Perennial Ryegrass ( Perennial Ryegrass ( Lolium perenne Lolium perenne L.) Improvement Through L.) Improvement Through Cisgenics® Cisgenics® Sathish Puthigae Sathish Puthigae
May 11, 2015
Perennial Ryegrass (Perennial Ryegrass (Lolium Lolium perenneperenne L.) Improvement Through L.) Improvement Through
Cisgenics®Cisgenics®
Sathish PuthigaeSathish 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 Pastoral Genomics is funded by Meat and Wool NZ, Fonterra, AgResearch, Deer Industry NZ, FRST and Dairy InSightIndustry 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 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
Data from DEXCEL 2002Data 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 MarchPastu
re g
row
th,
herd
dem
an
d(k
g D
M/h
a/d
ay)
Drought/Temptolerance
Flowering
Pasture Growth
Herd Demand
Cisgenics®Transgenics
Cisgenics® – what?Definitions and considerations
A cisgenic® organism is a transgenic organism
A cisgenic® organism is a transgenic organism…where elements of the organism’s own genome are used in
place of elements from other genomes
No elephant genes in daisies, no toad genes in potatoes
There are many things that cannot be done via this approach; transgenic option needed now and
perhaps always
– e.g. pharma plants; pesticide tolerance; most biomaterials
Mostly spoken of in plants
Cisgenics® - why?Common perceptions of GM
– Revulsion at inter-species transfer
– Scientists tinkering with natural order
– Perception of whatever-it-takes approach
– The offer to the public is a take-it-or-leave-it one
NZ-specific
– Unique social set-up of NZ – partnership and stewardship
– HSNO requires ERMA to consider environmental, toxicological, economic, social, cultural and ethical risks and issues - could cisgenics® be favourably assessed for some of these?
NB: Risk is not inversely proportional to safety for some
Precise, parsimonious, neat, aesthetically pleasing
Would a cisgenic® product be…
A GMO?
– Yes (except, perhaps, in the US)
LMO?
– Yes, due to methods used, unless someone lobbies for an exception
GRAS?
– Yes-maybe, because no new untested components. Would have to be a test case
Cisgenic®NO ADDITIVES, GUARANTEED
Cisgenic®NO ADDITIVES, GUARANTEED
101%Ryegrass
101%Ryegrass
Tear here
An NZ First!
Cisgenics® in the science literature:One idea, many expressions
Flavour Recombinable unit Unique aspects
Intragenics(Conner et al.)
Recognizable (via BLAST) fragments of
endogenous genome in any order
Likely to be able to do almost anything transgenics can do
“Cisgenics”(Schouten et al.)
Cassette-sized fragments of sexually-
compatible genome e.g. whole gene with own regulatory elements,
introns etc.
Like WideHyb approach; difficult to detect (create call for sequence-specific tags to identify GMO?)
Precision Breeding
(Rommens et al.)
TBA Combination of Intra- and Cisgenics®
Cisgenics®(Hanley et al.)
Functional units of endogenous genome
(promoters, CDS, etc. in new combinations – we would reserve the right
to omit introns)
Intermediate between Schouten and Conner. Achievable in part today;
more later.
Traditional crop breeding
Wide hybrids and inducedmutations GM crops
with no foreign DNA
GM cropswith foreign
vectorsGM crops with
transgenes
GM crops with synthetic
genes
Continuum in GM approaches
Biotech assisted GM ryegrass cultivars
Cisgenics®Cisgenics®
GeneThresher® SAGETM
84%
Methylated repeats
Nuclear genespace
Organelle genomes
The ryegrass genome
EST’s, 15 years on, how do they do?
