Transcript Bill Powell - SUNY-ESF - Forest Health Initiative

FHI Biotechnology Approaches
Marker-aided
breeding
Clonal Testing
New varieties
Transgenics
Genome
Sequencing
GE trees
SUNY-ESF team summer 2011
Chuck Maynard, Co-PI
Bill Powell, Co-PI
Linda McGuigan – TC lab manager (80% TACFNY)
Kathleen Baier – technician (100% FHI)
Andy Newhouse – technician (50% FHI/50% BRAG)
Lilibeth Northern – technician (100% FHI)
Amelia Bo Zhang – PhD grad (CPBR)
Allison Oakes – MS grad (TACFNY)
Kristen Russell Steward – MPS grad (FHI)
Aaron Barrigar – Undergrad (BRAG)
Mike Cook – Undergrad (BRAG)
Jessica Miller – Undergrad (BRAG)
Ashoor Howil – Undergrad (volunteer)
Objectives/deliverables
• Held “Early Screening” workshop (1st year)
– Dana Nelson, Susan McCord, & Bill Powell
– May 17-19, 2010, Asheville, NC
• Supplemental FHI grant
– Develop an early screening assay (2nd & 3rd year)
– Summary below
Combined
data from 5
experiments
48-52
inoculations
per leaf type
Error bars =
1 SEM
T-test: P<
0.0001
(Andy Newhouse)
Leaf assay with Transgenic Events:
Darling 4 (OxO), Hinchee 1 (OxO + ESF39)
(Andy Newhouse)
Darling 4 and Darling 5 inoculations this summer,
small stem assays to determine levels of
resistance & confirm leaf assays
Confirmed gene
expression
(Andy Newhouse)
Objectives/deliverables
• Establish field site in New York with eight older vector
constructs (300 trees minimum)
– Currently have five sites planted with two more planned
• Lafayette Rd Experiment Station (Syracuse, NY) - 278 transgenic trees (23
events)
• Heiberg Forest (Tully, NY) – 30 transgenic trees (six events)
• Zoar Valley (near Buffalo, NY) – 61 transgenic trees (six events)
• Lasdon Park (near NYC, NY) – 30 transgenic trees (six events)
• Biofuel Center Demo Plot (near Raleigh, NC) – 20 transgenic trees ( four
events)
– Total of 419 transgenic American chestnut trees planted
– Approximately 40 this spring and many more this fall
• Estimated over 500 will be planted by end of year 2
– USDA APHIS BRS permit #10-357-118r
Objectives/deliverables
• Molecular characterization (gene expression &
insert copy number)
– Example with the Oxalate Oxidase (OxO) events
OxO Expression in TC shoots: 35S-OxO Line
Relative OxO expression,
Example of lowest
to highest:
Normalized Fold Expression
250
200
150
100
Wirsig = 1
Darling 4 = ~400x
50
0
Ellis 1
3-7
(~200x compared to Darling 4)
Normalized
Fold copies
AZ-2SX1-8 = ~80,000x
1-7
1-6
3-8
2-8
#1
3-11
1-8
3-11
1-8
OxO Copy #: 35S-OxO Line
3
2
1
0
Ellis 1
3-7
1-7
1-6
3-8
2-8
#1
(Amelia Bo Zhang)
Objectives/deliverables
– Test selected events for Phytophthora resistance
• Steve Jeffers, Clemson
• USDA APHIS BRS transportation notice # 11-125-101n
• First two events sent with clonal controls (10 trees each)
Gene expression of ESF39 antimicrobial
peptide in root tissues of Hinchee 1 & 2
events
4.00
3.50
Relative Expression
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Hinchee 1
Hinchee 2
No significant difference in relative
expression between the two events (p =
0.8117, α = .05, t = 0.2463, df = 8)
Objectives/deliverables
• Supplemental FHI grant, add two early flowering gene constructs
from Steve Strauss’ lab to the transformation pipeline to determine if
they can induce early flowering in the greenhouse. (2nd & 3rd year)
7 events with heat shock promoter, 1 event with constitutive promoter
Trees with
desirable traits
Early flowering
transgenic tree
cross
Segregate out ~50% of plants with transgene
Early flowering
F1, BC1, BC2, etc.
Non-transgenic
Objectives/deliverables
(additional deliverables not specified in original FHI grant)
• Clone candidate genes (CGs) from full-length cDNA library from Chinese
chestnut stems (up to 30 total).
– Collaborations with Penn State, Clemson, USFS, UGA, & SUNY-ESF
• Adding the new 2nd Generation CG vectors to the transformation pipeline
– UGA & SUNY-ESF
Chinese chestnut CG, putative ID
Chinese chestnut CG, putative ID
1
β-1,3 glucanase
9
Ethylene-response transcription factor
2
CBS domain protein
10
Cysteine proteinase inhibitor
3
UDP glucosyltransferase
11
Lipid transfer protein SSH
4
Thaumatin-like protein
12
SKDH (Shikimate dehydrogenase)
5
DAHP synthase (DHS1)
13
Myo-inositol-1 phosphate synthase
6
Acid phosphatase
14
Triacylglycerol lipase
7
Laccase / diphenol oxidase
15
ACC oxidase
8
Proline-rich protein
16
Germin-like protein (working)
Objectives/deliverables
(non-supplemental, additional deliverables
not specified in original FHI grant)
• Clone putative Phytophthora (Ink disease) resistance
enhancing genes from same cDNA library.
Chinese chestnut CG, putative ID
1
RPH1 (Phytophthora resistance)
2
NPR3/4 (Phytophthora resistance) (working)
Suggestion
Darling 4 event is an excellent choice for deregulation & freedom to operate test case.
Good results from leaf assays,
conformation of enhanced
resistance this summer.
Even if only partial
enhancement, it would be useful
to the breeding program if
deregulated
It breaks down oxalic acid produced by the fungus, taking away one of the
pathogen’s main weapons. No antifungal activity, so likely less regulation.
A similar enzyme gene is up for deregulation in transgenic peanut (Virginia
Tech. petition APHIS petition #10-070-01p) for Sclerotinia Blight Resistance.
Chinese chestnut has a uniquely expressed germin-like protein from the
same gene family as OxO.
Driven by a vascular promoter (VspB from soybean) for more controlled expression.
So far, no differences in mycorrhizal associations, insect feeding, and plant
colonization in BRAG environmental impact studies.
Highlights
• Leaf assays are being developed as an early, non-destructive assay for blight
resistance
– First transgenic events are looking promising
• Currently have over 400 transgenic trees planted representing 23 events. Will
have over 500 trees & more events by end of year.
– Most planted with the help of the general public
– The number of events/year is greatly expanding
– Next spring planting at the Bronx Botanical Garden
• Near where the blight was first described
– Testing insert copy number, gene expression, & leaf assays
• Have cloned 18 “cisgenic” candidate genes from Chinese chestnut
– 16 for Chestnut Blight and 2 for Ink disease (Phytophthora root rot)
– Most are in the transformation pipelines at SUNY-ESF or UGA
• Are testing early (continuous) flowering genes to enhance breeding
Thank you for your support