From QTL to QTG: Are we getting closer?

Sagiv Shifman and Ariel Darvasi

The Hebrew University of JerusalemThe Hebrew University of Jerusalem

Presentation OutlinePresentation Outline

Overview of fine QTL mapping strategiesOverview of fine QTL mapping strategies

Inbred strain sequence/haplotype information for Inbred strain sequence/haplotype information for fine QTL mappingfine QTL mapping

Yin-Yang crosses: A framework for applying inbred Yin-Yang crosses: A framework for applying inbred strain sequence/haplotype information to fine map strain sequence/haplotype information to fine map QTLsQTLs

Simulation analysis using Celera’s sequence Simulation analysis using Celera’s sequence information of 4 inbred strains (C57, A/J, DBA, 129)information of 4 inbred strains (C57, A/J, DBA, 129)

The DifficultiesThe Difficulties

GeneticsGenetics Genotype/phenotype correlationGenotype/phenotype correlation RecombinationRecombination

FunctionalFunctional The “end-game” (knockout, transgenic, The “end-game” (knockout, transgenic,

mutation analysis, RNAi, etc.)mutation analysis, RNAi, etc.)

Fine Mapping StrategiesFine Mapping Strategies

Genomewide-based strategies:Genomewide-based strategies: Large scale BC, F2, half sibs, etc.Large scale BC, F2, half sibs, etc. Advanced Intercross Lines (AIL)Advanced Intercross Lines (AIL) The heterogeneous stock (HS)The heterogeneous stock (HS)

Locus-based strategies:Locus-based strategies: Selective phenotypingSelective phenotyping Recombinant progeny testingRecombinant progeny testing Interval specific congenic strains (ISCS)Interval specific congenic strains (ISCS) Recombinant inbred segregation test (RIST)Recombinant inbred segregation test (RIST)

Sample size requiredSample size required

Experimental Experimental designdesign

N for N for detectiondetection

N for mapping N for mapping into 1cMinto 1cM

FF221001006,0006,000

BCBC10010010,00010,000

RI StrainsRI Strains4040500500

Advanced intercross lines (AIL)Advanced intercross lines (AIL)

Semi-random intercrossing

P F1 F2 F3 Ft

CI = CICI = CIF2 F2 / (t/2)/ (t/2)

AIL - experimental resultsAIL - experimental results

HDL – QTLWang et al. 2003

The heterogeneous stock (HS)The heterogeneous stock (HS)

Eight-way cross of:

C57BL/6, BALB/C, RIII, AKR, DBA/2, I, A/J C3H

Established 30 years ago (~60 generations)by

McClearn et al.

HS - Experimental resultsHS - Experimental results

Open field activity

Talbot et al. 1999

0 0.2 0.4 0.6 0.8 1.0 1.2 1.40 0.2 0.4 0.6 0.8 1.0 1.2 1.4

cMcM

Locus-based strategies:Selective phenotyping (SPh)Selective phenotyping (SPh)

Theoretical basis: Only recombinants

increase mapping accuracy for a

detected QTL.

Procedure: Only individuals

recombinant at a QTL-containing

interval are subsequently phenotyped.

SPh - Experimental resultsSPh - Experimental results

0

2

4

6

0 20 40 60 80cM

LO

D

Lesions density

Paigen et al. BC

SPh-BC

Recombinant progeny Recombinant progeny testingtesting

QTLQTL

Males, recombinant at an interval of interest, are progeny tested to check which QTL allele was retained.

Interval specific congenic strains Interval specific congenic strains (ISCS)(ISCS)

QTLQTL

ISCS ISCS are produced by a series of backcrosses and intercrosses

Recombinant inbred segregation Recombinant inbred segregation test (RIST)test (RIST)

P1 RI P2

x x

F1,1 F1,2

F2,1 F2,2

QTLQTL

RIST - Experimental resultsRIST - Experimental results

F21 F22

C57L AKRAKXL-16

P=0.41

D2MIT64

D2MIT200

P=0.02

B. TaylorA. Darvasi

Obesity QTLObesity QTL

In silico mapping of complex disease-related traits in mice

Grupe et al. 2001

Comment:

Chesler et al. 2001Darvasi 2001

Park et al. 2003

Wiltshire et al. 2003

Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse

Multiple Cross and Inbred Strain Haplotype Mapping of Complex-Trait Candidate Genes

Unexpected complexity in the haplotypes of commonly used inbred strains of laboratory mice

Yalcin et al. 2004

QTL detection with two inbred lines (P1 and QTL detection with two inbred lines (P1 and P2) P2) ↓↓

