1

Many undergrads, high school

students, collaborators, and

many volunteers…

Current research team:

Bill Powell (Director)

Chuck Maynard (Co-Director Emeritus)

Linda McGuigan (TC lab manager)

Allison Oakes (Post doctoral fellow)

Kaitlin Breda (Admin assistant)

Jeff Zarnowski (Field/Greenhouse Man.)

Andrew Teller (Research analyst)

Patricia Fernandes (Visiting scholar)

Andy Newhouse (PhD grad student)

Tyler Desmarais (MS grad student)

Dakota Matthews (MS grad student)

Yoks Bathula (MS grad student)

Xueqing Xiong (MS grad student)

Erik Carlson (MS grad student)

Hannah Pilkey (MS grad student)

Masoumeh Khodaverdi (grad student)

The work of well over 100 people over 28 years

American Chestnut Research & Restoration ProjectSUNY College of Environmental Science & Forestry

(not-for-profit, restoration program with TACF since 1990)

‘Darling’ American chestnut

*

*

*

*

at TACF 2018

*

After over a century of unsuccessful attempts at combating the blight,

what are the choices for restoration?

Asian species or (F1) hybrids

are OK for ornamentals or crops,

Not for restoration

American chestnut

C. dentata

European chestnut

C. sativa

Chinese chestnut

C. mollissima

Japanese chestnut

C. crenata

Options for responding to blight:Which involve risks?

• Do nothing (active decision with consequences!)

• Breeding (Asian chestnuts tolerate blight)

– Plant hybrids

– Backcrossing

• Mutagenesis

• Biocontrol / Hypovirulence

• Genetic engineering

Consider:

GE risks or perceptions relative to traditional methods

Not regulated

Regulated

1/16 Chinese chestnut genes:

10 pages or 2,375 words It was very exciting at

that season to roam the

then boundless chestnut

woods of Lincoln, …Henry David Thoreau, “Walden: or

Life in the Woods,” 1899

Making very small changes, adding only2 words

X

CC AC

Breeding & Transgenics:(Both viable options & both have advantages & disadvantages)

Chestnut has ~ 38,000 gene pairs

blight resistant

> 99.999% American chestnut

- Not related to gluten

- Doesn’t kill fungus: Unlikely to develop

resistance to oxalate oxidase. Fungus

coexists with tree, like Chinese chestnut and

some oaks (saprophyte).

Oxalate oxidase (OxO) from wheat

-Naturally found in many other plants,

mosses, and fungi

Oxalate oxidase (OxO) from wheat

Buster Blight Charlie Chestnut

Isolation of C. parasitica from small stem assay cankers

Example of Darling 54 cankers

Tree & fungus co-exist.

Isolation of C. parasitica 52 DPI

Darling 54 Darling 54

Qing

CULTIVATED FOOD PLANTS

Common Name Scientific Name Reference or NCBI Gene ID

Peanut Arachis hypogaea (Wang et al. 2010)

Oat Avena sativa (Lane et al. 1991)

Beet Beta vulgaris (Obzansky and Richardson 1983)

African oil palm Elaeis guineensis (Rusli, Idris, and Cooper 2015)

Strawberry Fragaria ananassa (Dahiya et al. 2010)

Barley Hordeum vulgare (Sugiura et al. 1979)

Banana Musa paradisica (Anjum, Sundaram, and Rai 2014)

Rice Oryza sativa (Carrillo et al. 2009)

Date palm Phoenix dactylifera NCBI: LOC103698783

Peach & Apricot Prunus spp. (Liang et al. 2010)

Sorghum Sorghum bicolor (Satyapal and Pundir 1993)

Spinach Spinacia oleracea (Laties 1950)

Cacao Theobroma cacao (Gesteira et al. 2007)

Wheat Triticum aestivum (B. G. Lane et al. 1993)

Corn Zea maize (Vuletić and Šukalović 2000)

Natural Sources of OxO (Foods)

Natural Sources of OxO (Wild)

