The Hardwood Genomics Project: Outcomes
and Future Directions after Four Years of
Resource Development
Meg Staton
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of Environmental Stress
Responses in North American Hardwoods
• February 1, 2011 – January
31, 2015
• Creating genomic resources
for hardwood trees
• Current and increasingly
devastating forest threats:
invasive pests and pathogens,
climate change
• Enable population,
evolutionary and
conservation genetics
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
The Team
Penn State University
John Carlson, PI
Teodora Best, Research Associate
Nicole Zembower, Technician
Di Wu, PhD Student
Nick Wheeler, Manager
University of Notre Dame
Jeanne Romero-Severson, Co-PI
Dan Borkowski, PhD Student
Arpita Konar, PhD Student
Andrea Noakes, PhD Student
Lauren Fiedler, Technician
Olivia Choudhary
Michigan Tech University
Oliver Gailing, Co-PI
Sandra Owusu, PhD Student
Sudhir Khodwekar, PhD Student
Scott Schlarbaum, Co-PI
Ami Sharp, Research Associate
Jason Hogan, Research Associate
James Simons, Research Associate
Margaret Staton, Bioinformatics,
Jack Davitt, Research Associate
Nathan Henry, Research Associate
Thomas Lane, Research Associate
University of Missouri
Mark Coggeshall, Co-PI
Christopher Heim, MS student
Clemson University
Haiying Liang, Co-PI
Chis Saski, Director of CUGI
Tatyana Zhebentyayev, Research Associate
Ketia Shumaker, PI
University Tennessee
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Tulip Poplar
Sweetgum
Honeylocust
Northern Red Oak
Black Walnut
Sugar Maple
Blackgum
Green Ash
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Transcriptome Libraries
Stress treatments on full or half
sib seedlings
• Ozone treatments
–
–
•
Wounding
–
–
•
Leaf, twig
BW,GA,BG, TP, SM, SG
Heat, drought, cold
–
–
•
all species except HL
4 levels of ozone across 4
time points
Leaf, petiole, root
GA, HL, SM, SG
Reproductive or other tissues
–
RO, BW, GA
Goals
• Marker development
• Differential expression
• Conserved stress response
pathways across trees
Tulip Poplar
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Transcriptome Sequencing
Species
# Libraries
Raw Data
Transcripts
Green Ash
55
85Gb
107,611
Northern Red Oak 23
41Gb
51,662
Black Walnut
31
41Gb
78,834
Black Gum
16
6Gb
55,630
Honeylocust
5
2Gb
56,845
Tulip Poplar
28
5Gb
53,346
Sweetgum
43
20Gb
127,406
Sugar Maple
67
29Gb
128,406
TOTAL
268
229Gb
659,740
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Transcriptome Informatics
• Identification of SSRs
and primers
• Functional
annotation
– BLAST
– InterProScan
– GO terms
• Differential gene
expression (DESeq2)
Species
# SSRs
Green Ash
341
Northern
Red Oak
472
Black Walnut 680
Black Gum
486
Honeylocust
282
Tulip Poplar
363
Sweetgum
1,332
Sugar Maple
1,068
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC Libraries
RE
# Clones
Average insert size
Coverage
Northern Red Oak
QRNRBa
QRNRBb
HindIII
BstYI
27,648
46,080
110 kb
120kb
4.0X
7.3X
(760Mb genome) (760Mb genome)
Black Walnut
JNC_Ba
HindIII
27,648
130kb
4.5X
(800Mb genome)
JNC_Bb
BstYI
27,648
120kb
4.0X
(800Mb genome)
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC Sequencing
Functional groups
Genes Targeted
Flowering pathway homologs (Arabidopsis)
12
Epigenetic control/chromatin modification
12
Hormone regulation
7
Biotic/abiotic stress
25
Cell division/chromosome segregation
4
Miscellaneous (mainly unknown function)
12
MTP: Chestnut blight QTLs (Cbr1, Cbr2, Cbr3)
24
Total
Finished BACs Total Finished
(Single Contig)
Bases
96
G+C
N50
Northern Red
Oak
77
7,320,181
36%
114,670
Black Walnut
69
8,610,043
35%
120,554
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Full Sibling
Mapping Populations
•
•
•
Goals
– Genetic mapping
– Long term genetic resource
– Phenotyping over many years and locations
– QTL mapping
Two locations
– Missouri University Center for Agroforestry
– Tennessee Tree Improvement Program
