Genotype to Phenotype:Understanding the Association between Natural Genetic Variation and Complex Traits in Forest Trees

Collect seed from many trees

Grow families in a common environment

Measure many adaptive traits

Traits vs source

environment

Douglas-Fir of Western OR and WA

December Minimum Temperature

-10 -8 -6 -4 -2 0 2 4 6

Com

bina

tion

of V

aria

bles

, Prim

arily

Gro

wth

-5

-4

-3

-2

-1

0

1

2

3

St. Clair, J.B., Mandel, N.L., and Vance-Borland, K.W. 2005. Genecology of Douglas-fir in western Oregon and Washington. Anal. Bot. 96:1199-1214.

GIS

Douglas-fir common garden study

Fall cold damage

r = 0.79Qst = 0.68

Related to winter temperature, frost dates, latitude, and to a lesser extent elevation and summer aridity.

Genomic Approaches to Complex Trait Dissection• Quantitative Trait Locus (QTL) Mapping • Association Mapping

Pinus taeda (loblolly pine) Pseudotsuga menziesii (Douglas-fir)

ABc

aBC

aBC

ABc

aBC

aBc

Abc

Abc

Abc

abC

abC

Abc

Abc

aBC

A

B

c

abC

aBc

Abc

Quantitative Trait Locus Mapping

ABC

abc

F1

ABC

abc

F1

X

abc

abc

ABC

ABC

Parent 3 Parent 4

X

HEI

GH

T

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

ewsglwsg

vol%emfa

lmfaecwc

lcwc

DETECTIO

NVERIFI

CATION

RELATED

UNRELATED

Aco_10.0

PtIFG_3012_4312.715.0

PtIFG_2150_A19.619.9 PtIFG_2885_B20.1

estPtIFG_8569_a29.5PtIFG_2538_B30.2

PtIFG_2564_A40.3PtIFG_1A7_A42.6estPtIFG_9022_a43.1PtIFG_2536_146.5PtIFG_1A7_D46.8