WILD PLANTS AND FUNGI

Common Name Scientific Name Reference or NCBI Gene/Protein ID

Goatgrass Aegilops tauschii subsp. tauschii NCBI: LOC109742630

Spiny amaranth Amaranthus spinosus (Goyal, Thakur, and Pundir 1999)

Ramie Boehmeria nivea (Xuxia et al. 2012)

Bougainvillea Bougainvillea spectabilis (Srivastava and Krishnan 1962)

Stiff brome Brachypodium distachyon NCBI: LOC100841795

Rubber bush Calotropis procera (Freitas et al. 2017)

Insulin plant Costus pictus (Sathishraj and Augustin 2012)

Perennial ryegrass Lolium perenne (Davoine et al. 2001)

Azalea Rhododendron mucronatum (Sakamoto et al. 2015)

Castor bean Ricinus communis NCBI: LOC107261123

Wild einkorn (wheat

progenitor)

Triticum urartu NCBI: EMS64919.1

Narrowleaf cattail Typha angustifolia NCBI: ASM56683.1

Mosses 6 spp. (Laker, Hofmann, and Meeuse 1980)

Split-gill mushroom Schizophyllum commune NCBI: SCHCODRAFT_15706

Dermatophytic fungus Trichophyton rubrum NCBI: TERG_03492

Oxalate oxidase assay

Very important to have

negative controls

without oxalate

Run time:

2 hours for wheat germ

1-12 hours for chestnut(NRC GE Tree webinar)

Easy assay,

Difficult solution prep.

Examples of OxO leaf disk assays(- OA left, + OA right)

Ellis 1 (NT) Darling 38 (T) Darling 28 (T)

Darling 11 (T) Darling 311 (T) Darling 18 (T)

Zhang et al. 2013

Confirm with

PCR

Repeator

OxO protects plant from oxalic acid (OA)OA leaf disk assay (no fungus)

Green – living tissues

Brown – dead tissues

(Dakota Matthew’s assays)

N=10

Lower bar = more tolerantWild type (Ellis 1)

American chestnut

Chinese chestnut has no

Oxalate oxidase, so…

Oxalate-CoA ligase

Oxalyl-CoA decarboxylase

Formyl-CoA hydrolase

Formate dehydrogenase

OA tolerance trait

N=10

‘Qing’

Chinese chestnut

Chinese chestnut tolerance to oxalic acid

0

10

20

30

40

50

60

70

80

90

100

Alleghenychinquapin

Ellis 1American chestnut

Hong KongChinese chestnut

NankingChinese Chestnut

QingChinese chestnut

Pe

rce

nt

Ne

cro

tic

Are

a

50mM Oxalic Acid Soak 24hr

‘Darling’ AC

WT AC

N=10 leaf disks from each tree

Lower bar = more tolerant

Chinese chestnut tolerance to blight

American Chestnut Chinese Chestnut

Inheritance of blight tolerancePollination with transgenic pollen

Inheritance of blight tolerance“mother tree”

Inheritance of blight tolerancePollination with transgenic pollen

Inheritance of blight tolerancePollination with transgenic pollen

Inheritance of blight toleranceBags opened in lab

Small stem inoculations (Preliminary)

D58 T1s from tissue culture

(Andy Newhouse)

Field small stem (~1.0 cm diameter) assays with EP15576 days post inoculation (example from 2008)

Chinese chestnut seedling American chestnut seedling

2-year-old Darling T1 seedlingsthree D58, one D54, OxO minus siblings

18 DPI (still early), EP155

Initial inoculation~1.0 – 1.3 cm diameter stems

Approximate

area

W x H

2

D58 16001, OxO positive D58 16006, OxO negative

2-year-old Darling T1 full sibling seedlings

OxO positive – 18 Days Post Inoculation (DPI)

(8/16/18 )

Starting diameter 11.76 mm Staring diameter 12.66 mm

D58 16001, OxO positive D58 16006, OxO negative

2-year-old Darling T1 full sibling seedlings

OxO positive – 30 Days Post Inoculation (DPI)

(8/28/18 )

2-year-old Darling T1 sibling seedlings

OxO positive – 74 Days Post Inoculation (DPI)

(9/12/18 )

D58 16001, OxO positive

front back

D58 16006, OxO negative

front back

Darling 58 similar to Chinese chestnut at ~ 10 weeks DPI

D58 16001

OxO positive

74 DPI

Chinese chestnut (seedling from previous slide)

76 DPI

Tests for regulatorsExamples

Darling 58 insertion site in chestnut genome

Darling 54 found to be inserted in an intron of a predicted gene

Set aside pending further testing. Focusing on Darling 58.

