genotype to phenotype -...
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Genotype to PhenotypeGenomic Discovery for Applications in Tree Breeding and Forest Health Management
Develop and Apply ForestHealth Diagnostic Tools
Pinus albicaulis(Whitebark pine)
Pinuslambertiana(Sugar Pine)
Genecology of cold-hardiness in coastal Douglas-fir
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 5
A sampler of genecological literature pertaining to cold-hardiness:
1973
2006
ABc
aBC
aBC
ABc
aBC
aBc
Abc
Abc
Abc
abC
abC
Abc
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aBC
A
B
abC
c
aBc
Abc
Quantitative Trait Locus Mappin
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F1
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Parent 3 Parent 4
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HEIGHT
GENOTYPEBBBbbb
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Bb BbBbBB BB BBbb bb bb
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Parent 1 Parent 2
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Knott et al. (1997) TAG 84:810-820
Cold-hardiness QTLs in Douglas fir
Publication Phenotype No. QTLs
Jermstad et al. (2001, TAG 102:1142-1151)
Bud flush timing 33
Jermstad et al. (2001, TAG 102:1152-1158)
fall/spring cold-hardiness 11/15
Jermstad et al. (2003, Genetics 165:1489-1506)
many 4-11QTL x environment
Wheeler et al. (2005, Mol. Breed. 15:145-156)
spring cold-hardiness 10 (assoc. of 17 candidate genes to QTLs)
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 8
Linkage versus Association
several generations
X
X
XX
X
X
X
Natural population (= multiple genetic backgrounds)
Mapping pedigree
A favourable mutation
LG
SNP markers in linkage disequilibrium and can be used for family selection
A1
A2 B2
A1
A2 B2
B1 B1A Q1
T Q2
Q2
Q1
G
C A G
T C
QTL Genotype
Q1Q1
Q1Q2
Q2Q2
Phenotypic Value
Tree 1 - Discovery Tree 2 - Application
x x
Components of a Candidate Gene Based Association Study
Association Population - large (500-1000) clonal
Phenotyping - precisionCandidate Gene Selection - function,
expression, QTL co-locationSNP Discovery and GenotypingAnalyses
Two confounding factors to address
population structurecontrol of Type I error
Advantages of Candidate Gene Based
Association Studies in Conifers
Evolutionarily old, undomesticatedLarge random mating, unstructured populationsDirect determination of haplotype from
megagametophytePrecise evaluation of phenotypeRapid decay of linkage disequilibrium
Neale and Savolainen (2004)Trends in Plant Science 9:325-330
Linkage Disequilibrium in loblolly pine
0
0.2
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1
0 500 1000 1500 2000 2500 3000Distance between sites (bp)
r2
D = pA1B1-pA
1pB
1
r2 =pA1pA2
pB1pB2
D2
Linkage disequilibrium, LD, is the nonrandom association of alleles at linked loci.
Heritabilities for cold-hardiness traits
Trait Heritability (h2)
Bud flush 0.87
Bud set 0.70
Second flushing 0.45
Fall hardiness 0.19
Winter hardiness 0.11
Spring hardiness 0.77
Spring frost damage 0.56
Howe et al. 2003. Can. J. Bot. 81: 1247-1266.
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 14
Cold-hardiness in plants
– Stabilization of plasma membrane to physical damage (intercellular ice) and dehydration induced lesions.
– Stabilization of proteins, which can be denatured at low temperatures.
– Protection against reactive oxygen species (ROS) at low temperatures.
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 15
Genes with pleiotropic effects
• ABA-responsive genes are often 20-fold overexpressed in drought-tolerant phenotypes and 4-fold overexpressed in cold-hardiness phenotypes.
– cf. Gilmour and Thomashow (1991, Plant Mol. Biol. 17: 1233-1240) for a dissenting opinion for the direct role of ABA in cold-hardiness.
• Dehydrins - often associated with drought-tolerance (Eveno et al. 2008, Mol. Biol. Evol. 25:417-437), but also have been shown to be associated with cold-hardiness in angiosperms (Close 1997, Plant Physiol. 100: 291-296.)
