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GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA

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Presentation on theme: "GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA"— Presentation transcript:

1 GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA

2 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)

3 Sebastián Gómez Talquenca 1- Clonal selection
Sanitary selection Genetic selection

4 Sanitary selection National regulatory framework since 2002
Two levels of material Certified Standard

5 Certified stock ELISA test Indexage Wood grafting Green grafting
3 years at field nursery 2 years in tunneling nursery Green grafting

6 Certification support
Development of new diagnostics techniques Identification and characterization of local virus isolates

7 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

8 Qualitative parameter
Quality vs. yield Qualitative parameter A B 18 7 15 19 16 12 8 17 6 20 14 10 9 13 11 4 1 5 2 3 berry size C ºBrix media pH anthocianas (color) D? media 50 100 150 200 250 300 350 Yield

9 Clonal Selection of Malbec in Mendoza
Clones group A

10 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)

11 Financial support Clonal selection Clonal evaluation
National project INTA Clonal evaluation Regional project INTA Clonal homologation Certification National project INTA, CONICET project

12 2.b- Assessment of genetic diversity by molecular markers
Dra. Liliana Martínez Facultad de Ciencias Agrarias Universidad Nacional de Cuyo Mendoza, Argentina 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.

13

14 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.

15

16 RESULTS Moscatel de Alejandría x Criolla Chica Torrontés Riojano
Torrontés Sanjuanino Moscatel Amarillo

17 “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.

18 Polimorphism in clones 1 and 15 Rest show the same allele pattern
19 clones Malbec 6 loci: VVMD7, VVMD27, VVMD31, VVS2, ssrVrZAG62 y ssrVrZAG79 RESULTADOS Polimorphism in clones 1 and 15 Rest show the same allele pattern

19 Diferentiation of Syrah clones
Few polimorphism VrZAG 62 pb VrZAG79 pb

20 SSAP (Specific Sequence Amplified Polymorphism) Idem AFLP, except
Selective amplification: primer retrotransposon Vine-1 For clones identification

21 In Colaboration with University of
SSAP retrotransposon Vine-1 In Colaboration with University of Ljubljana, Slovenia

22

23 Negra Corriente Tacna Negra Corriente Majes Negra Corriente NCI Torrontés Sanjuanino Torrontés Mendocino Moscatel Mendocino Torrontés Riojano Italia Moquegua Moscatel Rosado Buredeos Tacna Pedro Giménez Criolla Grance Cirolla Chica Mollar Majes Rosada Vitor Italia Majes Italia Tacna Quebtanta Mollar ICA Buredeos Borgoña Moscatel Malbec Uvina Figure 1. Pattern alleles of Argentinean and Peruvian criollas varieties using VrZAG79 loci.

24

25 Comercial vineyards analysis with microsatellites markers
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.

26 PhD thesis of Cecilia Cesari
“Morphological and molecular characterization of Botryosphaeria spp. isolated from different vineyard of Argentina PhD thesis of Cecilia Cesari

27 (Internal Transcribed Spacer)
ITS (Internal Transcribed Spacer) Ribosomals Genes Highly conserved genes : 18S, 5.8S y 28S ITS Sequences Hundreds and thousands copies in the genome Appropiate length to Sequence. Highly variable regions among species

28 Projects Dra. Cecilia Agüero
3- Genetic transformation 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

29 Evaluation of sequences that codify signal peptides
from grapevines xylem sap proteins 20 25 37 60 75 100 150 250 ‘Chardonnay’ Xylem Sap Proteins Fig 1.

30 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),

31 NtPRp27-like protein 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

32 Evaluation of sequences that codify signal peptides
from grapevines xylem sap proteins Figure 2. Embriogenic Callus from inmature anther cultured in vitro in PIV media. Figure 3. Somatic embryos from ‘Sultanina’ in WPM media (left) and plantlets transfered to ½ MS media for further development (right).

33 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

34 Genetic Transformation
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

35 Methods In vitro multiplication of PCR-positive transgenic lines [presence of gene (X1-425, X1-496, X1-694 and X1+UTR-540)], ‘Sultanina’ no transformed controls, ‘Pedro Giménez’, ‘Criolla Chica’ and ‘Cereza’ plants. Microplants hardening in hydroponic and greenhouse conditions.

36 Nutritive solution composition (Long Ashton)
Methods Hydroponics Table 1. Nutritive solution composition (Long Ashton)

37 Botrytis tolerance analysis of Vitis vinifera cv
‘Sultanina’ transformed with pear PGIP RB CaMV35S-pgip-ocs3’ CaMV35S-uidA-3’term CaMV35S-nptII-tml3’ LB Schematic representation of the binary plasmid pDU

38 Untransformed Control
Expression of pear pgip gene in leaves infected with Botrytis cinerea slowed down the expansion of the lesions Untransformed Control gus transgenic gus/pgip transgenic Exp 1 (Sep 2002) 10.2 ( 0.53) 8.2 ( 0.32) 6.8 ( 0.21) Exp 2 (Nov. 2002) 9.8 ( 0.59) 8.9 ( 0.21) 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) Botrytis cinerea infection of control (left) and transgenic grape leaves (right) Agüero et al Molecular Plant Pathology 6 (1): Agüero et al Vitis 45(1):1-8.

39 4- Table grape breeding program
INTA EEA Rama Caída 4- Table grape breeding program Silvia Ulanovsky – Ruben Osorio – Eliana García Application of in vitro culture and molecular markers Goals Seedlessness Big berry size Extreme ripening time Muscat flavour Loose and uniform bunches

40 Obtention of new varieties
Application of in vitro culture Traditional breeding Seeded females Seedless pollinators Seedless females Seedless pollinators X X Seed traces Normal seeds Laboratory Inmersion in water In vitro culture Germination Greenhouse Germination Acclimatization to in vivo conditions Transfer to the field

41 Field evaluations Second selection plot: 10 plants for each genotype selected First selection plot: plant for each genotype EEA Rama Caída 5.160 seedlings EEA San Juan 15 genotypes multiplied 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

42 Construction of genetic maps QTL analysis
M.R.: Moscatel Rosado, R.: Ruby Seedless, C.: Consensus QTL analysis Seed fresh weight Seed dry weight Berry weight

43 CURRENT and FUTURE PROJECTS
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

44 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

45 ¡ THANK YOU VERY MUCH !


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