Botrytis tolerance analysis of Vitis vinifera cv 'Sultanina'

tunisianbromidrosisInternet και Εφαρμογές Web

5 Φεβ 2013 (πριν από 4 χρόνια και 8 μήνες)

132 εμφανίσεις


GENETIC and PLANT BREEDING of
GRAPEVINE in ARGENTINA

1
-

Clonal selection:


Sebastián Gómez Talquenca (INTA)

2
-

Use of molecular marker to


characterize and assess genetic


diversity:


Liliana Martínez (UNCuyo)

3
-

Genetic transformation:


Cecilia Agüero (UNCuyo)

4
-

Table grape breeding program:


Silvia Ulanovsky (INTA Rama Caída)

Sebastián Gómez Talquenca

1
-

Clonal selection


Sanitary selection




Genetic selection


Sanitary selection


National regulatory framework since
2002


Two levels of material


Certified


Standard


Certified stock


ELISA test


Indexage


Wood grafting


3 years at field nursery


2 years in tunneling nursery


Green grafting

Certification support


Development of new diagnostics
techniques


Identification and characterization
of local virus isolates

Clonal selection

Done


19 Malbec clones with agronomical
and enological evaluation in one place



Several head of clone selected for


Syrah, Cabernet Sauvignon, Chardonnay,
Semillon, Sauvignon Blanc, Barbera,
Bonarda, Tempranillo, Torrontés Riojano


Quality vs. yield

Yield

Qualitative
parameter


berry size



ºBrix



pH

media

media



anthocianas

(color)

18

7

15

19

16

12

8

17

6

20

14

10

9

13

11

4

1

5

2

3

0

50

100

150

200

250

300

350

A

B

C

D?

Clonal Selection of Malbec in Mendoza

Clones group A

Clonal selection

Doing


Agronomical and enological evaluation of
Syrah (12 clones)


Preliminary agronomical and enological
evaluation of Semillón


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


Implantation of Malbec evaluation trials in
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 Martínez

Facultad de Ciencias Agrarias

Universidad Nacional de Cuyo

Mendoza, Argentina

lmartinez@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 Riojano




Torrontés Sanjuanino




Moscatel Amarillo



“Polymorphism detection in ‘Malbec’ clones

using microsatellites markers”



Martínez, L.; García Lampasona, S., Agüero, C. ; Cavagnaro,P

and Masuelli, R.





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


RESULTADOS

Polimorphism in clones 1 and 15


Rest 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

SSAP






In Colaboration with University of

Ljubljana, Slovenia

retrotransposon

Vine
-
1

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


Comercial vineyards analysis with
microsatellites 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 to

Sequence.


Highly variable regions

among species

Ribosomals

Genes


Highly conserved genes :


18S, 5.8S y 28S

ITS

(Internal Transcribed Spacer)


Projects Dra. Cecilia Ag
üero


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
At
NHX1



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




ATGAAGATATGGGGACTGCGTTTGTTCCCTTTAATGCTCTTAGCTATAGGTGGCGCCTTTGCACAA
GAGCAATGTGGAAGGCAAGCCGGTGGAGCATTATGTTCAGGAGGGCTGTGTTGTAGCCAATATG
GTTACTGTGGCAGCACTTCTGCCTACTGCTCCACTGGCTGTCAGAGCCAATGTCCTTCTGGTGGT
TCCCCTTCTACTCCCTCCACTCCAACCCCAACTCCCAGTGGCGGCGGTGGGGATATTAGTTCTCTC
ATTAGCAAATCACTATTTGATGAAATGTTGAAGCACCGCAATGATGCTGCTTGCCCCGGCAAGGG
CTTCTACACTTACGAAGCTTTCATTTCTGCTGTTAAGTCCTTTGGAGGTTTTGGAACGACTGGTGA
CACCAACACTCGGAAAAGAGAGATTGCTGCCTTTCTGGCTCAAACTTCACATGAAACCACAGGT
GGTTGGGCATTGCTCCAGATGGACCATATGCATGGGGATATTGCTTCCTTCGGGAACAGGGCAAC
CCTGGAGACTACTGTGTTGCCAATCAACAATGGCCATGCGCTTCTGGTAAAAAATATTATGGCCG
AGGTCCCATCCAAA
TTTCATACAACTACAACTACGGTCCAGCAGGAAAAGCCATA



MKIWGLRLFPLMLLAIGGAFAQEQCGRQAGGALCSGGLCCSQYGYCGSTSAYCSTGCQSQCPSGG
SPSTPSTPTPTPSGGGGDISSLISKSLFDEMLKHRNDAACPGKGFYTYEAFISAVKSFGGFGTTGDTN
TRKREIAAFLAQTSHETTGGWALLQMDHMHGDIASFGNRATLETTVL
PINNGHALLVKNIMAEVPS
KFHTTTTTVQQEKP


