Genotype to Phenotype - Middle East Technical...
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Genotype to Phenotype Genomic Based Tools to Manage Forest Tree Populations in Response to Climate Change Genomic Based Tools to Manage Forest Tree Populations in Response to Climate Change
Transcript of Genotype to Phenotype - Middle East Technical...
Genotype to PhenotypeGenomic Based Tools to Manage Forest Tree Populations in Response to Climate ChangeGenomic Based Tools to Manage Forest Tree Populations in Response to Climate Change
“I like trees because they seem more resigned to the waythey have to live than other things do”
Willa Cather 1913~ Willa Cather 1913
Potentilla glandulosa from three different elevations planted at three different elevationsat three different elevations
(from Clausen, Keck and Hiesey 1940)
e 0 m
Timbe
rlin
El.
3,03
0
to 0 m
T E
Nat
ive
Mat
her
El.
1,40
N
ford
5 m
dead
TimberlineStanford Mather
Stan
fEl
. 35
dead
Grown atEl. 3,030 mEl. 35 m El. 1,400 m
Clinal Patterns of Adaptation in Balsam Fir
Estimated allele frequencies for eight allozyme loci in four subpopulations of balsam fir on Mt. Moosilauke, New
Hampshire
0,500
1,000
e Fr
eque
ncie
s 6-PgdPgmlGot1Got2Got3G i2
0,000
0,500
1464 1312 1159 854
Alle
le Gpi2Lap1Lap2
Relationship of temperature optimum for net photosynthetic CO2 uptake to elevational origin of
balsam fir seedlings. Points represent 1464 1312 1159 854
Elevation (Meters)
Neale DB, Adams WT (1985) Allozyme and mating-system variation in balsam fir across a continous
balsam fir seedlings. Points represent determinations on individual pots.
um (oC)
2 up
take
yelevational transect. Canadian Journal of Botany 63, 2448-2453.
ture optim
uynthetic CO2
Fryer JH, Ledig FT (1972) Microevolution of photosynthetic temperature optimum in
n (ft)
Tempe
rat
for ph
otosy
photosynthetic temperature optimum in relation to elevational complex. Canadian Journal of Botany 50, 1231-1235.
Traits that are Controlled by Single Genes
Genomic Approaches to Complex Trait Dissection
• Quantitative Trait Locus (QTL) Mapping • Association MappingAssociation Mapping
Pinus taeda Pseudotsuga menziesii Populus trichocarpa(loblolly pine) (Douglas-fir) (black cottonwood)
Quantitative Trait Locus Mappingabc
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Genecology of Phenology and Cold-hardinessin Coastal Douglas firin Coastal Douglas-fir
Trait h2Trait 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
H t l 2003 C J B t 81Howe et al. 2003. Can. J. Bot. 81: 1247-1266.
Phenology and Cold-hardiness QTLs in Douglas firgy g
Publication Phenotype No. QTLs
J t d t l (2001 B d fl h ti i 33Jermstad 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. spring cold-hardiness 10 ( ,Breed. 15:145-156)
p g(assoc. of 17 candidate genes to QTLs)
Linkage versus Association
XX
A favourable mutation
several generations
XX
XX
X
X
N l l i (M i di Natural population (= multiple genetic backgrounds)
Mapping pedigreeLG
Association Genetics in Conifers
0 100 200 300 400 500 600 700 800 900 1000 1100
-41 622 725 813 1079
Association Genetics in Conifers
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 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
1n2n
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
1
0.4
0.6
0.8
r2
2
Large and Random Mating Population
0
0.2
0 500 1000 1500 2000 2500 3000
Distance between sites (bp)
Cold induced Proteins in Douglas fir EST LibrariesCold-induced Proteins in Douglas-fir EST Libraries
• Candidate genes in Arabidopsis from Lee et al. (2005, Plant Cell 17: 3155-
939( ,3175)
• Total with tBLASTx scores 553• Total with tBLASTx scores < e-10 from Douglas fir EST libraries
553
• Automated and manual primer design for Sanger
i
378resequencing
• Final selection121
Eckert et al. 2009. Genetics 183: 289-298
Patterns of Linkage Disequilibrium
Intragenic LD:
1. Extends upwards of 1 kb
2. Higher than previously reported in other conifers
Intergenic LD:g
1. Prevalent among genes on the same linkage group.same linkage group.
2. Limited to proximal genes.
Eckert et al. 2009. Genetics 183: 289-298
Candidate Genes Consistent with Selective SweepsCandidate Genes Consistent with Selective Sweeps
Gene Product ResultCompound DHEW test
GRAM-containing/ABA-responsive protein PD = 0 001 PH < 0 001 PEW = 0 080GRAM containing/ABA responsive protein PD 0.001, PH < 0.001, PEW 0.080cold-regulated plasma membrane protein PD = 0.009, PH = 0.050, PEW = 0.035
dehydrin-like protein PD = 0.072, PH = 0.083, PEW = 0.042luminal binding protein PD = 0.034, PH = 0.148, PEW = 0.076
Polymorphism-to-divergencecyclosporin A-binding protein k = 0.32
GRAM-containing/ABA-responsive protein k = 0.58transcription regulation protein k = 0 41transcription regulation protein k = 0.41
Ka/Ks
thaumatin-like protein Ka/Ks = 14.45auxin-responsive family protein Ka/Ks = 5.97p y p
bicoid-interacting 3 domain containing protein Ka/Ks = 4.94pentatricopeptide (PPR) containing protein Ka/Ks = 4.27
Eckert et al 2009 Genetics 183: 289-298Eckert et al. 2009. Genetics 183: 289 298
SNP GenotypingSNP Genotyping
GTApplication SNPs n
GT(>0.25) CR CG
Linkage mapping 384 192 295 0.96 37
Association mapping 384 706 277 0.92 94
GT = 0.86
47 91 54
GT = 0.58GT 0.58
44 92 54
Association Genetics
• 30 associations
12 did t
• SNP genotypes track environmental gradients in the same way as the phenotypes to which they are associated.
7 ith t ll l f diff ti ti• 12 candidate genes
• Mostly additive effects
• 7 genes with strong allele frequency differentiation across the Cascade Crest• Direct relation to phenotypes is confounded with structure
Eckert et al. 2009. Genetics 182: 1289-1302
Patterns of Adaptive Molecular Genetic DiversityPatterns of Adaptive Molecular Genetic Diversity
Neutral Genotype Phenotype Non-neutral and associated with phenotype
Resequencing and SNP di (7 424 )discovery (7,424 genes)
High-throughput computational solutions developed for bioinformatic SNP determination and sequence analysis
Phenotyping:W d Q litWood QualityDisease ResistanceDrought-ToleranceGene ExpressionMetabolites
Genotype 1-2 SNPs per Candidate Gene on a 7600 Illumina Infinium chip
Sequencing, Assembly, & Analysis of 10 BAC clones
ADEPT2 SNP discovery
Materials and Methods• Range-wide diversity panel of 18 megagametophytes + 5 primer validation samples
i i i
• Sanger sequencing (Agencourt Biosciences)
• Sequence Analysis and SNP calling (PineSAP, cf. Wegrzyn et al. 2009, Bioinformatics)
Total number of EST ContigsF UGA d UMN l t i
40,000Total number of unique
EST ti
20,500Filter out contigs similar by
blast analysis to other sequences in this dataset
High throughput primer design pipeline
at Agencourt Biosciences
Bioinformatic assessment
From UGA and UMN clustering EST contigs
Number of EST contigs for whichPrimers were successfully designed
14,000
Number of validated
7,900Primer Validationby test squencing
and BLAST to EST Number of Candidate genes represented
7,850 ResequencingIn diversity panel
primer pairs contig databaseIn SNP discovery panel resequencing
