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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 characterize and assess genetic diversity: 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 n Sanitary selection n Genetic selection

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

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

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

7 Clonal selection Done n 19 Malbec clones with agronomical and enological evaluation in one place n Several head of clone selected for –Syrah, Cabernet Sauvignon, Chardonnay, Semillon, Sauvignon Blanc, Barbera, Bonarda, Tempranillo, Torrontés Riojano

8 Quality vs. yield Yield Qualitative parameter berry size ºBrix pH media anthocianas (color) A B C D?

9 Clonal Selection of Malbec in Mendoza Clones group A

10 Clonal selection Doing n Agronomical and enological evaluation of Syrah (12 clones) n Preliminary agronomical and enological evaluation of Semillón n Implantation of Bonarda, Tempranillo and Torrontés evaluation trials in one place n Implantation of Malbec evaluation trials in multiple places (homologation of clones)

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

12 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

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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.

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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 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

19 Diferentiation of Syrah clones Few polimorphism VrZAG pb VrZAG pb

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

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

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

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25 Comercial vineyards analysis with microsatellites markers 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 Morphological and molecular characterization of Botryosphaeria spp. isolated from different vineyard of Argentina PhD thesis of Cecilia Cesari

27 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)

28 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

29 Chardonnay Xylem Sap Proteins Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins Fig 1.

30 Chi1b ATGAAGATATGGGGACTGCGTTTGTTCCCTTTAATGCTCTTAGCTATAGGTGGCGCCTTTGCACAA GAGCAATGTGGAAGGCAAGCCGGTGGAGCATTATGTTCAGGAGGGCTGTGTTGTAGCCAATATG GTTACTGTGGCAGCACTTCTGCCTACTGCTCCACTGGCTGTCAGAGCCAATGTCCTTCTGGTGGT TCCCCTTCTACTCCCTCCACTCCAACCCCAACTCCCAGTGGCGGCGGTGGGGATATTAGTTCTCTC ATTAGCAAATCACTATTTGATGAAATGTTGAAGCACCGCAATGATGCTGCTTGCCCCGGCAAGGG CTTCTACACTTACGAAGCTTTCATTTCTGCTGTTAAGTCCTTTGGAGGTTTTGGAACGACTGGTGA CACCAACACTCGGAAAAGAGAGATTGCTGCCTTTCTGGCTCAAACTTCACATGAAACCACAGGT GGTTGGGCATTGCTCCAGATGGACCATATGCATGGGGATATTGCTTCCTTCGGGAACAGGGCAAC CCTGGAGACTACTGTGTTGCCAATCAACAATGGCCATGCGCTTCTGGTAAAAAATATTATGGCCG AGGTCCCATCCAAATTTCATACAACTACAACTACGGTCCAGCAGGAAAAGCCATA MKIWGLRLFPLMLLAIGGAFAQEQCGRQAGGALCSGGLCCSQYGYCGSTSAYCSTGCQSQCPSGG SPSTPSTPTPTPSGGGGDISSLISKSLFDEMLKHRNDAACPGKGFYTYEAFISAVKSFGGFGTTGDTN TRKREIAAFLAQTSHETTGGWALLQMDHMHGDIASFGNRATLETTVLPINNGHALLVKNIMAEVPS 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),

31 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

32 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

33 Table 2. Embryogenic callus number produced in 2005 Cultivar Anther number Embryogenic callus number Malbec Sultanina Torrontés riojano Table 3. Embryogenic callus number produced in 2006 Cultivar Anther number Embryogenic callus number Malbec Sultanina Torrontés riojano Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins

34 Salt tolerance Genetic Transformation Pre-embryogenics callus from anthers co- cultured with Agrobacterium tumefaciens LBA 4404 RB pCaMV 35S-nptII-tNOSpCaMV35S-AtNHX1-tNOS LB

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

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

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

38 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 Molecular Plant Pathology 6 (1): Agüero et al 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) 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)

39 INTA EEA Rama Caída 4- Table grape breeding program 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

40 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 X Laboratory X

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

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

43 n Clonal selection of Torrontés Riojano, Bonarda Argentina. n Mass selection of Pedro Giménez Mendoza y Neuquén. n Characterization of genetic potential of Criolla´s and others grapes by Marker Assisted Selection (MAS) colour salt tolerance fungus tolerance CURRENT and FUTURE PROJECTS

44 Clonal selection To do n Develop the protocol for clones homologation n Homologate the evaluated clones (Malbec and Syrah) n Evaluate the selected clones of others varieties n Perform selection in new varieties

45 ¡ THANK YOU VERY MUCH !


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