NSF Plant Genome Program: Association Genetics and Natural Variation of Complex Traits in Pine

David Neale & Chuck Langley – UC DavisGary Peter, John Davis, Dudley Huber, Matias Kirst, George Casella – U FloridaBarry Goldfarb & Bailian Li – North Carolina State Carol Loopstra & Tom Byram – Texas A&M

ADEPT 2: Team MembersUC Davis

• David Neale – Project Leader – SNP discovery, genotyping, association testing, databases

• 2 PhD students• Bioinformatics• Chuck Langley-

Population geneticist– Association testing & population genetics

ADEPT 2: Team MembersUF

• George Casella– Association testing & new

methods for multilocus testing• John Davis

– Disease phenotyping & association testing

• Dudley Huber– Association testing in structured

populations• Matias Kirst

– Gene expression phenotyping & association testing

• Gary Peter– Wood property phenotyping &

association testing, UF PM

• 4 Graduate Students– Disease

• Tania Quesada – Gene expression– Wood properties

• Xiaobo Li, SFRC;– Association testing pipeline &

methods• Jessica Li, Statistics

ADEPT 2: Team MembersTAMU

• Carol Loopstra– Gene expression phenotyping

• Tom Byram– Forest genetics

• 2 PhD students

NCSU• Barry Goldfarb

– Verification population & wood property phenotyping

• Bailian Li– Forest genetics

• 1 PhD student– Patrick Cumbie

• Nick Wheeler– Outreach

Over Arching Goal & Approach• Goal

– Discover genes/alleles that control wood property, disease resistance, and gene expression traits in loblolly pine

• Approach– Association genetics is a population level survey used to

identify trait-marker relationships. – Association genetics takes advantage of the evolutionary

history of recombination within a species, and that all of the phenotypic and genotypic variation within a species is present in a single experiment.

Timeline

• 4 Year project 2005-2009• Official project start July 2005• Resequencing 2006-2007• SNP Genotyping 2008• Disease phenotyping 2006-2008• GE phenotyping 2006-2008• Wood phenotyping 2006-2008• Association testing 2008-2009

Databases

Phenotypic Data

Genetic Architectureof Traits

Identification of Genes thatControl Complex Traits

Genotypic Data

Allele Discovery

Candidate Alleles

Candidate Gene Selection

Allele Selection

Primer Design & Genomic Sequencing

Primer

Allele

Phenotype

SNP Genotyping

Information forGene & Allele

Selection

Genotype

Genetic Analyses

Gene ExpressionAnalyses

Association Tests

TraitPheno-typing

Sequence

Databases

UC Davis

UF & TAMU

UF & NCSU

UF

NCSU

UFUC Davis

Assoc. Pop.

Community Deliverables

• Primer database with 10,000 tested primers• Genomic sequences of 5000 genes• SNP database for 5000 genes• Genotypic data for CCLONES & NCSU for ~1000

SNPs/haplotypes• Phenotypic data for CCLONES & NCSU populations • Novel association testing methods• Candidates for additional functional studies

• Wood Properties

• Water Deficit

• Disease Resistance

Specific Deliverables • Objective 1: Bioinformatics – To identify 5,000 target candidate genes

for wood property and disease resistance traits through the integration of loblolly pine genomic resources and the development of primer databases for SNP discovery, SNP genotyping, and RT-PCR.

• Deliverables:

• List of candidate genes including gene annotation information.

• Online primer database (TreeGenes).

Specific Deliverables • Objective 2: Discover alleles for 5,000 candidate genes using a high-

throughput sequencing pipeline at Agencourt Bioscience Corporation.

• Deliverables:

• Resequencing data (6924 genes) including data deposition to public databases (NCBI GenBank and TreeGenes).

o Chromatogramso Alignmentso SNP calls

• Custom software for DNA sequence manipulation and summarization.

• Population genetic summaries of data by locus.

Specific Deliverables• Objective 3: To estimate the extent of linkage disequilibrium in genomic

regions around a small number (~10) of candidate genes and discover 5’ regulatory (promoter) SNPs.

• Deliverables:• Sequences of 10 BACs.• Custom software for BAC annotation in non-model organisms.• BAC-specific SNPs and linkage disequilibrium (LD) estimates across

large physical distances.

Specific Deliverables

• Objective 4: Association Genetics – To detect and verify associations between SNPs in ~1000 candidate genes and a suite of whole plant wood property phenotypes, disease resistance phenotypes and gene expression phenotypes.