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



ATGGCTAGTAGGCACATTGTCCTTCTCTCTTGTTTTGTATTCCTAGCAGCCCAGCATGGGATCCAAGCAG
TCGAGTATGAGGTCACCAACAATGCTGGAAGCAGCGCCGGTGGCGTCCGATTCACAAATGAAATCGGA
ATCCCGTACAGCAGGCAGACACTAGTATCTGCCACCGACTTCATATGGGGGGTCTTCCAACAGAACACT
CCAGAAGAGAGAAAAACGGTTCAGAAAGTGAGTCTGATAATTGAAAACATGGACGGAGTAGCCTATGC
TTCCAACAATGAGATTCATGTCAACGCCAACTACATCGGAAGCTACTCAGGCGATGTGAAGACTGAATT
CACTGGGGTGCTTTACCATGAGATGACACACATTTGGCAGTGGAATGGCAACGGACAGACTCCGGGGG
GACTAATAGAGGGAATTGCCGATTATGTGAGGTTGAAGGCTAACTATGCCCCCAGCCACTGGGTGCAAC
CTGGGCAAGGGAACCGTTGGGACCAGGGCTATGATGTTACAGCTCGATTTCTGGACTACTGCAACAGCC
TTAGAAATGGGTTTGTAGCAGAACTCAACAAGAAGATGAGAAGTGGGTACAGTGCAGACTTCTTCGTG
GAGCTTCTGGGGAAGACAGTTGATCAGCTGTGGACTGACTATAAGGGCTA



MASRHIVLLSCFVFLAAQHGIQAVEYEVTNNAGSSAGGVRFTNEIGIPYSRQTLVSATDFIWGVFQQNTPE
ERKTVQKVSLIIENMDGVAYASNNEIHVNANYIGSYSGDVKTEFTGVLYHEMTHIWQWNGNGQTPGGLIE
GIADYVRLKANYAPSHWVQPGQGNRWDQGYDVTARFLDYCNSLRNGFVAELNKKMRSGYSADFFVELL
GKTVDQLWTDYKG*


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’

1200

1440

840

38

39

26

Table 3.
Embryogenic callus number produced in
2006


Cultivar

Anther
number

Embryogenic
callus number

‘Malbec’

‘Sultanina’

‘Torrontés riojano’

480

780

420

76

46

22

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
-
npt
II
-
tNOS

pCaMV35S
-
AtNHX1
-
tNOS


LB

Methods


Hydroponics

Table 1
.

Nutritive solution

composition
(
Long
Ashton)

Concentración
final
Macroelementos
mg/l
MgSO
4
,7H
2
O
370
KH
2
PO
4
218
K
2
H PO
4
70
K NO
3
303
NH
4
NO
3
160
Ca(NO
3
), 4 H
2
O
826
Microelementos
MnCl
2
, 4 H
2
O
1,8
CuSO
4,
5 H
2
O
0,176
ZnSO
4,
7 H
2
O
0,219
H
3
BO
3
2,861
MoO
24
(NH
4
)
6,
4 H
2
O
0,258
Na
2
-EDTA
0,03726
FeSO
4
.7H
2
O
0,0278
RB

CaMV35S

-
pgip
-
ocs3’

CaMV35S
-
uid
A
-
3’term

CaMV35S
-
npt
II
-
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 Caída


4
-

Table grape breeding program

Application of
in vitro culture

and molecular markers


Goals


Seedlessness


Big berry size


Extreme ripening time


Muscat flavour


Loose and uniform bunches



Silvia Ulanovsky


Ruben Osorio


Eliana García


Obtention of new varieties


Traditional breeding

Application of

in vitro

culture



Seeded females





Seedless females





Normal seeds


Seed traces



Inmersion in water


In vitro

culture



Germination


Greenhouse

Germination

Acclimatization to
in vivo

conditions



Transfer to the field


Seedless pollinators


Seedless pollinators

X

Laboratory

X

Field 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 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 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 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 X X X X X X X X X

Second selection plot:
10 plants for each
genotype selected

EEA Rama Caída

5.160 seedlings


EEA San Juan

15 genotypes multiplied


Construction of


genetic maps

M.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, Bonarda
Argentina.



Mass selection of Pedro Giménez Mendoza y Neuquén.



Characterization of genetic potential

of
Criolla
´
s and
others grapes by

Marker Assisted Selection (MAS)




colour




salt tolerance




fungus tolerance







CURRENT and FUTURE
PROJECTS

Clonal selection

To do


Develop the protocol for clones
homologation


Homologate the evaluated clones
(Malbec and Syrah)


Evaluate the selected clones of
others varieties


Perform selection in new varieties


¡ THANK YOU VERY MUCH !