perennial ryegrass (lolium perenne l.) improvement through cisgenics®

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

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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


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


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


GTAC


GTAC


GTAC AAAAAAAAATTTTTTTTT


Divide in half; ligate to linkers A and B

MA

GN

ET

MA

GN

ET


CATG

GTAC


CATGGTAC


CATG

GTAC


CATG

GTAC


CATG


CATG


Cleave with tagging enzyme; Blunt end

GGATGCATG◊◊◊◊◊◊◊◊◊◊CCTACGTAC ◊◊◊◊◊◊◊◊◊◊


GGATGCATG □□□□□ □□□□□CCTACGTAC □□□□□ □□□□□

GGATGCATG□□□□□ □□□□□CCTACGTAC □□□□□ □□□□□



Ligate
































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


Concatanate ditags

Clone concatanated ditags and sequence


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


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


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

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1020

3040

5060

70

10

00

10

9

11

23

60

21

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11

29

50

31

6

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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

perennial ryegrass (lolium perenne l.) improvement through cisgenics®

Documents

ryegrass genes

ryegrass genome database

cisgenics schouten

cisgenics transgenics

clover genes

compatible genome

rice genome

ryegrass tear