Non-target Comparison Studies(Other species controls for context)

Consistently: No enhanced risks compared to trad. breeding• Mycorrhizal colonization of transgenic roots (GH, field)• Tadpole growth & development with transgenic leaves• Bee feeding/use of transgenic pollen• Nutrition of transgenic nuts• Caterpillar feeding on transgenic leaves (+ tri-trophic)• Aquatic insect growth/survival on transgenic leaves• Transgenic leaf decomposition rates• Native seed germination through transgenic leaf litter• Native plant abundance near transgenic trees• Growth rates, form, etc.• Metabolomics (similarity of small molecules)• Respiration/photosynthesis• Transgenic inheritance from transgenic pollinations,

survival/expression/growth/blight resistance of offspring

Transgene did not affect

respiration or photosynthesis of T1

chestnuts in an orchard setting

Light

response

curves of

attached

leaves

Rlight

Rdark

Full light

response

curves

Data at low

light: leaf

respiration in

dark and

light via Kok

method

Dr. John Drake and Cindy Duong

VariableP-value of transgene

effect

Respiration in the dark 0.2

Respiration in the light 0.6

Light-saturated photosynthetic rate 0.3

Quantum yield 0.7

Light compensation point 0.3

Light saturation point 0.9

(P-value < 0.05 significant difference)

(slide by Andy Newhouse)

Tadpole Survival: Cox Proportional Hazard Model

Increasing Survival Hazard

SM – Sugar Maple

AB – American beech

D4 – Transgenic

NT – WT American chestnut

HY – Hybrid chestnut

CC – Chinese chestnut

Tadpole Growth & Development(Results without supplemental food)

Goldspiel, HB, Newhouse, AE, Gibbs, JP, and Powell, WA. 2018. Effects of

Transgenic American Chestnut Leaf Litter on Growth and Survival of Wood

Frog Larvae. Restoration Ecology (DOI: 10.1111/rec.12879)

American

chestnuts

USDA-APHIS-BRSSteps & estimated time to decision

Access to petition,

First public input

USDA-APHIS-BRSSteps & estimated time to decision

Likely EIS

9 months

(PPRA)

Environmental Impact Statement (EIS)

USDA-APHIS-BRSSteps & estimated time to decision

EIS

EIS

~17 months

Future submissions

EPAStill deciding if we are regulated

under FIFRA?

– If not, then done with this process

(But still regulated as any other plant)

– If so, we will seek an exemption (because a pesticide registration will

not work for a restoration tree)

Example – annual fees ~$60,000 and we are a not-for-profit, who pays

– Time unknown, but optimistic (efforts to streamline process)

FDA

Consultation – food & feed safety

• Subset of information USDA petition

• Addition to normal nutritional labeling

of nuts for food or feed sale

– Add to ingredients “wheat oxalate oxidase”

• Because OxO gene comes from wheat

• If OxO came from corn or rice, no label needed

• Not science based, just a rule

– We will request a labeling exemption• Because OxO is not a wheat allergen

• 120 day process

• Estimated ~15 months review time after submission

A unique opportunity with the Darling

lines of blight resistant

American chestnut:

Rescuing the surviving

genetic diversity.