• Deliverables:• Illumina 7600 SNP genotyping chips for loblolly pine (UCD).• Genotyping results for three association populations and two pedigrees (UCD).• Phenotypes across five categories:

o Gene expression (TAMU)o Metabolites (UCD)o Wood quality (UF)o Drought-tolerance (NCSU)o Disease resistance (UF)

• Genotype-phenotype associations.

Deliverables TimelineObjective 1:• 100% - List of candidate genes including gene annotation information.• 100% - Online primer database (TreeGenes).Objective 2:• 100% - Resequencing data acquisition, base calling, alignments and SNP

identification.• 67% - Data deposition to public repositories (dbSNP remains).• 100% - Development of custom software for DNA sequence manipulation and

summarization (3 publications: 2 in print or press; 1 in review).• 33% - Population genetic summaries by locus.

Deliverables TimelineObjective 3:• 100% - Acquisition of DNA sequences for 10 BACs.• 33% - Assembly and annotation of 10 BACs. • 33% - Development of custom software for BAC annotation in non-model organisms.• 0% - BAC-specific SNPs and linkage disequilibrium (LD) estimates across large physical

distances.Objective 4:• 100% - Design of loblolly pine Illumina genotyping chip (~7600 SNPs).• 100% - Genotyping results for three association and two linkage mapping populations.• 90% - Phenotypes across five categories.• 20% - Genotype-phenotype associations.

http://dendrome.ucdavis.edu

http://dendrome.ucdavis.edu/treegenes

http://dendrome.ucdavis.edu/interface

http://dendrome.ucdavis.edu/TGPlone

ADEPT2 BioinformaticsProject Resources

• PineSAP – High throughput Pine Alignment and SNP calling Pipeline– Alignments, SNP calls, Genotype file generation, and Data Submissions

• DNASam – High throughput sequence analysis tool• ADEPT2 Project Website

– Portal to research summaries, software packages, and collaborative tools

• Dendrome Plone – Content Management System– Full content management for analysis and collaborations– Full management of public database accounts and user permissions

• TreeGenes Database – 10 modules to store genotypic and phenotypic data

• DiversiTree – extensive workspace interface to enable users to query information from TreeGenes

241,796 reads submitted to Trace Archive

Trace Archive Accessions:2072163883-2072419182

6,178 PopSets submitted via Sequin to NCBIAccessions: FJ043059-FJ147084

Dendrome Plone – ADEPT2

Dendrome

Dendrome Website and Related Web Resources Encompasses 6,754 Pages

Detailed Usage Statistics Available @ http://dendrome.ucdavis.edu/awstats/awstats.pl

DiversiTree Interface

Select data type to search

Retrieve search results

Display one or more entries from search

Display linked data

Create lists

Site Statisticswww.forestrycareers.org

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Site Hits

• Forestrycareers.org is a recruitment tool for the forestry and natural resource disciplines

• It has been marketed through NAUFRP (National Ass. Univ. with Forestry and Nat. Res. Programs

• Number of visitors and site hits has increased continuously since it was rolled out in June, 08

“I just wanted to let you know I use this web site all the time!  It really is a great resource, and I am so thankful

that it was created!”  Student and Careers Services Center, University of Georgia.

“Please let those who were associated with putting the forestry and natural resources web site together that they did a wonderful job.  The Cal Poly NRM Dept. will link to it

from our NRM web site.  Please pass along our appreciation for a job well done!!!”

“This is a great addition to our recruitment efforts for the college”

Virginia Tech

Selected Feedback on Recruitment Website

Collaborators Institution Species/Project

Shirley Luckhart UCDavis Mosquito

Nicholas Wheeler NCSU Fagaceae

Patricia Faivre Rampant INRA-URGV Populus nigra

Janice Cooke University of Alberta Mountain Pine Beetle

Rich Shuren & Brian Stanton Greenwood Resources Populus nigra

Shannon Dillon CSIRO Pinus radiata

Outi Savolainen & Sonja Kujala University of Oulu Pinus sylvestris

Santiago Gonzalez Martinez ICIFOR - INIA Pinus pinaster & Populus alba

Delphine Grivet ICIFOR - INIA Pinus pinaster

Kathleen Jermstad IFG Pinus lambertiana & Pseudotsuga menziesii

Glenn Howe OSU Pseudotsuga menziesii

Marta Scalfi IASMA Picea abies

Dudley Huber UF Pinus elliottii

Kermit Ritland UBC Picea glauca

Martin Lascoux Uppsala University Picea abies

Cristophe Plomion INIA Pinus pinaster

Rich Cronn USFS-PNW North American white pines

Aaron Liston OSU North American white pines

John Syring Linfield College North American white pines