dbEST release 062207; Summary by Organism – 22 June 2007; UniGene count 19 July 2007
1 27
6 6
92
ES
T e
ntr
ies
1 21
1 4
18
ES
T e
ntr
ies
Arabidopsis thaliana Oryza sativa
Nu
mb
er
of
Un
iGen
e e
ntr
ies
From: Sorghum Genome Sequencing by Methylation Filtration Bedell JA, et al. PLoS Biology Vol. 3, No. 1, e13 doi:10.1371/journal.pbio.0030013
% G
enes
Tag
ged
0 100 200 300 400 500 600 700 800 900
Reads (x1000)
Methyl filtration
EST
EST’s vs GeneThresher®100
90
80
70
60
50
40
30
20
10
0
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’
Number of Sequences 528 528 539539
– GeneThresher® 511 987– EST 16 552
Number of Contigs 80 16280 162– Average Contig length (bp)
964– Average number of sequences per Contig 3
Number of Singletons 189 189 697697
– Average Sequence length (bp) 507
ESTs, Contigs & Singletons with polyA features ~25 25 000000
Ryegrass Genome Database
Syntenial tracking against rice genome
• Genes• Promoters• Regulatory Introns• Active transposons• Gene linked markers
>8,500 microsatellite markers(linked to non-methylated DNA)
>1,000’s Single Nucleotide Polymorphisms
GeneThresher™ helped us mine some…
Biotech assisted GM ryegrass cultivars
Cisgenics®Cisgenics®
GeneThresher® SAGETM
AAAAAAAAACATG
AAAAAAAAACATG
AAAAAAAAAAAAAA
CATG
AAAAAAAAAAAAAA
CATG
AAAAAAAAAAAAAA
CATG
AAAAAAAAAAAAAA
CATG
Isolate mRNA/total RNA from tissue of choicecDNA primed with oligo(dT) magnetic beads
AAAAAAAAAAAAAA
CATGTTTTTTTTTTTTTTT
AAAAAAAAAAAAAA
CATGTTTTTTTTTTTTTTT
AAAAAAAAAAAAAA
CATGTTTTTTTTTTTTTTT
AAAAAAAAAAAAAA
CATGTTTTTTTTTTTTTTT
AAAAAAAAACATG
TTTTTTTTT
AAAAAAAAACATG
TTTTTTTTT
MA
GN
ET
Transcriptomics: SAGE™ SCIENCE
Digest cDNA with a 4 base cutter (anchoring enzyme)
MA
GN
ET
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
GTAC AAAAAAAAATTTTTTTTT
GTAC AAAAAAAAATTTTTTTTT
Divide in half; ligate to linkers A and B
MA
GN
ET
MA
GN
ET
GTAC AAAAAAAAATTTTTTTTT
CATG
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
CATGGTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
CATG
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
CATG
GTAC
TTTTTTTTTTTTTTTAAAAAAAAAAAAAA
CATG
GTAC AAAAAAAAATTTTTTTTT
CATG
Transcriptomics: SAGE™ SCIENCE
Cleave with tagging enzyme; Blunt end
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
Ligate
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
Transcriptomics: SAGE™ SCIENCE
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□
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
Transcriptomics: SAGE™ SCIENCE
Concatanate ditags
Clone concatanated ditags and sequence
Transcriptomics: SAGE™ SCIENCE
SAGE™: Serial Analysis of Gene Expression
1113311
Compare with other libraries
In planta functional genomics
Genes Promoters
Rice/Ryegrass
Not limited to studying known genes
Free from cross-hybridization problem
Sensitive, if sequenced deeply
Accurate because of dimer-formation prior to amplification
Advantages of SAGE™
Ryegrass SAGE™ Library: Snapshot
Application of SAGE™ in ryegrass functional genomics
SAGE™
Genes
High-throughput expression analysis• microarray (Agilent)
Functional analysis• model system (rice)• ryegrass
GeneThresher®
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
… … now to assemble a Cisgenic® ryegrassnow to assemble a Cisgenic® ryegrass
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)
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 for New Zealand in 75 years (data from NIWA)
• in Arabidposis enhanced VP levels lead to increased plant fitness
• transgenic ryegrass tested
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
• Ortholog of AVP1 found in perennial ryegrass
• Ortholog was not expressed under water-stress
Drought tolerance - Vacuolar Pyrophosphatase
a
b
cg
d
e
f h
Drought tolerant Cisgenic® perennial ryegrass cultivars
Two different first introns probably do not play a significant role in drought tolerance
Planning to conduct a replicated trial for drought tolerance using T0 plants
Test if expression of vp1 by a drought-inducible promoter is better than constitutive expression
Vacuolar Pyrophosphatase - refinements
Drought tolerant Cisgenic® perennial ryegrass cultivars
Genes developed with PG IP – ORF4 & ORF12
Drought tolerance – Transcription factors
Increased biomass Cisgenic® perennial ryegrass cultivars
• Germinate seeds produced by T0 perennial ryegrass events
• Examine for transgenic progeny
• Conduct a replicated trial with transgenic and null progeny plants
RICE
RICE
Wild
typ
e
ORF54 T1 plants
0
1020
3040
5060
70
10
00
10
9
11
23
60
21
0
11
29
50
31
6
11
29
50
31
7
11
29
50
31
8
11
29
50
32
0
Sho
ot D
ry M
atte
r (g
)
Gene discovered in our functional genomics programme in rice
Elements to be validated
• 3’ UTR
• Selection marker elimination/cisgenic® selection marker
Target to produce Cisgenic® compliant perennial ryegrass Target to produce Cisgenic® compliant perennial ryegrass
thereafterthereafter
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: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