estPtIFG_48_a58.3estPaINRA_PAXY13_a59.5estPtIFG_464_a62.2

PtIFG_1633_a66.0

PtIFG_48_178.4estPtIFG_8939_aPtIFG_3006_183.4PtIFG_1918_h83.8

86.186.3

PtIFG_1623_A90.9estPtIFG_66_a92.8

94.6PtIFG_1626_a95.4

PtIFG_2986_A102.7PtIFG_1D11_A104.0

PtIFG_1165_a121.1

6Pgd_11140.7

estPpaINRA_AS01C10-1_a154.6

LG 2

PtIFG_2006_C0.0estPtIFG_1934_a0.3PtIFG_2145_13.4

PtIFG_2068_A7.8PtIFG_2897_d10.4PtIFG_975_312.2

estPtIFG_8500_a18.8

PtIFG_138_B24.1

estPtNCS_22C5_a30.1PtIFG_2588_132.5estPtNCS_C612F_a33.8

PtIFG_2718_344.8

PtIFG_2745_154.2PtIFG_1918_357.4

59.5

estPtIFG_8612_a64.2PtIFG_2090_267.6

69.4PtIFG_1636_370.1

78.2

PtIFG_2988_2183.6

PtIFG_2718_186.8

estPtIFG_2889_a95.7

PtIFG_2889_2198.9

estPtIFG_8781_a104.1

PtIFG_2145_76107.4PtIFG_2145_5109.0

113.4 PtIFG_1D9_2113.6116.2

LG 3

C4H-1

Pta14A9

SAMS-1

DETECTIO

N

VERIFICATIO

NRELATEDUNRELATED

DETECTIO

NVERIFI

CATION

RELATED

UNRELATED

PtIFG_2819_12PtIFG_653_dPtIFG_2086_13PtIFG_1626_c

PtIFG_2697_A

PtIFG_2006_A

estPtINCS_20G2_aestPtIFG_9053_aestPtIFG_8843_aPtUME_Ps3_A

estPtIFG_8537_a

estPtIFG_2253_aestPpINR_AS01G01_aestPtIFG_1576_aPtIFG_2253_A

PtIFG_2782_31

PtIFG_1457_b

estPtIFG_9198_aestPtIFG_8496_a

PtIFG_2146_31

PtIFG_2441_1estPtIFG_107_aPtIFG_2931_bestPtNCS_6N3E_aPtIFG_2393_1PtIFG_2931_A

PtIFG_851_1

LG 1

LAC

GlyHMT

PtNCS_CAD-08_b

SCALE

0 cM

10 cM

Brown et al. 2003 Genetics164:1537-46

Alpha tubulin-10.0

Pm1011_a3.7Pm1147_a4.2Pm1011_b9.1

Pm1052_j29.4

Pt2356_d45.1

EF-160.0Pm1486_a62.0Pm1383_a68.5CABBP_170.0Pm1174_a77.0

40S_RPS2103.0DER1-like106.0Pm1592_a106.1CABBP_2110.0UGT117.0Pt2006_b118.1Pm1496_a121.5ACRE146131.0TBE138.0Pt2291_g146.3

LG1

Pm1504_b0.0

Pm1052_c16.6

Pm1611_b26.7Pm1301_a33.3

Pm0343_a66.4F3H76.0Pm0123_a78.6MAD80.0

Pm1052_a94.1

Pm1090_a147.2

LG2

Pt2957_a7.0

LEA-II20.0

Pm1480_a_MMIP38.0Pt2553_a43.2PRS47.0MT-like48.0

ANT70.0SAHH75.0Pm1486_e79.6Formin-like88.0

LG4

Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2

QTL mapping and positional candidate genes

Spring cold hardiness (buds)

Fall cold hardiness (needles)

Fall cold hardiness (stem)

Fall cold hardiness (buds)

Bud flush

Spring cold hardiness (stem)

Spring cold hardiness (needles)EF-1 (translation elongation factor-1) cold-inducedCABBP1 (chlorophyll a/b-binding protein type 1) downregulated under the water deficitDER1-like (degradation of misfolded proteins) possibly cold-inducedCABBP2 (chlorophyll a/b-binding protein type 2) downregulated under the water deficitF3H (flavanone-3-hydroxylase) upregulated under the water deficitLEA-II (late embryogenesis abundant type II) dehydrin-like protein cold-inducedMT-like (metallothionein-like protein) stress-induced; downregulated under the water deficitSAHH (S-adenosyl-L-homocysteinas hydrolase) upregulated under the water deficit

Jermstad et al. 2001a,b, 2003Wheeler et al. 2004

Association Mapping

recombination throughevolutionary history

present-daychromosomesin natural population

*TG

*TA

CG

CA*TG

CA

ancestral chromosomes

*TG

Advantages of Candidate Gene BasedAssociation 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

Gymnosperms

Pinus

Angiosperms

Populus

Arabidopsis

Eudicots

MonocotsOryza

Ferns, mosses

Non-seed plants

330 – 225 Mybp

450 – 300 Mybp

300 – 220 Mybp

250 – 120 Mybp

The upper bounds (i.e., the older numbers) are based on DNA sequence data; the lower bounds are based on the fossil record. [Not drawn to scale.]

Phylogenetic Relationship of Vascular Plants

Reference:

Raven et al. 1992. Biology of Plants. Worth Pub.Martin et al. 1993. Molecular phylogenies in angiosperm evolution. Mol. Biol. Evol. 10:140-162. Eckenwalder. 1996. Systematics and evolution of Populus. In: Biology of Populus. Nat. Res. Counc. Canada. p. 7-32.