Strategies
– Controlled pollination
– Paternity exclusion
• Open pollination, then parentage
analysis
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Full Sibling Mapping Populations
Host
Species
DNA Collected
No. Seedlings
Honeylocust
Yes
226+ half sibs (149 full sibs so far)
Northern Red Oak
Yes
339 full sibs (PE confirmed)
Black Walnut
Yes
323 full sibs (PE confirmed)
Tulip Poplar
Yes
212 full sibs (controlled cross)
Green Ash*
Yes
328 full sibs (controlled cross)
*Produced by Jennifer Koch, FS Northern Research Station
Herbarium samples are being obtained for all parent trees
• vouchers will be deposited at the Dunn-Palmer Herbarium at MU
• imaged and uploaded to the TROPICOS website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Genetic Map Construction
• Northern Red Oak
– 509 full sibs
• 37 genomic SSRs
• 71 EST-SSRs
– 250 full sibs
• GBS: double digest RAD
tag strategy
• Up to 7,994 SNPs
useable
– Map currently has
SSRs + 500 RAD tag
SNPs
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Genetic Map Construction
• Black walnut
– 300 full sibs submitted for GBS
• Honeylocust
– 96 full sibs submitted for GBS
• Tulip poplar
– 196 EST-SSRs
– 365 full sibs
• Green ash
– 370 full sibs
– DNA extraction complete
– SSR optimization underway
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Phenotyping
• Follow on to the current funded study
• Northern Red Oak
– Full sib population established at UT
– Replicated clonal orchard: 339 sibs plus parents were grafted and established
at Missouri, with each tree represented 2-4 times
• Phenotyping in 2013, 2014
–
–
–
–
leafing date
bud burst
leaf morphology
leaf N content
• Future phenotyping
– stomatal density
– insect defoliation
– marcescence
• All phenotypic data was based on Quercus robur descriptors, previously
defined by French colleagues*
*http://w3.pierroton.inra.fr/CartoC
hene/index.php?page=pheno_carac
ter
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Leafing Dates and Bud Burst
Dormant
Stage I
Stage II/
Budburst
Stage III
Stage IV
Stage V
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Software Development
• Sylvan Photomeasure software
• Collaborating with Dr. David Larsen
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1.
2.
3.
4.
5.
6.
Develop Deep EST Sequence Resources
BAC Libraries and BAC Sequencing
Mapping Populations
Genetic Map Construction
Phenotyping and QTL Map Construction
Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Continuation of HWG
• Continues to be updated regularly
• New Jbrowse and webApollo coming this
month
• Expand from a project website to a community
level resource
• Plan to integrate with Galaxy to offer data
analysis
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Future Directions
Resources
Hypothesis-Driven Science
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
NSF Advisory Board
Penn State University
John Carlson, PI
Teodora Best, Research Associate
Nicole Zembower, Technician
Di Wu, PhD Student
Nick Wheeler, Manager
University of Notre Dame
Jeanne Romero-Severson, Co-PI
Dan Borkowski, PhD Student
Arpita Konar, PhD Student
Andrea Noakes, PhD Student
Lauren Fiedler, Technician
Olivia Choudhary
Michigan Tech University
Oliver Gailing, Co-PI
Sandra Owusu, PhD Student
Sudhir Khodwekar, PhD Student
University Tennessee
Scott Schlarbaum, Co-PI
Ami Sharp, Research Associate
Jason Hogan, Research Associate
James Simons, Research Associate
Margaret Staton, Bioinformatics,
Jack Davitt, Research Associate
Nathan Henry, Research Associate
Thomas Lane, Research Associate
Bert Abbott
Steve DiFazio
Robert Mangold
Ron Sederoff
Doug Soltis
University of Missouri
Mark Coggeshall, Co-PI
Christopher Heim, MS student
Clemson University
Haiying Liang, Co-PI
Chris Saski, Director of CUGI
Tatyana Zhebentyayev, Research
Associate
Ketia Shumaker, Co-PI
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org