FHI Biotechnology Approaches

1. FHI Biotechnology Approaches New varieties Clonal Testing Marker-aided breeding Transgenics Genome Sequencing GE trees

2. 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)

3. 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)

4. Leaf assay with Transgenic Events:Darling 4 (OxO), Hinchee 1 (OxO + ESF39) (Andy Newhouse)

5. Darling 4 and Darling 5 inoculations this summer, small stem assays to determine levels of resistance & confirm leaf assays Confirmed gene expression (Andy Newhouse)

6. 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

7. Objectives/deliverables • Molecular characterization (gene expression & insert copy number) • Example with the Oxalate Oxidase (OxO) events Relative OxO expression, Example of lowest to highest: Wirsig = 1 Darling 4 = ~400x AZ-2SX1-8 = ~80,000x (~200x compared to Darling 4) (Amelia Bo Zhang)

8. 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 No significant difference in relative expression between the two events (p = 0.8117, α = .05, t = 0.2463, df = 8)

9. 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

10. 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

11. Objectives/deliverables(non-supplemental, additional deliverables not specified in original FHI grant) • Clone putative Phytophthora (Ink disease) resistance enhancing genes from same cDNA library.

12. 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.

13. 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