Large and random mating populations

FBRC - Association Mapping Population

Used for SNP discovery

– 32 parents• 64 families

– ~1400 clones

22 11 441212

1313

0 100 200 300 400 500 600 700 800 900 1000 1100

-41 622 725 813 1079

F1 R1A F2 R2-12 548 575 990

39

58

76

84

108

124

126

136

147

172

207

441

456

474

510

644

647

651

667

696

703

731

756

792

828

835

842

847

861

870

878

890

917

926

949

963

HAPLOTYPE

1 G T G T G C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C1 G T G T G C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C1 G T G T G C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C1 G T G T G C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C1 G T G T G C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C1 G T G T G C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C

2 G T G T C C A G G A A A G C C T T G T T T A A C G A T C C A C G G C G C2 G T G T C C A G G A A A G C C T T G T T T A A C G A T C C A C G G C G C2 G T G T C C A G G A A A G C C T T G T T T A A C G A T C C A C G G C G C

3 G T G T C C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C3 G T G T C C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C3 G T G T C C A G G A G G G C C T T G T T T A A A G A T C C A C G G C G C

4 G T G T G C A G G G G G G C C T T G T T T A A A G A T C C A C G G C G C

5 G T G T G C A G G A G A T C C C G G T G T A A C G A - C T T T G G C G C5 G T G T G C A G G A G A T C C C G G T G T A A C G A - C T T T G G C G C5 G T G T G C A G G A G A T C C C G G T G T A A C G A - C T T T G G C G C

6 C T G T G C A G G A G A T C C C G G T G T A A C G A - C T T T G G C G C6 C T G T G C A G G A G A T C C C G G T G T A A C G A - C T T T G G C G C

7 C T G T G C A G G A G A T C C C G G T G T A G C G A - C T T T G G C G C8 C T C T G C A G G A G A T C C C G G T G T A A C G A - C T A T G G C G C

9 G A G T G C A G G A G G G C T T T C T T G A G C G G T T T A C T G A G T9 G A G T G C A G G A G G G C T T T C T T G A G C G G T T T A C T G A G T9 G A G T G C A G G A G G G C T T T C T T G A G C G G T T T A C T G A G T

10 G A G T G C A G G A G G G C T T T C T T G T G C G G T T T A C T G A G T11 G A G T G C A G G A G A G C T T T C T T G A G C G G T T T A C T G A G T12 G A G T G C A G G A G G G C T T T C T T G A A C G G T T T A C T G A G T

13 G A G T G C A G G A G G G T T T T C C T G A G C G G T T T A C T G A G T13 G A G T G C A G G A G G G T T T T C C T G A G C G G T T T A C T G A G T

14 G A G G A C A G G A G G G C C T T G T T T A A C G A T C C A C G G T G C

15 G A G T G C G A G A G G G C C T T G T T T A G C T G T T T A C G C C C T16 G A G T G G A G A A G G G C C T T G T T T A G C T G T T T A C G C C C T

Direct determination of haplotypes - AGP6

1n2n

0.000

0.002

0.004

0.006

0.008

0.010

0.012

human

soyb

ean

C. jap

onica

loblol

ly pin

eDou

glas-f

irDro

soph

ila

maize

θT

106 loci

56

21

2412

7142

Nucleotide Diversity Among Species

The decay of LD 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-pA1

pB1

r2 = pA1pA2

pB1pB2

D2

Linkage disequilibrium, LD, is the nonrandom association of alleles at linked loci.

