mildew and treated with ethephon. Plant Pathol. 48 (3), 325-336

ATGGCTAGTAGGCACATTGTCCTTCTCTCTTGTTTTGTATTCCTAGCAGCCCAGCATGGGATCCAAGCAGTCGAGTATGAGGTCACCAACAATGCTGGAAGCAGCGCCGGTGGCGTCCGATTCACAAATGAAATCGGAATCCCGTACAGCAGGCAGACACTAGTATCTGCCACCGACTTCATATGGGGGGTCTTCCAACAGAACACTCCAGAAGAGAGAAAAACGGTTCAGAAAGTGAGTCTGATAATTGAAAACATGGACGGAGTAGCCTATGCTTCCAACAATGAGATTCATGTCAACGCCAACTACATCGGAAGCTACTCAGGCGATGTGAAGACTGAATTCACTGGGGTGCTTTACCATGAGATGACACACATTTGGCAGTGGAATGGCAACGGACAGACTCCGGGGGGACTAATAGAGGGAATTGCCGATTATGTGAGGTTGAAGGCTAACTATGCCCCCAGCCACTGGGTGCAACCTGGGCAAGGGAACCGTTGGGACCAGGGCTATGATGTTACAGCTCGATTTCTGGACTACTGCAACAGCCTTAGAAATGGGTTTGTAGCAGAACTCAACAAGAAGATGAGAAGTGGGTACAGTGCAGACTTCTTCGTGGAGCTTCTGGGGAAGACAGTTGATCAGCTGTGGACTGACTATAAGGGCTA

MASRHIVLLSCFVFLAAQHGIQAVEYEVTNNAGSSAGGVRFTNEIGIPYSRQTLVSATDFIWGVFQQNTPEERKTVQKVSLIIENMDGVAYASNNEIHVNANYIGSYSGDVKTEFTGVLYHEMTHIWQWNGNGQTPGGLIEGIADYVRLKANYAPSHWVQPGQGNRWDQGYDVTARFLDYCNSLRNGFVAELNKKMRSGYSADFFVELLGKTVDQLWTDYKG*

Cramer,G.R. and Cushman,J.C. (2002) An expressed sequence tag database for abiotic stressed leaves of Vitis vinifera var. Chardonnay. Unpublished

NtPRp27-like protein

Figure 3. Somatic embryos from ‘Sultanina’ in WPM media (left)

and plantlets transfered to ½ MS media for further development (right).

Figure 2. Embriogenic Callus from inmature anther cultured in vitro in PIV media.

Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins

Table 2. Embryogenic callus number produced in 2005

Cultivar Anther number

Embryogenic callus number

‘Malbec’‘Sultanina’‘Torrontés riojano’

12001440840

383926

Table 3. Embryogenic callus number produced in 2006

Cultivar Anther number

Embryogenic callus number

‘Malbec’‘Sultanina’‘Torrontés riojano’

480780420

764622

Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins

Salt tolerance Genetic Transformation

Pre-embryogenics callus from anthers co- cultured with Agrobacterium tumefaciens LBA 4404

RB pCaMV 35S-nptII-tNOS pCaMV35S-AtNHX1-tNOS LB

MethodsHydroponics

Table 1.

Nutritive solution composition (Long

Ashton)

Concentración final

Macroelementos mg/ lMgSO4,7H2O 370KH2PO4 218K2H PO4 70K NO3 303

NH4NO3 160Ca(NO3), 4 H2O 826

MicroelementosMnCl2, 4 H2O 1,8CuSO4, 5 H2O 0,176ZnSO4, 7 H2O 0,219H3BO3 2,861

MoO24(NH4)6, 4 H2O 0,258Na2-EDTA 0,03726FeSO4.7H2O 0,0278

RB CaMV35S-pgip-ocs3’ CaMV35S-uidA-3’term CaMV35S-nptII-tml3’ LB

Schematic representation of the binary plasmid pDU94.0928

Botrytis tolerance analysis of Vitis vinifera cv ‘Sultanina’ transformed with pear PGIP

Expression of pear pgip gene in leaves infected with Botrytis cinerea slowed down the expansion of the

lesions

Botrytis cinerea infection of control (left) and transgenic grape leaves (right)

•Agüero et al. 2005. Molecular Plant Pathology 6 (1): 43-51.•Agüero et al. 2006. Vitis 45(1):1-8.

 

  Untransformed Control

gus transgenic

gus/pgip transgenic

Untransformed Control

gus transgenic

gus/pgip transgenic

Exp 1 (Sep 2002) 10.2 ( 0.53)   10.2 ( 0.53) 8.2 ( 0.32)   6.8 ( 0.21)

Exp 2 (Nov. 2002) 9.8 ( 0.59)

8.9 ( 0.21)

  9.8 ( 0.59)

7.4 ( 0.33)

9.4 ( 0.6)

12.0 ( 0.51)

  7.9 ( 0.23)

5.7 ( 0.89)           

           

Thompson Seedless Chardonnay

Exp 3 (March 2003) 9.4 ( 0.37)

10.9 ( 0.8)

INTA EEA Rama CaídaINTA EEA Rama Caída 4- Table grape breeding program4- Table grape breeding program

Application of in vitro culture and molecular markers

GoalsSeedlessnessBig berry sizeExtreme ripening timeMuscat flavourLoose and uniform bunches

Silvia Ulanovsky – Ruben Osorio – Eliana García

Obtention of new varietiesObtention of new varieties

Traditional breeding Application of in vitro culture

Seeded females Seedless females

Normal seedsSeed traces

Inmersion in water •In vitro culture

• Germination

Greenhouse

Germination Acclimatization to in vivo conditions

Transfer to the field

Seedless pollinatorsSeedless pollinatorsX

Laboratory

X

Field evaluationsField evaluations

First selection plot: 1 plant for each genotype

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X XX X X X X X X X X X X X X X XX X X X X X X X X X X X X X XX X X X X X X X X X X X X X XX X X X X X X X X X X X X X XX X X X X X X X X X X X X X X

X X X X X X X X X XX X X X X X X X X X

Second selection plot: 10 plants for each genotype selected

EEA Rama Caída5.160 seedlings

EEA San Juan15 genotypes multiplied

Construction of genetic mapsM.R.: Moscatel Rosado, R.: Ruby Seedless, C.: Consensus

02 04 05 02 04 05 02 04 05

Seed fresh weight

Seed dry weight

Berry weight

QTL analysis

Clonal selection of Torrontés Riojano, Clonal selection of Torrontés Riojano, Bonarda Argentina.Bonarda Argentina.

Mass selection of Pedro Giménez Mendoza y Mass selection of Pedro Giménez Mendoza y Neuquén.Neuquén.

Characterization of genetic potentialCharacterization of genetic potential of of Criolla´s and others grapes byCriolla´s and others grapes by Marker Marker Assisted Selection (MAS)Assisted Selection (MAS)

colourcoloursalt tolerance salt tolerance fungus tolerancefungus tolerance

CURRENT and FUTURE CURRENT and FUTURE PROJECTSPROJECTS

Clonal selectionClonal selectionTo doTo do

Develop the protocol for clones Develop the protocol for clones homologationhomologation

Homologate the evaluated clones Homologate the evaluated clones (Malbec and Syrah)(Malbec and Syrah)

Evaluate the selected clones of Evaluate the selected clones of others varietiesothers varieties

Perform selection in new varietiesPerform selection in new varieties

¡ THANK YOU VERY MUCH !