Perennial Ryegrass ( Lolium perenne  L.) Improvement Through Cisgenics® Sathish Puthigae
Dedicated to the memory of  Oluf   L .  Gamborg   (1924–2007)  SIVB 2005 Lifetime  Distinguished  Achievement Award Winner
Pastoral Genomics is funded by Meat and Wool NZ, Fonterra, AgResearch, Deer Industry NZ, FRST and Dairy InSight Mission
Our Meat, Dairy, Wool and Deer industries rely on productive pasture for their international low-cost and high-quality pos...
Data from DEXCEL 2002 Condensed tannins Availability of Ryegrass in New Zealand Winter   Spring     Summer   Autumn Feed d...
Cisgenics ® Transgenics
Cisgenics ®  – what? Definitions and considerations <ul><li>A cisgenic® organism is a transgenic organism </li></ul><ul><l...
Cisgenics® - why? <ul><li>Common perceptions of GM </li></ul><ul><ul><li>Revulsion at inter-species transfer </li></ul></u...
Would a cisgenic® product be… <ul><li>A GMO? </li></ul><ul><ul><li>Yes (except, perhaps, in the US) </li></ul></ul><ul><li...
Cisgenics® in the science literature: One idea, many expressions Combination of Intra- and Cisgenics® TBA Precision Breedi...
Continuum in GM approaches Traditional crop breeding Wide hybrids and induced mutations   GM crops with no foreign DNA GM ...
Biotech assisted GM ryegrass cultivars Cisgenics® GeneThresher®  SAGE TM
The ryegrass genome 84% Methylated repeats Nuclear genespace Organelle genomes
EST’s, 15 years on, how do they do? dbEST release 062207; Summary by Organism – 22 June 2007; Uni Gene count  19 July 2007...
From: Sorghum Genome Sequencing by Methylation Filtration Bedell JA, et al. PLoS Biology Vol. 3, No. 1, e13 doi:10.1371/jo...
The ryegrass genome is comprised of islands of genes nestled among oceans of repetitive junk DNA*.   Genomic subclones are...
<ul><li>Number of Sequences   528 539 </li></ul><ul><ul><li>GeneThresher ®     511 987 </li></ul></ul><ul><ul><li>EST     ...
Syntenial tracking against rice genome
<ul><li>Genes </li></ul><ul><li>Promoters </li></ul><ul><li>Regulatory Introns </li></ul><ul><li>Active transposons </li><...
Biotech assisted GM ryegrass cultivars Cisgenics® GeneThresher®  SAGE TM
Transcriptomics: SAGE ™  SCIENCE AAAAAAAAA CATG AAAAAAAAA CATG AAAAAAAAAAAAAA CATG AAAAAAAAAAAAAA CATG AAAAAAAAAAAAAA CATG...
Transcriptomics: SAGE ™  SCIENCE Digest cDNA with a 4 base cutter (anchoring enzyme) MAGNET GTAC TTTTTTTTT TTTTTT AAAAAAAA...
Transcriptomics: SAGE ™  SCIENCE Cleave with tagging enzyme; Blunt end GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC   ◊◊◊◊◊◊◊◊◊◊ GGATGC...
Transcriptomics: SAGE ™  SCIENCE GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC   ◊◊◊◊◊◊◊◊◊◊ GGATGCATG  □□□□□ □□□□□ CCTAC GTAC   □□□□□ □□...
Clone concatanated ditags and sequence Transcriptomics: SAGE ™  SCIENCE Concatanate ditags
SAGE ™ :  S erial  A nalysis of  G ene  E xpression 11 1 3 3 1 1 Compare with  other libraries In planta   functional geno...
<ul><li>Not limited to studying known genes </li></ul><ul><li>Free from cross-hybridization problem </li></ul><ul><li>Sens...
Ryegrass  SAGE ™  Library: Snapshot
Application of  SAGE ™  in ryegrass functional genomics SAGE ™ Genes <ul><li>High-throughput expression analysis </li></ul...
Agrobacterium-mediated transformation technique developed using the variety Tolosa is  now extended to varieties Impact (d...