Estimating QTL map locationEstimating QTL map location↓↓

Sequence based fine mappingSequence based fine mapping↓↓

Selecting an optimal new inbred strain (Pi)Selecting an optimal new inbred strain (Pi)↓↓

Yin-Yang crossesYin-Yang crosses

Yin-Yang Crosses : A framework for Yin-Yang Crosses : A framework for Multiple Cross Inbred Strain Haplotype Multiple Cross Inbred Strain Haplotype

Mapping Mapping

QTL mapped in a AxB crossQTL mapped in a AxB cross

A B

Sequence/Haplotype Sequence/Haplotype InformationInformation

A B

Yin-Yang CrossesYin-Yang Crosses

A B

C

Simulation AnalysisSimulation Analysis

Initial cross between A/JxC57Initial cross between A/JxC57

22,814 SNPs, each at its turn, simulated as the QTN22,814 SNPs, each at its turn, simulated as the QTN

The strain closest to being half similar to A/J and half to C57 The strain closest to being half similar to A/J and half to C57 was selected first for Yin-Yang crosses (DBA or 129)was selected first for Yin-Yang crosses (DBA or 129)

The forth strain was subsequently introducedThe forth strain was subsequently introduced

At each stage the number of the remaining SNPs that can At each stage the number of the remaining SNPs that can be the QTN, and the size of the QTL containing interval be the QTN, and the size of the QTL containing interval were estimatedwere estimated

Mapping Resolution: Chromosome Mapping Resolution: Chromosome 16 16

2 strains3 strains4 strains

Low resolution region

High resolution region

11 12 13 14 15

0.0

00

.02

0.0

40

.06

Number of SNPs = 30 Interval= 2.1 Mb

Location (Mb)

pro

po

rtio

n o

f S

NP

s

11 12 13 14 15

0.0

00

.05

0.1

00

.15

Number of SNPs = 12 Interval= 1 Mb

Location (Mb)

pro

po

rtio

n o

f S

NP

s

11 12 13 14 15

0.0

0.1

0.2

0.3

0.4

Number of SNPs = 5 Interval= 0.4 Mb

Location (Mb)

pro

po

rtio

n o

f S

NP

s

57 58 59 60 61

0.0

00

.02

0.0

4

Number of SNPs = 2318 Interval= 4 Mb

Location (Mb)

pro

po

rtio

n o

f S

NP

s

57 58 59 60 61

0.0

00

.02

0.0

40

.06

Number of SNPs = 1696 Interval= 3.6 Mb

Location (Mb)

pro

po

rtio

n o

f S

NP

s

57 58 59 60 61

0.0

00

.02

0.0

40

.06

Number of SNPs = 1696 Interval= 3.6 Mb

Location (Mb)

pro

po

rtio

n o

f S

NP

s

Mean Reduction in Interval Mean Reduction in Interval Length Length

23

4

5

2

1

54%51%

37%44%

42%

30% 31%30%

21%

Number of Strains

Initial Lentgh (Mb)

Distribution of Mapping Resolution with 4 Inbred Distribution of Mapping Resolution with 4 Inbred Strains Strains

Pro

port

ion0 1 2 3 4 50

.00

0.0

40

.08

0 1 2 3 4 50.0

00

.10

0 1 2 3 4 50.0

00

.10

0.2

0

Interval length

ConclusionsConclusions Using inbred strain sequence/haplotype information Using inbred strain sequence/haplotype information

combined with multiple crosses (Yin-Yang crosses) can combined with multiple crosses (Yin-Yang crosses) can efficiently aid QTL fine mapping efficiently aid QTL fine mapping

With 4 strains most regions exhibit modest mapping With 4 strains most regions exhibit modest mapping resolution. resolution.

The significant haplotype variation observed in 4 strains The significant haplotype variation observed in 4 strains may suggest that with a large number of strains in hand may suggest that with a large number of strains in hand significant resolution can be achieved.significant resolution can be achieved.

With sequence information in hand, resolution maps and algorithms With sequence information in hand, resolution maps and algorithms can be established to guide the researchers to the optimal strain can be established to guide the researchers to the optimal strain selection strategy and provide the resolution expected for their selection strategy and provide the resolution expected for their region of interest.region of interest.

A large number of sequenced strains will allow the actual selection A large number of sequenced strains will allow the actual selection of a very small number of strains for additional crosses.of a very small number of strains for additional crosses.

So, Do We Need the 1K RI So, Do We Need the 1K RI Set?Set?

YESUntil we have the 1K RI set any QTL mapping strategy is like riding a bike: it’s fun and we can get very far – Once we get something with an engine though, we will feel the difference!

… we are still missing an engine for the “end-game” – Genetics is not everything