Genetics and plant breeding in Australia CSIRO Vitis breeding: Dr. Rob Walker Power of genomic approaches: Dr. Chris Ford A u s t r a l i a ’ s G r o w i n g F u t u r e Grapevine Breeding Viticultural Seminar Argentina May 2007 Dr. Rob Walker, CSIRO Plant Industry Grapevine breeding in Australia CSIRO is working with Australia’s grape industries to develop new and improved grapevine varieties • Table grapes • Dried grapes • Wine grapes • Rootstocks Tablegrape breeding and evaluation Table grapes National program Breeding by CSIRO Evaluation in WA, Qld and NT by state agencies Aiming for seedlessness, excellent taste, ripe and uniform maturity, large and uniform berry size and good production and postharvest characteristics. Top-working M1 vines from Range of colours, sizes, textures and flavours new selections irradiated buds Red Bright red early White muscat Late white Crisp white seedless seedless seedless seedless seedless Millennium Muscat™ Seeded Very early Muscat flavour Domestic market Berry Weight (g) 5-7 Berry Dimensions mm (diam/lgth) 22 / 25 Bunch Weight (g) 250 - 600 Bunch Number 20 - 45 Yield (kg) 8 - 18 Optimum Maturity (oBrix) 17 Sugar/Acid ratio >32 Magic Seedless™ Black Early Seedless Berry Weight (g) 4-8 Berry Dimensions mm (diam/lgth) 19 / 25 Bunch Weight (g) 450 - 900 Bunch Number 20 - 30 Yield (kg) 10 - 14 Optimum Maturity (oBrix) 17.0 Sugar/Acid ratio > 30 Dried grapes Emphasis is on rain tolerant, low browning sultana types which are disease resistant and show consistent fruitfulness Involves: In-ovulo embryo rescue Top-working for accelerated evaluation Evaluation under mechanised systems (world’s best practice) Comparison with comparator varieties Range of selections under evaluation Heritability and expected genetic gain °Brix Seedlessness Berry weight Yield / vine 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 2 Heritability (h ) Genetic gain (%) Rootstocks Germplasm – Vitis spp. Crosses Interspecific families Creation and recombination of Ease of propagation genetic variability Graft compatibility Nematode tolerance 1000s Screening techniques, selection & inheritance Mineral discrimination Viticultural performance • Grafted vine vigour Assessment of selections • Phylloxera tolerance as grafted vines • Yield 75 • WUE & drought tolerance Regional semi-commercial • Carbohydrate partitioning evaluation Fruit quality Wine quality Commercial performance GxE 4 Industry adoption Rootstocks for quality wine Current Focus Stock-scion compatibility New rootstocksPBR recently released to industry for evaluation Merbein 5489 Selected for: Merbein 5512 low to moderate vigour Merbein 6262 Salt tolerance and water use efficiency ‘reduced’ potassium uptake enhanced wine quality tolerance of phylloxera and nematodes SALT SUSCEPTIBLE SALT TOLERANT Winegrapes Emphasis on varieties suited to Australian conditions, particularly warm-climate regions. Selection for yield performance, improved grape juice composition and wine quality. January 2000 releases: TyrianPBR, CiennaPBR and RubiennePBR Compared with (parent) Cabernet Sauvignon, all 3 have improved yields, higher juice titratable acidity, higher wine colour density and total anthocyanins and have higher colour hue (brighter wine). Pre-breeding • Genes and traits (CSIRO Plant Industry, Adelaide) – Colour and tannins – Grape berry development and ripening – Fruit flavour and aroma – Fungal pathology – Carotenoids, hormones and flavour • Rapid flowering genotypes and breeding efficiency Growing these grapes... ... to make this wine... ... to respond to this consumer Gene expression and vine performance Microarray mapping of gene expression (Mark Thomas and Chris Davies CSIRO Plant Industry Adelaide) Future – microarrays will enable :- Comparison of gene expression in: - high versus low quality - warm versus cool climate - full versus restricted irrigation International Grapevine Genome Program Multinational research initiative to use a genomics approach to discover and determine the function of all grapevine genes. Key people and inputs • Table grapes (Peter Clingeleffer and Steve Sykes) • Dried grapes (Peter Clingeleffer, Steve Sykes and Steve Swain) • Rootstocks (Peter Clingeleffer, Steve Sykes, Rob Walker and Tim Jones) Genetics and plant breeding in Australia CSIRO Vitis breeding: Dr. Rob Walker Power of