Lecture 20: From Phenotype to Genes and Back

Oct 30, 2006

Last Time

Tension zones and mosaic zones

Introduction to QTL analysis

Today

QTL examples and limitations

Linkage Disequilibrium and Association studies

Reproductive isolationPre-mating barriers to gene flow• Geographic• Ecological• Phenological• Behavioral• Mechanical

Post-mating barriers to gene flow• Gamete incompatibility• Sperm competition• Hybrid inviability• Hybrid sterility• Hybrid breakdown

Slide courtesy of T. Bradshaw

Bumblebee-pollinated Hummingbird-pollinatedPink RedWide corolla opening Narrow, tubular corolla1-2μl nectar 40-100μl nectar

Slide adapted from T. Bradshaw

QTL for Reproductive isolation in Mimulus

Sea level to 2000 m1600-3000 m

M cardinalis OR 01M cardinalis CA 02M cardinalis CA 03

M cardinalis CA 04M cardinalis CA 11

M cardinalis Mx 13M cardinalis CA 14

M cardinalis CA 05M cardinalis CA 06

M cardinalis CA 07M cardinalis CA 15

M cardinalis CA 10M cardinalis CA 09

M lewisii WA 1M lewisii WA 3

M lewisii OR 4 1M lewisii OR 4 2

M lewisii MT 6M lewisii OR 5

M lewisii N CA 7 1M lewisii N CA 7 2

M lewisii N CA 8M lewisii CA 1 1

M lewisii CA 1 2M lewisii CA 3

M lewisii CA 4M lewisii CA 5 1

M lewisii CA 5 3M lewisii CA 5 2

M lewisii CA 2M lewisii CA 6

M lewisii CA 7M lewisii WA 2

M eastwoodiae CO 2M eastwoodiae UT 4

M eastwoodiae UT 3M verbenaceus AZ 2

M verbenaceus UT 3M verbenaceus UT 4M nelsonii Mx 1

M rupestris Mx 1M parishii CA 2

M parishii CA 5M parishii CA 3

M parishii CA 6M bicolor CA 2

M filicaulis CA 2

0.01 changes

76

99

100 9672

87

95

94

97

79

58

69

100

60

8060

51

100

85 93

8978

100

66

6978

10064

97

lewisiiRockies; Cascades

lewisiiSierra Nevada

cardinalis

Paul BeardsleyNeighbor-joining478 AFLPs

Mimulus section Erythranthe (7-8 spp.)

77

Slide courtesy of T. Bradshaw

Absolute contribution (AC) to component of reproductive isolation (RI):

Co po e s o ep oduc eisolation between M. lewisii

and M. cardinalis

Geography and ecology58.8%

Pollinator40.3%

Post-mating0.9%

Ramsey, J., Bradshaw, H.D., Jr., & Schemske, D.W. (2003) Evolution 57:1520Slide adapted from T. Bradshaw

Based on botanical surveys, controlled crosses, hybrid fitness, and field observations of pollination

Mimulus Floral Characteristics• Interspecific cross

between species with different pollination syndromes

• Mapped QTL for floral traits

M. lewisii M. cardinalisF1

F2

Trait Linkage group

PVE Mode of action

83%

21%

42%

32%

41%

69%

33%

Stamen length AL 47% add

50%

Carotenoids (yellow) DC L > C

Anthocyanins (red/purple) DC L > C

Petal width EL L > C

Corolla width AL L > C

Corolla projected area CC C > L

Petal reflexing AL L > C

Nectar volume B add

Pistil length EL add

Major QTLs in Mimulus

Slide courtesy of T. Bradshaw

Courtesy of T. Bradshaw

• Planted F2 population in zone of sympatry between parent species (Yosemite National Park)

• Measured pollinator visits• Measured characteristics of

each F2 plant

Mimulus map

YUP

• Can a single QTL have a large effect on pollinator choice?

• How to prove this?Slide courtesy of T. Bradshaw

Near-isogenic lines (NILs)

lewisii

cardinalis

F1

F2

F2

xC

xL xL xL

xC xC

NIL2

NIL1

Slide courtesy of T. Bradshaw

yupYUP

Bumblebees

N=1090

yupYUP

Hummingbirds

N=201

Slide courtesy of T. Bradshaw

M. lewisii NILs

yupYUP

Bumblebees

N=180

yupYUP

Hummingbirds

N=3738

Slide courtesy of T. Bradshaw

M. cardinalis NILs

Visitation rate ratio

BEEYUP:yup

5.2 5.8 74.1

HUMMERyup:YUP

1.2 68.0 1.1

• NIL results consistent with F2 results• A mutation at the YUP locus increases visitation by the new pollinator ~70-fold,

and this response is symmetrical: instant reproductive isolation?

Slide courtesy of T. Bradshaw

QTL LimitationsLimited genetic base: QTL may only apply to the two individuals in the cross!

Genotype x Environment interactions rampant: some QTL only appear in certain environments

Huge regions of genome underly QTL, usually hundreds of genes

How to distinguish among candidates?