Illumina- BeadStation500G-BeadLab Platform

~150,000 data points per week at UCD Genome Center

•• Wood PropertiesWood Properties

•• Water DeficitWater Deficit

•• Disease ResistanceDisease Resistance

Wood Property Traits in Pinewood specific gravity

microfibril angle

S1

S2

S3

secondarywall

primarywall

cell wall chemistry

lignin

hemicellulose

cellulose

early late

0 500 1000 1500 2000 2500 3000 3500 bp

I II III IV V VI

coding sequence

untranslated sequence

cad-n1 mutation

Cad null sequence mutation : Gene position and protein alteration

codon240 245 250

WT G A A - - G C A G C A G A G A G C C T A G A T T A C A T A A T G G A C ……E A A E S L D Y I M D

cad-n1 G A A A A G C A G C A G A G A G C C T A G A T T A C A T A A T G G A C ……E K Q Q R A *

Gill et al. 2003 Plant Biotech. J. 1:253-506

Genetic association between SNPs and wood propiertytraits in loblolly pine

Marker effect Trait

Wood-age

type Gene SNP N

F P R2

FDR

Q-value

ewsg transition sams-2 M44 403 6.7595 0.0013 0.0327 0.0630

all age cad M28 409 7.7480 0.0005 0.0347 0.0228

PCA cad M28 366 6.5945 0.0015 0.0351 0.0742

lw transition lp3-1 Q5 431 7.9007 0.0004 0.0357 0.0248

ewmfa transition α-tubulin M10 374 8.3766 0.0040 0.0221 0.0062

PCA α-tubulin M10 370 13.508 0.0003 0.0355 0.0078

Gonzalez-Martinez et al., Genetics, in press

Water Use Efficiency

Stable carbon isotope discriminationin foliage, in two sites (Cuthbert & Palatka).

Strong family structure (partial diallel), including 15-24 offspring from 61 families.

-1-0.8-0.6-0.4-0.2

00.20.40.60.8

1

carb

on is

otop

e

Cuthbert Palatka

-0.8-0.6-0.4-0.2

00.20.40.60.8

Genotype by family for DHN1-S2

Trai

tSNP x Phenotype Associations for WUE in Loblolly Pine

Dehydrin and SOD Showed Associations at both Sites

Orthogonal modelp -value n

Cuthbert

DNH1 S2 0.003 487

SODchl S9 0.087 349

WRKY-like S9 0.035 228

Palatka

CCoAOMT S10 0.056 763

EIN2 S1 0.062 352

LP5 S28 0.015 371

SODchl S9 0.049 421

Disease Resistance CandidatesGENE Functional

ClassificationNUMBER O

ALLELESFRAGMENT

SIZESNP

FREQUENCPOLYMORPI

SITESSINGLETO

SITES

THETA Nucleotide Diversity

TAJIMA'SD SIGNIFICANC

PCNA Diff. Exp. 29 537 1:60 9 3 0.00647 -1.31419 NSPR4 Diff. Exp. 32 520 1:58 9 3 0.0043 -1.58467 NSEIN2 Diff. Exp. 26 504 1:252 2 1 0.00104 -0.3186 NSLDOX-A Diff. Exp. 31 501 1:34 15 4 0.00769 -0.6668 NSCESA3 Diff. Exp. 32 606 1:202 3 2 0.00134 -1.22285 NSNAC1 Diff. Exp. 31 737 1:39 19 4 0.00634 0.05368 NSCYP450 Diff. Exp. 32 520 1:52 10 7 0.0054 -2.04767 S*TERPS Diff. Exp. 31 528 1:48 11 6 0.00668 -0.67478 NSpAEOMT Reg.Seq-promoter 32 985 1:70 14 7 0.00357 0.23064 NSpCHI Reg.Seq-promoter 27 598 1:15 39 9 0.01797 -0.85635 NSpPCBER Reg.Seq-promoter 32 556 1:37 15 9 0.0083 -1.41894 NSEREBP Trans.Factor 32 809 1:48 17 6 0.00746 0.56236 NSERF1A Trans.Factor 29 321 1:54 6 2 0.00476 0.43475 NSERF1B Trans.Factor 32 628 1:48 13 2 0.00522 0.82406 NSGATABP2 Trans.Factor 32 913 1:65 14 4 0.00385 -0.65376 NSMYB2 Trans.Factor 31 605 1:121 5 3 0.00208 -1.62694 NSMYB3 Trans.Factor 29 423 1:38 11 10 0.00662 -2.29003 S*BHLH62 Trans.Factor 32 411 1:51 8 6 0.00494 -1.77195 NSAXR Trans.Factor 32 433 1:433 1 - 0.00058 -0.77374 NSSCARECROW Trans.Factor 32 530 1:41 13 6 0.00609 -0.61075 NSSETB Trans.Factor 32 577 1:115 5 3 0.00271 -1.01029 NSSETC Trans.Factor 32 479 1:21 23 14 0.01202 -1.82611 S*WRKY1 Trans.Factor 32 357 1:44 8 2 0.00568 -0.04948 NSWRKY2A Trans.Factor 29 443 1:22 20 10 0.0115 -0.62025 NSWRKY2B Trans.Factor 32 379 1:22 18 9 0.01179 -0.65195 NS4CL Phen.prop PW 32 437 1:50 11 1 0.0561 1.47425 NSC3H Phen.prop PW 32 1254 1:60 21 15 0.0043 -1.9588 S*CCOAOEMT Phen.prop PW 31 551 1:40 14 2 0.00668 2.0229 S*COMT2 Phen.prop PW 31 1206 1:80 15 6 0.00358 0.50414 NSCOMT4 Phen.prop PW 32 444 1:111 4 2 0.00254 -1.59159 NSPAL1 Phen.prop PW 31 415 1:70 6 2 0.00363 -0.88514 NS

