Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
The Hardwood Genomics Project: Outcomes and Future Directions after Four Years of
Resource DevelopmentMeg Staton
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
The TeamPenn 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 AssociateJason Hogan, Research AssociateJames 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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Transcriptome Libraries
Stress treatments on full or half sib seedlings• Ozone treatments
– all species except HL– 4 levels of ozone across 4
time points
• Wounding– Leaf, twig– BW,GA,BG, TP, SM, SG
• Heat, drought, cold– 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
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
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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC Libraries
Northern Red Oak Black Walnut
QRNRBa QRNRBb JNC_Ba JNC_Bb
RE HindIII BstYI HindIII BstYI
# Clones 27,648 46,080 27,648 27,648
Average insert size 110 kb 120kb 130kb 120kb
Coverage4.0X 7.3X 4.5X 4.0X
(760Mb genome) (760Mb genome) (800Mb genome) (800Mb genome)
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC SequencingFunctional 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 96
Finished BACs(Single Contig)
Total Finished Bases 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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Full Sibling Mapping PopulationsHost 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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
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
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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
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/CartoChene/index.php?page=pheno_caracter
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Leafing Dates and Bud Burst
Stage II/Budburst
Dormant Stage I
Stage IVStage III Stage V
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
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
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Deliverables
1. Develop Deep EST Sequence Resources
2. BAC Libraries and BAC Sequencing
3. Mapping Populations
4. Genetic Map Construction
5. Phenotyping and QTL Map Construction
6. Website
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://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
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
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 AssociateJason Hogan, Research AssociateJames 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
Chris Saski, Director of CUGI
Tatyana Zhebentyayev, Research Associate
Ketia Shumaker, Co-PI
Bert AbbottSteve DiFazioRobert MangoldRon SederoffDoug Soltis