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GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA

GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA. 2- Use of molecular marker to characterize and assess genetic diversity: Liliana Martínez (UNCuyo). 1- Clonal selection: Sebastián Gómez Talquenca (INTA). 3- Genetic transformation: Cecilia Agüero (UNCuyo).

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GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA

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  1. GENETIC and PLANT BREEDING of GRAPEVINE in ARGENTINA

  2. 2- Use of molecular marker to characterize and assess genetic diversity: Liliana Martínez (UNCuyo) 1- Clonal selection:Sebastián Gómez Talquenca (INTA) 3- Genetic transformation: Cecilia Agüero (UNCuyo) 4- Table grape breeding program: Silvia Ulanovsky (INTA Rama Caída)

  3. Sebastián Gómez Talquenca1- 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 • 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 selectionDone • 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 4 1 11 10 9 14 6 13 3 19 20 12 5 17 2 18 16 8 7 15 0 50 100 150 200 250 300 350 Yield Quality vs. yield A B berry size C • ºBrix media • pH • anthocianas • (color) D? media

  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. Dra. Liliana Martínez Facultad de Ciencias Agrarias Universidad Nacional de Cuyo Mendoza, Argentina lmartinez@fca.uncu.edu.ar 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.

  12. 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.

  13. RESULTS Moscatel de Alejandría x Criolla Chica Torrontés Riojano Torrontés Sanjuanino Moscatel Amarillo

  14. “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.

  15. 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

  16. Diferentiation of Syrah clones Few polimorphism VrZAG 62 189 195 pb VrZAG79 242 248 pb

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

  18. 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.

  19. 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.

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

  21. 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

  22. 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

  23. 250 • 150 • 100 • 75 • 60 • 37 • 25 • 20 Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins ‘Chardonnay’ Xylem Sap Proteins Fig 1.

  24. 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

  25. 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

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

  27. Evaluation of sequences that codify signal peptides from grapevines xylem sap proteins Table 2. Embryogenic callus number produced in 2005 Table 3. Embryogenic callus number produced in 2006

  28. RB pCaMV 35S-nptII-tNOS pCaMV35S-AtNHX1-tNOS LB Salt tolerance Genetic Transformation Pre-embryogenics callus from anthers co- cultured with Agrobacterium tumefaciens LBA 4404

  29. MethodsHydroponics Table 1. Nutritive solutioncomposition (Long Ashton)

  30. Botrytis tolerance analysis of Vitis vinifera cv ‘Sultanina’ transformed with pear PGIP RB CaMV35S-pgip-ocs3’ LB CaMV35S-nptII-tml3’ CaMV35S-uidA-3’term Schematic representation of the binary plasmid pDU94.0928

  31. Untransformed Control gus transgenic gus/pgip transgenic Untransformed Control gus transgenic gus/pgip transgenic Thompson Seedless Chardonnay 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) Exp 3 (March 2003) 9.4 ( 0.37) 10.9 ( 0.8) 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.

  32. 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

  33. Obtention of new varieties Application of in vitro culture Traditional breeding Seedless females Seedless pollinators Seedless pollinators Seeded females 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

  34. Field evaluations Second selection plot: 10 plants for each genotype selected First selection plot: 1 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 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 XX 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

  35. Construction of genetic maps M.R.: Moscatel Rosado, R.: Ruby Seedless, C.: Consensus 02 04 05 02 04 05 QTL analysis • Seed fresh weight • Seed dry weight • Berry weight 02 04 05

  36. 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 byMarker Assisted Selection (MAS) colour salt tolerance fungus tolerance

  37. Clonal selectionTo 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

  38. ¡ THANK YOU VERY MUCH !

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