genomic approaches: Dr. Chris Ford Genomics and post-genomics approaches to understanding grape berry composition Christopher M Ford School of Agriculture Food and Wine The University of Adelaide Grapevine biotechnology Classical era – ‘genetic modification’ Technically difficult with grapevines Widespread opposition to GM crops Post-genomics – a ‘systems approach’ All genes identified ( + physical mapping) Control of gene expression All proteins and metabolites identified varietal; developmental; stress-response Control of metabolic pathways - composition Grape berry acidity 2 major acids – tartaric, malic Malic acid – central metabolite Tartaric acid – unique, unusual and important Use of acids in winemaking $$s costs Future considerations – global warming… Context – physiology of TA and acid metabolism Pathways to tartaric acid COOH 1COOH 1 CO OH 2 OH 2 COOH COOH COOH HO HO 3 HO O A O OH OH 4 HO CHO COOH 3 HO HO HO 4 OH OH OH L-tartaric acid + C4/C5 5CHO HO 5 HO HO O cleavage 6 CH2OH CH2OH CH2OH CH2OH 6CH OH 2 L-Ascorbic 2-keto-L-gulonic L-idonic 5-keto-D-gluconic Glycoaldehyde acid acid acid acid B C2/C3 cleavage 3COOH 1COOH 4 OH 2 + HO 5 COOH 6 CH2OH Oxalic acid L-threonic acid The search for TA synthesis components Classical approaches – biochemical homologous genes grapevines as experimental systems… Molecular resources – analysis of transcripts – identity and abundance predicted enzymatic activity of encoded sequences Identification of candidate sequences 1. cDNA libraries are clustered based upon Bud, root Leaf Post-veraison veraison, pre- Root & leaf Pre-veraison Petiole, stem Flower Ripe berry Pre-veraison Veraison & shoot Flower veraison similarity of EST expression pattern leaf berry petiole, flower, bud, root 2. Unigenes, grouped on similarity of expression across all 55 cDNA libraries Cross referencing candidate genes for in-depth sequence analysis Identification of differentially expressed cDNAs: 565 candidate genes Limiting EST number to no less than 6 per TC narrowed the number of candidate genes: 87 candidate genes Domain and motif screening for oxidoreductase enzymes: 8 candidate genes Berries were sampled from 25 species of grapevine. Organic acid levels were compared for each species. 2.5 15 Tartaric acid Oxalic acid mg OA per berry 2.0 mg TA per berry 1.5 10 1.0 5 0.5 0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 range (species 1-25) range (species 1-25) Correlating biosynthesis with gene expression A. One species identified in this study makes NO tartaric acid B B. Gene expression studies showed that one candidate gene correlated with tartaric acid biosynthesis; other candidates were expressed in both TA and non TA accumulating grapevines Combined metabolic and transcriptional profiling to identify candidate tartrate biosynthetic genes Prov. Patent 503479 Overexpression in E. coli of the protein encoded by candidate gene assays and tag-facilitated purification allowed determination of its catalytic activity Enzymology revealed substrate specificity and 1.00 Sorbitol 2-keto L gulonate catalytic activity for a key 0.75 D-gluconate A 340 nm Water (no idonate) TA synthesis intermediate 0.50 Boiled enzyme Idonate and NADH 0.25 Buffer with no enzyme Idonate with NADP 0.00 0 5 10 15 Time (min) 0.25 Idonate with NADH 0.00 5 10 15 water (no substrate) -0.25 Time (min) no enzyme and 5-KG A 340 nm -0.50 ascorbate with NADH -0.75 NADPH with 5-KG -1.00 Boiled enzyme -1.25 -1.50 -1.75 Outcomes, and future directions • Continuing to investigate the pathway and regulation of TA synthesis • Integrating malate and tartaric acid metabolism • Understanding the metabolism of ascorbic acid during grape berry development The people… University of Adelaide: Seth DeBolt (PhD candidate 2003-2006) Vanessa Melino (PhD candidate 2005- ) Steve Tyerman, Matt Hayes Flinders University of South Australia: Crystal Sweetman (PhD candidate 2006- ) Crista Burbidge (PhD candidate 2007- ) Kathy Soole University of California (Davis) Doug Cook The funding This project was supported by the Australian Government’s Cooperative Research Centre Program and conducted by the Cooperative Research Centre for Viticulture. This work was supported by Australia’s grape growers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government.
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