Biased toward detection of large-effect loci

Need very large pedigrees to do this properly

QTL Vary by Year, Site, and PopulationLoblolly pine QTL measured in different years at same site, in different sites, and with a different genetic background

Stippled: not repeated across years

wood-specific gravity

% latewood

microfibril angle

Brown et al

Linkage Disequilibrium and Quantitative Trait Mapping

Linkage and quantitative trait locus (QTL) analysis

Need a pedigree and moderate number of molecular markersVery large regions of chromosomes represented by markers

Association Studies with Natural Populations

No pedigree requiredNeed large numbers of genetic markersSmall chromosomal segments can be localizedMany more markers are required than in traditional QTL analysis

Cardon and Bell 2001, Nat. Rev. Genet. 2: 91-99

Association Mapping

recombination throughevolutionary history

present-daychromosomesin natural population

*TG

*TA

CGCA

*TGCA

ancestral chromosomes

*TG

Slide courtesy of Dave Neale

Linkage DisequilibriumLD is nonrandom association of alleles in gametes

Usually driven by physical linkage

Epistasis also a possibility

Hedrick 2005

Ideally: With LD:

Estimating LD:

Substituting from Table 1

Problem: Upper limit of D set by genotype frequencies

e.g., x11=0.5, x22=0.5,

Dmax=0.25 butx11=0.1, x22=0.9,

Dmax=0.09

Solution: D' = D/Dmaxranges from -1 to 1

Can also use square of correlation coefficient between alleles:

r2= D2/(p1p2q1q2)

r' = r/rmax

Factors affecting LDFactor EffectRecombination rate Higher recombination lowers LDMating systems: selfing species High LDMating systems: outcrossing

speciesLow LD

Genetic isolation between lineages Increased LDPopulation subdivision Increased LDPopulation admixture Increased LDNatural and artificial selection Locally increased LDPopulation size Small populations have more LDBalancing selection Increased LDMutation rate High mutation rate decreases overall LD,

but LD around newly created mutated allele remains high until dissipated by recombination

Genomic rearrangements Rearrangements suppress local recombination leading to LD increase in the vicinity

Stochastic effects (chance) Increase or decrease

Rafalski and Morgante, TIG 2004

Effects of recombination rate on LD

Hedrick 2005

Decline in LD over time with different theoretical recombination rates (c)

Even with independent segregation (c=0.5), multiple generations required to break up allelic associations

Admixture, bottlenecks, and inbreeding create genome-wide linkage disequilibrium

(from Martinsen et al. 2001)(from Martinsen et al. 2001)

r² = 0.24

0.60 0.26

0.080.040.06

0.02

0.08

0.10

0.16

0.04

0.04

Population Admixture and LD

Candidate Gene Associations vs. Whole Genome Scans

If LD is high and haplotypeblocks are conserved, entire genome can be efficiently scanned for associations with phenotypes

Simplest for case-control studies (e.g., disease, gender)

If LD is low, candidate genes are usually identified a priori, and a limited number are scanned for associations

Biased by existing knowledgeUse "Candidate Regions" from high LD populations, assess candidate genes in low LD populations

P_2852_A157.3

P_2385_A

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LOW

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P_204_C0.0S8_328.8P_2385_C11.6T4_1012.1S15_8S5_3713.8T4_7S6_1215.5S8_2917.9P_2786_A S12_1820.4T1_1322.3T7_423.5T3_13 T3_36S17_2124.1S15_16T12_1525.3T2_3026.5S13_2029.5S1_2036.5T9_1 S1_1943.2S3_1350.5S1_2452.9S2_754.1P_575_A59.1T12_2260.6S2_3285.0T7_995.7S2_6107.8S13_16 T5_25121.4T5_12124.3T10_4129.0T1_26 T7_13135.7P_93_A148.6S4_20150.2S7_13 S7_12T12_4152.8S4_24T3_10S6_4154.1

S3_1163.4S6_20 S13_31T7_15171.3T2_31178.2S8_4180.8S8_28182.1O_30_A184.2T5_4193.5T3_17198.1T12_12206.8S5_29210.6P_2789_A219.9P_634_A S17_43226.5S17_33230.3S17_12232.7S4_19243.1

S17_26262.9

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QTL Candidate Region

Candidate Gene Identification

Human HapMap Project and Whole Genome Scans

LD structure of human Chromosome 19 (www.hapmap.org)1 common SNP genotyped every 5kb for 269 individuals9.2 million SNP in total

NATURE|Vol 437|27 October 2005

LD varies substantially across human genomeNATURE|Vol 437|27 October 2005

Average r2 for pairs of SNP separated by 30 kb in 1 Mb windows

LD affected by location relative to telomeresand centromeres, chromosome length, GC content, sequence polymorphism, and repeat composition

Highest and lowest levels of LD found in gene-rich regions

Next Time

Association study examples

Signatures of selection