1. From QTL to QTG: Are we getting closer? Sagiv Shifman and Ariel Darvasi The Hebrew University of Jerusalem

2. Presentation Outline Overview of fine QTL mapping strategies Overview of fine QTL mapping strategies Inbred strain sequence/haplotype information for fine QTL mapping Inbred strain sequence/haplotype information for fine QTL mapping Yin-Yang crosses: A framework for applying inbred strain sequence/haplotype information to fine map QTLs Yin-Yang crosses: A framework for applying inbred strain sequence/haplotype information to fine map QTLs Simulation analysis using Celera’s sequence information of 4 inbred strains (C57, A/J, DBA, 129) Simulation analysis using Celera’s sequence information of 4 inbred strains (C57, A/J, DBA, 129)

3. The Difficulties Genetics Genetics Genotype/phenotype correlation Genotype/phenotype correlation Recombination Recombination Functional Functional The “end-game” (knockout, transgenic, mutation analysis, RNAi, etc.) The “end-game” (knockout, transgenic, mutation analysis, RNAi, etc.)

4. Fine Mapping 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: Selective phenotyping Selective phenotyping Recombinant progeny testing Recombinant progeny testing Interval specific congenic strains (ISCS) Interval specific congenic strains (ISCS) Recombinant inbred segregation test (RIST) Recombinant inbred segregation test (RIST)

5. Sample size required Experimental design N for detection N for mapping into 1cM F2F2F2F21006,000 BC10010,000 RI Strains 40500

6. Advanced intercross lines (AIL) Semi-random intercrossing P F1F1 F2F2 F3F3 FtFt CI = CI F2 / (t/2)

7. AIL - experimental results HDL – QTL Wang et al. 2003

8. 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.

9. HS - Experimental results Open field activity Talbot et al. 1999 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 cM

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

11. SPh - Experimental results Lesions density Paigen et al. BC SPh-BC

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

13. Interval specific congenic strains (ISCS) QTL ISCS ISCS are produced by a series of backcrosses and intercrosses

14. Recombinant inbred segregation test (RIST) P1P1 RI P 2 xx F 1,1 F 1,2 F 2,1 F 2,2 QTL

15. RIST - Experimental results F 21 F 22 C57LAKRAKXL-16 P=0.41 D2MIT64 D2MIT200 P=0.02 B. Taylor A. Darvasi Obesity QTL

16. In silico mapping of complex disease-related traits in mice Grupe et al. 2001 Comment: Chesler et al. 2001 Darvasi 2001

18. 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

19. QTL detection with two inbred lines (P1 and P2) QTL detection with two inbred lines (P1 and P2)↓ Estimating QTL map location ↓ Sequence based fine mapping ↓ Selecting an optimal new inbred strain (Pi) ↓ Yin-Yang crosses Yin-Yang Crosses : A framework for Multiple Cross Inbred Strain Haplotype Mapping

20. QTL mapped in a AxB cross AB

21. Sequence/Haplotype Information AB

22. Yin-Yang Crosses AB   C

23. Simulation Analysis Initial cross between A/JxC57 Initial cross between A/JxC57 22,814 SNPs, each at its turn, simulated as the QTN 22,814 SNPs, each at its turn, simulated as the QTN The strain closest to being half similar to A/J and half to C57 was selected first for Yin-Yang crosses (DBA or 129) The strain closest to being half similar to A/J and half to C57 was selected first for Yin-Yang crosses (DBA or 129) The forth strain was subsequently introduced The forth strain was subsequently introduced At each stage the number of the remaining SNPs that can be the QTN, and the size of the QTL containing interval were estimated At each stage the number of the remaining SNPs that can be the QTN, and the size of the QTL containing interval were estimated

24. Mapping Resolution: Chromosome 16 2 strains 3 strains 4 strains

25. Low resolution region High resolution region

26. Mean Reduction in Interval Length

27. Distribution of Mapping Resolution with 4 Inbred Strains Proportion Interval length

28. Conclusions Using inbred strain sequence/haplotype information combined with multiple crosses (Yin-Yang crosses) can efficiently aid QTL fine mapping Using inbred strain sequence/haplotype information combined with multiple crosses (Yin-Yang crosses) can efficiently aid QTL fine mapping With 4 strains most regions exhibit modest mapping resolution. With 4 strains most regions exhibit modest mapping resolution. The significant haplotype variation observed in 4 strains may suggest that with a large number of strains in hand significant resolution can be achieved. The significant haplotype variation observed in 4 strains may suggest that with a large number of strains in hand significant resolution can be achieved. With sequence information in hand, resolution maps and algorithms can be established to guide the researchers to the optimal strain selection strategy and provide the resolution expected for their region of interest. With sequence information in hand, resolution maps and algorithms can be established to guide the researchers to the optimal strain selection strategy and provide the resolution expected for their region of interest. A large number of sequenced strains will allow the actual selection of a very small number of strains for additional crosses. A large number of sequenced strains will allow the actual selection of a very small number of strains for additional crosses.

29. So, Do We Need the 1K RI Set? YES Until 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