…  now to assemble a Cisgenic® ryegrass
Validation of elements required for Cisgenics® Transfer border sequences P-DNA - 2 different right border sequences identi...
Perennial ryegrass promoters - inducible (drought) - constitutive Validation of elements required for Cisgenics®
Drought tolerant Cisgenic® perennial ryegrass cultivars Genes for targeted traits Predicted temperature and precipitation ...
<ul><li>in Arabidposis enhanced VP levels lead to increased plant fitness </li></ul><ul><li>transgenic ryegrass tested </l...
Drought tolerance - Vacuolar Pyrophosphatase a b c g d e f h
Drought tolerant Cisgenic® perennial ryegrass cultivars <ul><li>Two different first introns probably do not play a signifi...
Drought tolerant Cisgenic® perennial ryegrass cultivars Genes developed with PG IP – ORF4 & ORF12 Drought tolerance – Tran...
Increased biomass Cisgenic ®  perennial ryegrass cultivars <ul><li>Germinate seeds produced by T 0  perennial ryegrass eve...
Elements to be validated <ul><li>3’ UTR </li></ul><ul><li>Selection marker elimination/cisgenic® selection marker </li></u...
Zac Hanley Shivendra Bajaj Catherine Bryant  Kerry Templeton Geoff Gill Margaret Biswas Kieran Elborough   David Whittaker...
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Perennial Ryegrass (Lolium perenne L.) Improvement Through Cisgenics®

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  • I wish to thank the organizers for giving me the opportunity to present before you our research accomplishments in perennial ryegrass to date. Before I do just that I would like to tell you who we are. Pastoral Genomics is the sole investment by the New Zealand pastoral industries into forage improvement through biotechnology. We are funded by Meat and Wool NZ, Fonterra, AgResearch, Deer Industry NZ, FRST and Dairy InSight. Our research focus is in the area of Cisgenics® and MAS in clover and ryegrass.
  • Our Meat, Dairy, Wool and Deer industries rely on productive pasture for their international low-cost and high quality positions. Biotechnology will give the greatest stepwise and sustainable improvement in pasture productivity. We will use our in-depth knowledge of pasture genomes to enhance conventional breeding. Pastoral Genomics will use ryegrass genes in ryegrass, clover genes in clover to capture the untapped genetic potential in pasture plants.
  • This slide shows the supply and demand for perennial ryegrass in New Zealand pastures. As anywhere else in the world, growth in spring and summer is almost twice during autumn and winter, a period when New Zealand cows are gestating and calving. The decrease in biomass can be attributed to the effects of drought and low temperature as well as the re-routing of energy by the plant from biomass to the production of floral tillers and seeds. Also, throughout the year there is a underlying need for increased condensed tannins to ensure animal health. Given this background it is easy for anyone to predict what our area of research priorities are.
  • Earlier on, I had mentioned that Pastoral Genomics has adopted Cisgenics® over transgenics as the preferred tool for plant improvement. I will now dwell a bit more and explain what is Cisgenics®
  • A cisgenic® organism is a transgenic organism with a subtle but critical difference. A cisgenic® organism is a transgenic organism where elements of the organism’s own genome are used in place of elements from other genomes. So, no elephant genes in daisies or toad genes in potatoes. There are many things that cannot be done via this approach, and transgenic option will be required now and perhaps always.
  • Why is that we are subscribing to cisgenics®? It is because of the common perceptions of GM crops held world-wide and also because of New Zealand-specific geo-political realities. We believe cisgenics® could be favourably assessed over transgenics under these constraints.
  • A cisgenic® product is a genetically modified organism (GMO) that is to be defined as a living modified organism (LMO) according to the Cartagena Protocol on Biosafety, but perhaps also one that may be generally recognized as safe (GRAS) organism.
  • There are various flavours of Cisgenics® in the literature and this table describes the vast majority of them.
  • Here I have adapted a slide from Crop&amp;Food that describes the progression of crop development to describe where cisgenics® lie.
