Genotype to Phenotype - CAForestPestCouncilcaforestpestcouncil.org/wp-content/uploads/2009/05/david-neale.pdf · Acknowledgements Oregon State University Glenn Howe Marilyn Cherry

Genotype to PhenotypeGenomic Discovery for Applications in Tree Breeding and Forest Health Management

Phenotype = Genotype + Environment

Adaptation

Breeding

Conservation

Bristlecone Pine

Develop and Apply ForestHealth Diagnostic Tools

Pinus albicaulis(Whitebark pine)

Pinuslambertiana(Sugar Pine)

Douglas fir (Pseudotsuga menziesii)

Eckert et al. Cold-hardiness in Douglas fir 27-Aug-2008 4

Genecology of cold-hardiness in coastal Douglas-fir


A sampler of genecological literature pertaining to cold-hardiness:

1973

2006

Traits that are Controlled by Single Genes

ABc

aBC

aBC

ABc

aBC

aBc

Abc

Abc

Abc

abC

abC

Abc

Abc

aBC

A

B

abC

c

aBc

Abc

Quantitative Trait Locus Mappin

ABC

abc

F1

ABC

abc

F1

X

abc

abc

ABC

ABC

Parent 3 Parent 4

X

HEIGHT

GENOTYPEBBBbbb

♦♦

♦♦♦

♦♦♦

♦

Bb

Bb BbBbBB BB BBbb bb bb

abc

abc

ABC

ABC

Parent 1 Parent 2

X

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)


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

0.4

0.6

0.8

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.


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.


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.)


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


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


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).


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


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.


Population structure and demography


Population structure is absent.

A population growth model may be appropriate.

Neutrality tests – How many genes?


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


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)


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


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


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


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


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


A gene with a large effect on many individual traits

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

1 51 101 151 201 251 301 351

Position (bp)

Mar

ker

Effe

ct

3’ UTRexon



Marker Effects along Environmental Gradients

Patterns of Adaptive Molecular Genetic Diversity


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.


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

Documents

Genotype to Phenotype - CAForestPestCouncilcaforestpestcouncil.org/wp-content/uploads/2009/05/david-neale.pdf · Acknowledgements Oregon State University Glenn Howe Marilyn Cherry