Sequence analysis assessmentDescription Count OutgroupDescription Count OutgroupCore Set 4861 ----------Outgroup-1 2975 Radiata pine (P. radiata)g ( )Outgroup-2 719 Sugar pine (P. lambertiana)
Analysis tools Database and web tools
Pi SAP D SAM Di iTPineSAP DnaSAM DiversiTree
http://dendrome.ucdavis.edu/interface/
E k t t l 2010 M l E l RW t l 2009 Bi i f ti
Li kLoblolly pine BAC assemblies:10 BACs
Data sets
Eckert et al. 2010. Mol. Ecol. Res.Wegrzyn et al. 2009. Bioinformatics
Linkage maps:
(Pinus taeda)
10 BACsLengths: ~60000-150000 bpTotal Length: ~950000 bp
~1800 gene loci mapped
~1900 cM total map distance
~0.80 loci/cM (range 1-10+)
Loblolly pine diversity and divergencey p y g
Population structure in loblolly pine
Using genotypes at 3900 SNPs and 23 nuclear microsatellites for 907 range-wide collections g g yp gin combination with the Bayesian clustering algorithms (STRUCTURE) and PCA 3 main genetic clusters were identified:
Eckert et al. in press. Genetics.
Deviations from neutrality in loblolly pine
Moderate levels of diversity
id d fRapid decay of LDLittle population differentiationdifferentiationSigns of selective sweeps:
• LG8• 5-10 genes. • Putative functions: abiotic and biotic stress response
Eckert et al. in press. Genetics.
Molecular Basis of Adaptive GeneticMolecular Basis of Adaptive Genetic Diversityy
Which SNPs have unusual correlations to aridity gradients (i.e. ratio of precipitation to
t ti l t i ti )?potential evapotranspiration)?
Eckert et al. in press Genetics.
Which SNPs have unusual correlations to multivariatecorrelations to multivariate climate gradients corresponding to temperature, growing degree-to temperature, growing degreedays, precipitation and aridity? Eckert et al. in press. Mol. Ecol.
Models Incorporating Population Structure Corrections
PCA – GLM: Bayesian GLMM:
Six highly significant associations (after Bonferroni corrections) to aridity Four
Core set of 48 SNPs correlated to temperature and precipitation with Bayes
Eckert et al. in press. Genetics. Eckert et al. in press. Mol. Ecol.
Bonferroni corrections) to aridity. Four are shown here. This is out of 3,938 SNPs tested.
temperature and precipitation with Bayes factors > 100. This is out of 1,730 tested.
CRSP - Comparative Re-Sequencing inRe Sequencing in
Pinaceae
Scientific Name Common Name Genome Size(Mb) SNPs Identified InvestigatorsScientific Name Common Name Genome Size(Mb) SNPs Identified Investigators
Pinus ellottii Slash Pine 24,200 2,879 Dudley Huber (University of Florida)
Pinus radiata Monterey Pine 26,500 2,474 Shannon Dillon (CSIRO)
Pinus pinaster Maritime Pine 30,300 2,981 Santiago Gonzalez Martinez & DelphinePinus pinaster Maritime Pine 30,300 2,981 Santiago Gonzalez Martinez & Delphine Grivet (ICIFOR - INIA)
Pinus sylvestris Scots Pine 24,600 3,803 Outi Savolainen & Sonja Kujala (University of Oulu)
Pinus lambertiana Sugar Pine 33 500 4 238 Kathie Jermstad (IFG)Pinus lambertiana Sugar Pine 33,500 4,238 Kathie Jermstad (IFG)
Pseudotsuga menziesii Douglas Fir 18,700 2,491 Kathie Jermstad (IFG)
Picea abies Norway Spruce 18,200 3,417 Marta Scalfi (IASMA)