  • The foundation for our cisgenics® approach is built upon two genomics technologies, namely GeneThresher®, i.e. methyl filtered genomic sequence database and SAGE™, i.e. serial analysis of gene expression.
  • The ryegrass genome is organised methylated so called junk DNA ( * red) non-methylated functional gene space shown in blue and green. dark green * shows the organelle genome content blue * the nuclear non-methylated genespace. Traditionally, EST technology has been used to access the functional genes and relies heavily according to their expression. GeneThresher® uses the methylation status of the functional gene sequences to isolate it in an unbiased manner.
  • As I had mentioned earlier, EST is the most widely used technology to isolate functional gene sequences. Despite a massive build-up of EST database over the past 15 years, in Arabidopsis and rice ~15% and ~10% of the genes have not been captured by this technology. In black cottonwood ( Populus trichocarpa ), whose genome was published last year, the draft analysis indicates 45 555 protein coding gene loci, of which only 54% were covered by the EST database comprising of 102 019 entries. Similarly, a preliminary g enome analysis in barel medic ( Medicago trunculata ) indicates the presence of 42 358 gene coding loci, but NCBI EST database of 236 819 entries indicate 38% contributing to the unigene database. However, in many organisms the EST database tends to be small and hence approximately 50% of the genes are not discoverable by standard EST projects.
  • A comparison between GeneThresher® and EST in gene space capture as demonstrated by the Sorghum project.
  • We invested in GeneThresher® in partnership with Orion genomics based in the US Looking closer at the ryegrass genome we see that there are islands of un-methylated genes (shown in blue) amongst methylated junk DNA shown in red. GeneThresher® based on the fragmentation of the genome and the selection of un-methylated DNA only for sequencing. Multiple hits per gene Random gene tagging equal chance of low expressed genes being identified as high Contig sequences put together
  • Here is a snapshot of what we have got in our genomic database.
  • This valuable resource was placed in Gramene! Database more than three years ago for the benefit of monocot researchers worldwide.
  • For marker assisted breeding we have characterised over 2,000 microsatellite markers and many thousands of SNP’s.
  • The foundation for our cisgenics® approach is built upon two genomics technologies, namely GeneThresher®, i.e. methyl filtered genomic sequence database and SAGE™, i.e. serial analysis of gene expression.
  • Here is what we got in our expression database.
  • P-DNA is the ryegrass ortholog of Agrobacterium T-DNA. The terminology is based on Simplot Plant Sciences publication in Plant Physiology. Rommens et al. 2004 Plant Physiol 135(1): 421-431
  • Why drought? Even in New Zealand, climate change is going to make water a scarce and erratic commodity.
  • A preliminary screen of VP events screened for drought tolerance in order to select the best 20% of the events. Many plants were examined for the engineered VP expression at the transcript level and a few at the protein level.
  • Perennial Ryegrass (Lolium perenne L.) Improvement Through Cisgenics®

    1. 1. Perennial Ryegrass ( Lolium perenne L.) Improvement Through Cisgenics® Sathish Puthigae
    2. 2. Dedicated to the memory of Oluf L . Gamborg (1924–2007) SIVB 2005 Lifetime Distinguished Achievement Award Winner
    3. 3. Pastoral Genomics is funded by Meat and Wool NZ, Fonterra, AgResearch, Deer Industry NZ, FRST and Dairy InSight Mission
    4. 4. Our Meat, Dairy, Wool and Deer industries rely on productive pasture for their international low-cost and high-quality positions. Biotechnology will give the greatest stepwise and sustainable improvement in pasture productivity. We will use our in-depth knowledge of pasture genomes to enhance conventional breeding. Pastoral Genomics will use ryegrass genes in ryegrass, clover genes in clover to capture the untapped genetic potential in pasture plants. Introduction
    5. 5. Data from DEXCEL 2002 Condensed tannins Availability of Ryegrass in New Zealand Winter Spring Summer Autumn Feed demand and supply 0 10 20 30 40 50 60 70 80 90 June September December March Pasture growth, herd demand (kg DM/ha/day) Drought/Temp tolerance Flowering Pasture Growth Herd Demand
    6. 6. Cisgenics ® Transgenics
    7. 7. Cisgenics ® – what? Definitions and considerations <ul><li>A cisgenic® organism is a transgenic organism </li></ul><ul><li>A cisgenic® organism is a transgenic organism…where elements of the organism’s own genome are used in place of elements from other genomes </li></ul><ul><li>No elephant genes in daisies, no toad genes in potatoes </li></ul><ul><li>There are many things that cannot be done via this approach; transgenic option needed now and perhaps always </li></ul><ul><ul><li>e.g. pharma plants; pesticide tolerance; most biomaterials </li></ul></ul><ul><li>Mostly spoken of in plants </li></ul>
    8. 8. Cisgenics® - why? <ul><li>Common perceptions of GM </li></ul><ul><ul><li>Revulsion at inter-species transfer </li></ul></ul><ul><ul><li>Scientists tinkering with natural order </li></ul></ul><ul><ul><li>Perception of whatever-it-takes approach </li></ul></ul><ul><ul><li>The offer to the public is a take-it-or-leave-it one </li></ul></ul><ul><li>NZ-specific </li></ul><ul><ul><li>Unique social set-up of NZ – partnership and stewardship </li></ul></ul><ul><ul><li>HSNO requires ERMA to consider environmental, toxicological, economic, social, cultural and ethical risks and issues - could cisgenics® be favourably assessed for some of these? </li></ul></ul><ul><li>NB: Risk is not inversely proportional to safety for some </li></ul><ul><li>Precise, parsimonious, neat, aesthetically pleasing </li></ul>
    9. 9. Would a cisgenic® product be… <ul><li>A GMO? </li></ul><ul><ul><li>Yes (except, perhaps, in the US) </li></ul></ul><ul><li>LMO? </li></ul><ul><ul><li>Yes, due to methods used, unless someone lobbies for an exception </li></ul></ul><ul><li>GRAS? </li></ul><ul><ul><li>Yes-maybe, because no new untested components. Would have to be a test case </li></ul></ul>An NZ First! Cisgenic® NO ADDITIVES, GUARANTEED 101% Ryegrass Tear here
    10. 10. Cisgenics® in the science literature: One idea, many expressions Combination of Intra- and Cisgenics® TBA Precision Breeding (Rommens et al.) Intermediate between Schouten and Conner. Achievable in part today; more later. Functional units of endogenous genome (promoters, CDS, etc. in new combinations – we would reserve the right to omit introns) Cisgenics® (Hanley et al.) Like WideHyb approach; difficult to detect (create call for sequence-specific tags to identify GMO?) Cassette-sized fragments of sexually-compatible genome e.g. whole gene with own regulatory elements, introns etc. “ Cisgenics” (Schouten et al.) Likely to be able to do almost anything transgenics can do Recognizable (via BLAST) fragments of endogenous genome in any order Intragenics (Conner et al.) Unique aspects Recombinable unit Flavour
    11. 11. Continuum in GM approaches Traditional crop breeding Wide hybrids and induced mutations GM crops with no foreign DNA GM crops with foreign vectors GM crops with transgenes GM crops with synthetic genes
    12. 12. Biotech assisted GM ryegrass cultivars Cisgenics® GeneThresher® SAGE TM
    13. 13. The ryegrass genome 84% Methylated repeats Nuclear genespace Organelle genomes
    14. 14. EST’s, 15 years on, how do they do? dbEST release 062207; Summary by Organism – 22 June 2007; Uni Gene count 19 July 2007 1 276 692 EST entries 1 211 418 EST entries Arabidopsis thaliana Oryza sativa Number of UniGene entries
    15. 15. From: Sorghum Genome Sequencing by Methylation Filtration Bedell JA, et al. PLoS Biology Vol. 3, No. 1, e13 doi:10.1371/journal.pbio.0030013 EST’s vs GeneThresher® % Genes Tagged 0 100 200 300 400 500 600 700 800 900 Reads (x1000) Methyl filtration EST 100 90 80 70 60 50 40 30 20 10 0
    16. 16. The ryegrass genome is comprised of islands of genes nestled among oceans of repetitive junk DNA*. Genomic subclones are generated by fragmenting DNA from ryegrass. Methyl filtration leaves behind only the ‘genespace’ *Rabinowicz et al . ‘ Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome’ in Nature Genetics 1999 23(11): 305-9 Project completed GeneThresher™ Methyl filtration leaves behind only the ‘genespace’
    17. 17. <ul><li>Number of Sequences 528 539 </li></ul><ul><ul><li>GeneThresher ® 511 987 </li></ul></ul><ul><ul><li>EST 16 552 </li></ul></ul><ul><li>Number of Contigs 80 162 </li></ul><ul><ul><li>Average Contig length (bp) 964 </li></ul></ul><ul><ul><li>Average number of sequences per Contig 3 </li></ul></ul><ul><li>Number of Singletons 189 697 </li></ul><ul><ul><li>Average Sequence length (bp) 507 </li></ul></ul><ul><li>ESTs, Contigs & Singletons with polyA features ~ 25 000 </li></ul>Ryegrass Genome Database
    18. 18. Syntenial tracking against rice genome
    19. 19. <ul><li>Genes </li></ul><ul><li>Promoters </li></ul><ul><li>Regulatory Introns </li></ul><ul><li>Active transposons </li></ul><ul><li>Gene linked markers </li></ul>>8,500 microsatellite markers (linked to non-methylated DNA) >1,000’s Single Nucleotide Polymorphisms GeneThresher™ helped us mine some…
    20. 20. Biotech assisted GM ryegrass cultivars Cisgenics® GeneThresher® SAGE TM
    21. 21. Transcriptomics: SAGE ™ SCIENCE AAAAAAAAA CATG AAAAAAAAA CATG AAAAAAAAAAAAAA CATG AAAAAAAAAAAAAA CATG AAAAAAAAAAAAAA CATG AAAAAAAAAAAAAA CATG Isolate mRNA/total RNA from tissue of choice cDNA primed with oligo(dT) magnetic beads AAAAAAAAAAAAAA CATG TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG TTTTTTTTT TTTTTT AAAAAAAAA CATG TTT TTTTTT AAAAAAAAA CATG TTT TTTTTT MAGNET
    22. 22. Transcriptomics: SAGE ™ SCIENCE Digest cDNA with a 4 base cutter (anchoring enzyme) MAGNET GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA GTAC AAAAAAAAA TTT TTTTTT GTAC AAAAAAAAA TTT TTTTTT Divide in half; ligate to linkers A and B MAGNET MAGNET GTAC AAAAAAAAA TTT TTTTTT CATG GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG GTAC TTTTTTTTT TTTTTT AAAAAAAAAAAAAA CATG GTAC AAAAAAAAA TTT TTTTTT CATG
    23. 23. Transcriptomics: SAGE ™ SCIENCE Cleave with tagging enzyme; Blunt end GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ Ligate GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□
    24. 24. Transcriptomics: SAGE ™ SCIENCE GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG ◊◊◊◊◊◊◊◊◊◊ CCTAC GTAC ◊◊◊◊◊◊◊◊◊◊ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ GGATGCATG □□□□□ □□□□□ CCTAC GTAC □□□□□ □□□□□ PCR Amplify CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG ◊◊◊◊◊◊◊◊◊◊ GTAC ◊◊◊◊◊◊◊◊◊◊ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ CATG □□□□□ □□□□□ GTAC □□□□□ □□□□□ Cleave with Anchoring Enzyme
    25. 25. Clone concatanated ditags and sequence Transcriptomics: SAGE ™ SCIENCE Concatanate ditags
    26. 26. SAGE ™ : S erial A nalysis of G ene E xpression 11 1 3 3 1 1 Compare with other libraries In planta functional genomics Genes Promoters Rice/Ryegrass
    27. 27. <ul><li>Not limited to studying known genes </li></ul><ul><li>Free from cross-hybridization problem </li></ul><ul><li>Sensitive, if sequenced deeply </li></ul><ul><li>Accurate because of dimer-formation prior to amplification </li></ul>Advantages of SAGE ™