18 in. DBH American chestnut

Manlius, NY

Unique feature of the ‘Darling’ American chestnut trees:

Rescuing genotypes surviving trees

Transgenic

American

chestnut

TACFNY LSC “Mother” Trees

Or surviving wild population

Or backcross trees

pollen

Regionally

adapted

Continue to

maximize

out-crossing

Allows:

Allelic rescue,

local adaptation,

and increases

genetic diversity

Genotypes

½ mother & ½ father

Parental

allelic

composition

Offspring

50% OxO & fully blight resistance

seed

40

Darling 58 (D58), Ellis 1 background

pollenAmerican Chestnut mother trees in 2018 (12 new AC lines):

WB275-27 (southern source)

Johnnybrook 40A (594 nuts)

Lasdon

Fert2T2

H1-12H66

Fert3T1

Brag 8G Lasdon

Crumhorn

Fert9T3

I64-J2

Pond

AC Seedling McCabe Plot

Fert9T2* (424 nuts)

Others for experimental use:

B3F3 (Fred’s selected)

F1 Hybrid (Chinese/American)

Allegheny Chinquapin

EC Marron du Var (European chestnut)

Outcross 2 (OC2)

In progress

Outcrossing Darling 58 American chestnut Lead EventProgress to Date on USDA Permitted Sites

D58+ #16020 T1

McCabe R39TA, NY mother tree Fert9T2, NY mother tree

Outcross 1 (OC1) D58+ #16001 T1

12+ new mother tree

backgrounds from 2

father trees.

Pollen from the

resulting OxO+

seedlings can make

OC3 generation.

Pending

Regulatory

approval,

this is what

may be

distributed for

breeding

from SUNY-

ESF

1703 nuts!

Will be used

for long-term

research sitesNew diverse crosses

each year

Reaching Diversification goals

• TACFNY “Mother Tree” program (citizen science)

– Promoting outcrossing to surviving trees and offspring

• Also working with Jared Westbrook & Sara Fitzsimmons (TACF) on broader controlled crosses (Chestnut magazine article)

– Conservation orchards

• We must coordinate our efforts

Nuanced diversification goals

Different levels of diversity for different uses

• Horticultural use: Arboretums, botanical gardens, historical sites, parks, yards, and small (<100 trees) private land plantings.

– Lower diversity: clones, OC1 (T1), and OC2 (T2)

– Example DED tolerant American elm are all a group of clones (Valley Forge, Jefferson, Princeton, etc.)

– First distribution

• Large scale forest restoration (>100 trees, most likely thousands)

– OC 3 and above (Jared’s example)

den

tata

bas

e

Selective breeding

• Diverse trees for forest ecosystem restoration

• Timber-type for woodlots

• Horticultural forms for yard and parks (Alan Hart)

• Nut crops – hybrids? – for agriculture

• Multiple use rotation agroforestry

Next generation:

From TACF Science Review2 southern lines, each with different promoters

2 northern lines, each with different promoters

= 3 new events

New promoter is win3.12

Wound and pathogen inducible

low continuous expression

higher expression after wounding

even higher expression in presence of pathogen

Transgenic OxO events with the win3.12 promoter

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Wilt on 7/24 Wilt on 8/21 Wilt on 8/24 Wilt on 8/29 Wilt on 9/1

Wilt Following EP155 Stem Inoculation

NTAC

QING

WX162

WX167

Preliminary stem inoculation data

Phytophthora and gall wasp

Breeding and GE

Stacking blight tolerance with PRR resistance

• Crossing Darling 58 with B3F2 & F3 with Phytophthora

resistance

– OxO + Chinese Phytophthora resistance

– Working with Dr. Jared Westbrook

– Also scions from Joe James

• B3F3 W5-31-13 R4C12

Stacked genes ready for testing

• Also have many genes and constructs from the FHI

project and other collaborations

• Oxalate oxidase + RPH1 events – From C. mollissima (Chinese chestnut)

– RPH stands for resistance to phytophthora

• Oxalate oxidase + stilbene synthase events – From grape

– Produces resveratrol (antioxidant & antimicrobial found in wine)

– Similar gene enhanced Phytophthora resistance in Papaya

• Visiting scholar from Portugal, Patricia Fernandes– Gnk2-like gene

• Putative resistance gene for Phytophthora cinnamomi infection.

Higher constitutive expression in C. crenata (Japanese chestnut)

than in C. sativa (European chestnut).

www.esf.edu/chestnut