The Hardwood Genomics Project: Outcomes and Future Directions after Four Years of Resource...

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 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







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







Tulip Poplar

Sweetgum

Honeylocust

Northern Red Oak

Black Walnut

Sugar Maple

Blackgum

Green Ash







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







Deliverables






6. Website







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







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







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







Deliverables






6. Website







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)







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







Deliverables






6. Website







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







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







Deliverables






6. Website







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







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







Deliverables






6. Website







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







Leafing Dates and Bud Burst

Stage II/Budburst

Dormant Stage I

Stage IVStage III Stage V







Software Development

• Sylvan Photomeasure software• Collaborating with Dr. David Larsen







Deliverables






6. Website







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







Future Directions

Resources Hypothesis-Driven Science







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

Date post:	18-Jul-2015
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