WHISP - White Pine Re-Sequencing ProjectGoal: SNP Discovery in North American members of the white pines toinvestigate evolutionary relationships among eleven species
200 genes will be resequenced in the following white pines:
Pinus strobus Pinus albicaulis Pinus aristata Pinus ayacahuitePinus monticolaPinus strobiformisPinus strobus Pinus albicaulis Pinus aristata Pinus ayacahuitePinus monticolaPinus strobiformis
Pinus balfouriana Pinus flexilisPinus lambertianaPinus longaevaPinus monophylla
Alpine Ecosystems in Changing Environments: Biodiversity Sensitivity and Adaptive Potentialod ve s ty Se s t v ty a d dapt ve ote t a
Pinus abies
Pinus cembraPinus cembra
Larix decidua
Pinus mugo
Abies alba
Resequencing of the candidate genes - 4 species: p
Pinus cembra PICE Pinus mugoPIMG
Larix decidua LADE Abies albaABAL- 12 individuals per species (megagametophytes)- 735 genes assigned to 3 categories according to their putative function:
candidate control demographic- several locations across the species distribution in European PinusPinus cembracembra Pinus mugoPinus mugop pMountains
inusinus ce b ace b a us ugous ugo
LarixLarix deciduadecidua Abi lbAbi lbLarix Larix deciduadecidua Abies albaAbies alba
All genes
ABAL LADE PICE PIMGGenes # 260 298 501 532
Polym. # 178 200 304 410
SNPs # 1044 1625 1748 3522
SNP freq. 106 79 118 63
Diversityy
Gene categories
ABAL LADE PICE PIMG
Candidate 196 192 317 335
Control 70 82 154 165
Demographic 21 24 30 42
Divergence
Sampling across Alpine Mountains- 3 sampling levels: from geographic level to local onep g g g p- 5 species: Abies alba; Larix decidua;
Pinus cembra; Pinus mugo; Picea abies800 trees per species- 800 trees per species
- 2 gradients: altitudinalsoil type
Clinal Patterns of Adaptation in Balsam Fir
Estimated allele frequencies for eight allozyme loci infour subpopulations of balsam fir on Mt. Moosilauke,New Hampshire
0,500
1,000
Freq
uenc
ies
Relationship of temperature optimum for net photosyntheticCO2 uptake to elevational origin of balsam fir seedlings.Points represent determinations on individual pots.
0,0001464 1312 1159 854
Alle
le
Neale DB, Adams WT (1985) Allozyme and mating-system variation in balsam fir across a continous elevational transect. Canadian Journal of Botany 63, 2448-2453.
p p
m (oC)
2 up
take
Elevation (Meters)
ture optim
umynthetic CO2
Elevation
Tempe
rat
for ph
otosy
Fryer JH, Ledig FT (1972) Microevolution of photosynthetic temperature optimum in relation to elevational complex. Canadian Journal of Botany 50, 1231-1235.
Elevation (ft)
Barry Goldfarb
AcknowledgmentsSteve McKeand
David NealeJill Wegrzyn
Chuck LangleyKristian Stevens
Barry GoldfarbNick WheelerBialian LiPatrick Cumbie
Dana NelsonBrad St. Clair
S eve c e dRoss WhettenFikret IsikJB JettJill Wegrzyn
Andrew EckertKathleen JermstadJohn Liechty
Kristian Stevens Brad St. Clair
John LiechtyKatie TsangBen FigueroaElly Chen
Betty WolfWei Tao
Gary PeterJohn DavisM i KiJeff Deany
Alex Voong Marc RubenfieldKerri StelattoPatricia Harris
Carol LoopstraTom Byram
Matias KirstDudley HuberGeorge Cassela
Jeff Dean
Tom ByramKonstantin Krutovsky
Glenn HoweG e oweDavid HarryNick Wheeler
Chung-Jui Tsai Mark DavisBrian Stanton
Funding:
