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
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at TACF 2018
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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