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									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
                                                                         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™

    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™


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 under mechanised
       systems (world’s best practice)
      Comparison with comparator
 Range of selections under
            Heritability and expected genetic gain



                                                 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
              Heritability (h )                                              Genetic gain (%)

                                                 Germplasm – Vitis spp.
                                                 Crosses  Interspecific families
  Creation and
recombination of                                 Ease of propagation
genetic variability                              Graft compatibility
                                                 Nematode tolerance
                       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

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

 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).

• 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
          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
 Pathways to tartaric acid
                                                                             COOH         1COOH
     CO                                                                        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
     CH2OH                 CH2OH               CH2OH           CH2OH                      6CH OH

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
     Oxalic acid           L-threonic acid
         The search for TA synthesis
   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


                                                                                                                      veraison, pre-
                                                                                                                      Root & leaf

                                                                                                      Petiole, stem

                                                                                                      Ripe berry


                                                                                                                      & shoot

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.


                             Tartaric acid                                                                                    Oxalic acid
                                                                                           mg OA per berry

mg TA per berry


                  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. Gene expression
                        studies showed that
                        one candidate gene
                        correlated with tartaric
                        acid biosynthesis;
                        other candidates were
                        expressed in both TA
                        and non TA
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   5         10     15
                                                                   Time (min)
                                                                                     Idonate with NADH
                                                               5         10     15   water (no substrate)
                                                                   Time (min)        no enzyme and 5-KG
                                       A 340 nm

                                                                                     ascorbate with NADH
                                                   -0.75                             NADPH with 5-KG
                                                   -1.00                             Boiled enzyme
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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