18207 1:48 379 160 0.00700697

Distribution of SNP alleles within CASE and CONTROL groups for 4 loci that increase Relative Risk of the Fusiform Rust Disease

estimated from 10GS phenotypes

10GS : 10-GALL SCORE

Significant Associations for BPC-LL(Broad Pitch Canker Lesion Length) According to the Model Test Results

Summary…..

• Candidate gene association genetics approach works nicely in forest trees

• Individual genes can be associated with complex traits

• Desirable alleles can be discovered for breeding and conservation

However…..

• Associations must be validated

• Size of effects of individual genes are small %PVE < 0.05

• Number of candidate genes in association screens must be increased

Re-sequencing 10K Genes

Information on Individuals

DNA

extractions

Sequences

Root

Megagametophyte

Xylem from 8 different genotypes

Phenylalanine

0

100

200

300

400

500

600

700

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

7C547B272B265A449A536A7C_avg547B_avg272B_avg265A_avg449A_avg536A_avg

Metabolomic Profiling

Whole Plant (wood, WUE, disease) and MolecularPhenotyping in Association Population of 500Unrelated Loblolly Pine Clones

Comparative Resequencing in the Pinaceae

Pine host HR Segregation

Whitebark A no Mexican white A no Foxtail A no Great Basin white A no Limber P no Sugar P yes Western white P yes Southwestern white P yes

Hypersensitive Response (HR)

Two forms of resistance observed:Monogenic (Major Gene Resistance)Polygenic (Partial Resistance)

Everything else…blight reactionbark reactionontogenic resistanceYFR (your favorite reaction)

Kinloch and Dupper 2002. Phytopathology 92:273-280

Fine Resolution Mapping of Cr1 SP_5701

OP_AD09_920OP_K01_1110BC_422_1450OP_AG05_610OP_D19_1120OP_AN10_590OP_AI03_650OP_E16_800OP_E12_1500OP_E12_1700OP_G16_950Cr1(MGR)OP_F03_810BC_432_1110

OP_T15_650

BC_090_725

BC_315_325

2.50.74.61.33.81.31.11.20.40.8

13.4

10.2

20.7

OP_E12_1700

OP_G16_950

Cr1 (MGR)

OP_F03_810

BC_432_1110

1.1

1.2

0.4

0.8

Converted RAPD markers to SCARmarkers (Sequence Characterized Amplified Region)

Use to screen BAC* library in loblolly pine. Sequence and identify all genes in region.

√

* A BAC library consists of large chunks of the genome

F1 x F1(intermediate)

Number and location of loci contributing to ‘slow-rust’ resistance

Size of effect of individual loci

Full genome map for positioning RGCs

1000 progeny at Happy Camp (~5 yrs old)

(intermediate)

Phenotype Genotype with EST markers from other conifers

Benefits:

QTL mapping of ‘slow-rust’ resistance

Genotype to Phenotype:Understanding the Association between Natural Genetic Variation and Complex Traits in Forest Trees