FHI Biotechnology Approaches

GenomeSequencing

Clonal Testing

Transgenics

Marker-aidedbreeding

New varieties

GE trees

SUNY-ESF team summer 2011Chuck 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

(Andy Newhouse)

Confirmed gene expression

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

(Amelia Bo Zhang)

Relative OxO expression,Example of lowest to highest:

Wirsig = 1

Darling 4 = ~400x

AZ-2SX1-8 = ~80,000x(~200x compared to Darling 4)

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)

No significant difference in relative expression between the two events (p =

0.8117, α = .05, t = 0.2463, df = 8)

Gene expression of ESF39 antimicrobial peptide in root tissues of Hinchee 1 & 2

events

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)

cross

Segregate out ~50% of plants with transgene

Non-transgenic

Trees with desirable traits

Early floweringtransgenic tree

7 events with heat shock promoter, 1 event with constitutive promoter

Early floweringF1, BC1, BC2, etc.

• 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

Objectives/deliverables(additional deliverables not specified in original FHI grant)

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)

• Clone putative Phytophthora (Ink disease) resistance enhancing genes from same cDNA library.

Objectives/deliverables(non-supplemental, additional deliverables

not specified in original FHI grant)

Chinese chestnut CG, putative ID

1 RPH1 (Phytophthora resistance)

2 NPR3/4 (Phytophthora resistance) (working)

SuggestionDarling 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