    28. 28. Ryegrass SAGE ™ Library: Snapshot
    29. 29. Application of SAGE ™ in ryegrass functional genomics SAGE ™ Genes <ul><li>High-throughput expression analysis </li></ul><ul><li>microarray (Agilent) </li></ul><ul><li>Functional analysis </li></ul><ul><li>model system (rice) </li></ul><ul><li>ryegrass </li></ul>GeneThresher ®
    30. 30. Agrobacterium-mediated transformation technique developed using the variety Tolosa is now extended to varieties Impact (diploid) and Banquet (tetraploid) Biotech assisted GM ryegrass varieties Adapted from: Bajaj et al. A high throughput Agrobacterium tumefaciens -mediated transformation method for functional genomics of perennial ryegrass ( Lolium perenne L.). Plant Cell Reports 2006 25(7): 651-659
    31. 31. … now to assemble a Cisgenic® ryegrass
    32. 32. Validation of elements required for Cisgenics® Transfer border sequences P-DNA - 2 different right border sequences identified - 1 left border sequence identified Borders tested using 2xCaMV35S::Hpt::35S 3’UTR as sample construct T-DNA Plants growing on Hygromycin selection medium T-DNA (control) P-DNA (ORF128)
    33. 33. Perennial ryegrass promoters - inducible (drought) - constitutive Validation of elements required for Cisgenics®
    34. 34. Drought tolerant Cisgenic® perennial ryegrass cultivars Genes for targeted traits Predicted temperature and precipitation for New Zealand in 75 years (data from NIWA)
    35. 35. <ul><li>in Arabidposis enhanced VP levels lead to increased plant fitness </li></ul><ul><li>transgenic ryegrass tested </li></ul>Licensed gene From: Gaxiola et al. Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump Proc Natl Acad Sci, USA (2001) 98(20): 11444-11449 <ul><li>Ortholog of AVP1 found in perennial ryegrass </li></ul><ul><li>Ortholog was not expressed under water-stress </li></ul>
    36. 36. Drought tolerance - Vacuolar Pyrophosphatase a b c g d e f h
    37. 37. Drought tolerant Cisgenic® perennial ryegrass cultivars <ul><li>Two different first introns probably do not play a significant role in drought tolerance </li></ul><ul><li>Planning to conduct a replicated trial for drought tolerance using T 0 plants </li></ul><ul><li>Test if expression of vp1 by a drought-inducible promoter is better than constitutive expression </li></ul>Vacuolar Pyrophosphatase - refinements
    38. 38. Drought tolerant Cisgenic® perennial ryegrass cultivars Genes developed with PG IP – ORF4 & ORF12 Drought tolerance – Transcription factors
    39. 39. Increased biomass Cisgenic ® perennial ryegrass cultivars <ul><li>Germinate seeds produced by T 0 perennial ryegrass events </li></ul><ul><li>Examine for transgenic progeny </li></ul><ul><li>Conduct a replicated trial with transgenic and null progeny plants </li></ul>Gene discovered in our functional genomics programme in rice RICE Wild type ORF54 T1 plants 0 10 20 30 40 50 60 70 1000109 112360210 112950316 112950317 112950318 112950320 Shoot Dry Matter (g)
    40. 40. Elements to be validated <ul><li>3’ UTR </li></ul><ul><li>Selection marker elimination/cisgenic® selection marker </li></ul>Target to produce Cisgenic® compliant perennial ryegrass thereafter
    41. 41. Zac Hanley Shivendra Bajaj Catherine Bryant Kerry Templeton Geoff Gill Margaret Biswas Kieran Elborough David Whittaker Jonathan Phillips Claudia Smith-Espinoza Nimali Withana Catherine Carter Anke Rintelmann Robert Winz Muhannad Al-Wahb Paul Bickerstaff Plus Researchers at: Orion Genomics, Dexcel, HortResearch, AgResearch, MetaHelix, University of Florida, Cold Spring Harbor Lab, University of Manchester, Crop&Food, Phytowelt, Eurogene, Scion (ForestResearch), University of Chile Acknowledgement

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