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 16
Combined Population and Quantitative Genetics Approach
1. Choice of candidate genes2. Selection of diversity panel3. Resequencing and sequence analysis4. Population genetic analysis5. Selection of SNPs for genotyping6. Association analyses between cold-
hardiness phenotypes from St. Clair et al. (2005) and genotypes for 384 SNPs typed in ~700 trees
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 17
Gene categories represented by Douglas fir ESTs
cold acclimation
maximum cold hardiness
cold deacclimation
nucleic acid binding
lipid bindingcarbohydrate binding receptor activity
protein binding
3949 ESTs 3701 ESTs 3684 ESTs
Cold-induced proteins in Douglas fir EST libraries
• Candidate genes in Arabidopsis from Lee et al. (2005, Plant Cell17: 3155-3175)
• Total with tBLASTx scores < e-10
from Douglas fir EST libraries
• Automated and manual primer design
• Final selection
939
553
378
121
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 19
Resequencing strategy
• Diversity panel: 24 trees from Krutovsky and Neale (2005)
• 121 candidate genes (250 bp -1100 bp fragments)– Agencourt Biosciences: 71– Neale lab (UCD): 50
• Outgroup: Pseudotsuga macrocarpa• Sequence analysis: PineSAP (Wegrzyn et al. 2008,
Bioinformatics in press).
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 20
Resequencing summary
S θW θπ Dxy
All 933 0.00450 0.00435 0.00852
Silent 732 0.00777 0.00756 0.01296
Syn 254 0.00791 0.00760 0.01413
Nonsyn 201 0.00214 0.00200 0.00580
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 21
Patterns of linkage disequilibrium
Intragenic LD:
1. Extends upwards of 1 kb
2. Higher than previously reported
Intergenic LD:
1. Prevalent among genes on the same linkage group.
2. Limited to proximal genes.
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 22
Population structure and demography
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 23
Population structure is absent.
A population growth model may be appropriate.
Neutrality tests – How many genes?
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 24
Tajima’s D:
4 positive (2)
11 negative (3)
Fay and Wu’s H:
8 positive (2)
6 negative (2)
Candidate genes consistent with selective sweeps
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 25
Candidate genes consistent with balancing and diversifying selection
Locus Gene product Result
ES424016.1 glutathione S-transferase H > 0(r = 0.34)
Pm_CL1400Contig1 beta-D-xylosidase D > 0(r = 0.09)
CN639130.1 MADS-box transcription factor
H > 0, D > 0(r = 0.29)
CN640289.1 serine hydroxymethyltransfersae
D > 0(r = 0.11)
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 26
GIS derived maps of parent trees in the common-garden population
Elevation and seed zones Julian day for family mean bud flush(Clair et al., unpublished)
The Phenotypes
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 28
21 cold-hardiness phenotypes:
4 phenology
3 cold-tolerance
14 growth
79% PVE total
The Genotypes
Application SNPs nGT
(>0.25) CR CG
Linkage mapping 384 192 295 0.96 37
Association mapping 384 706 277 0.92 94
47 91 54
GT = 0.86
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 29
44 92 54
GT = 0.58
How Many Unique Associations?
The 265 significant SNP-trait associations due to:
55 unique SNPs
39 unique candidate genes
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 30
Associated Genes – A Sampler
Locus Gene product Effect
Pm_CL61Contig1 cyclophilin r2 = 0.021-0.092
atub α-tubulin r2 = 0.023-0.044
f3h2 flavenoid 3-hydroxylase r2 = 0.027
Pm_CL988Contig1 thioredoxin-like protein r2 = 0.019-0.027
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 31
Cumulative Marker Effects
0.00 0.10 0.20 0.30 0.40 0.50 0.60
BB2
BS1
BS2
budcold
ndlcold
stmcold
diam
emean
emstd
ht1
ht2
rtwt
shwt
totwt
PCA1
PCA2
PCA3
Phen
otyp
ic T
rait
Marker Effect
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 32
A gene with a large effect on many individual traits
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1 51 101 151 201 251 301 351
Position (bp)
Mar
ker
Effe
ct
3’ UTRexon
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 33
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 34
Marker Effects along Environmental Gradients
Patterns of Adaptive Molecular Genetic Diversity
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 35
Summary
Only SNPs associated with at least one phenotypic trait:
• Varied along environmental gradients.
• Appeared to be under diversifying selection.
• Exhibited multivariate patterns of spatial distribution that coincided with temperature-related environmental gradients.
Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 36
Acknowledgements
Oregon State UniversityGlenn HoweMarilyn CherryDana Howe
University of California at Davis
David Neale
Brandon Tearse
Jill Wegrzyn
Jennifer Lee
USDA Forest ServiceValerie Hipkins (PSW Station)Kathie Jermstad (PSW Station)Robert Saich (PSW Station)Jennifer DeWoody (PSW Station)Brad St. Clair (PNW Station)
Other AffiliationsKostya Krutovsky (Texas A&M University)Nick Wheeler (Molecular Tree Breeding
Services, WA)
ACKNOWLEDGEMENTS