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QTN modulating the transcription rate of a chromosome domain encompassing PLAG1 control bovine stature. Michel Georges University of Liège Belgium Holstein-Friesian X Jersey intercross 500 traits Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 750 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Stature Stature Human: Paradigmatic Quantitative Trait h2 80% Quasi-infinitesimal architecture Dog: 5 loci explain nearly all the difference of stature between breeds. Cattle: Auroch: 2m=> domestic cattle: 1.1-1.5m Economically important trait h2 25-80% Many reported “QTL” Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions A QTL affecting stature maps to BTA14: HF x J F2 population A QTL affecting stature maps to BTA14: line-cross model 294 microsatellites A note on genome-wide thresholds Significant: Expected to be exceeded on average 1/20 genome scans Expected not to be exceeded in a proportion e^(1/20)=0.049 of genome scans Suggestive: Expected to be exceeded on average 1/1 genome scans Expected not to be exceeded in a proportion e^(1)=0.63 of genome scans Determine these thresholds by genome-wide permutation A note on F2 vs line-cross models Is it reasonable to assume that the Q and q alleles will be alternatively “fixed” in the alternate F0 lines? Hence, that the QTL genotype of all F1 animals is the same? Especially when this is not the case for the SNP or microsatellite markers. This has lead to many erroneous conclusions of imprinted QTL in animal genetics A note on F2 vs line-cross models A QTL affecting stature maps to BTA14: ½-sib model Across-family analysis – 1 QTL 8->56 μsat. A QTL affecting stature maps to BTA14: ½-sib model Within family analysis – effects Within family analysis – significance A note on bootstrapping Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Factors limiting mapping resolution Marker density Cross-over density Current (linkage) Historical (LD; limits) QTL detectance Within “blocks”, LD is a function if mutational sequence NOT distance LD doesn’t decay « simply » with distance D’<1 5 2 1 4 12 3 4 5 6 3 6 D’=1 The International HapMap Consortium. Nature 379: 1299 (2005) Factors limiting mapping resolution Marker density Cross-over density Current (linkage) Historical (LD; limits) QTL detectance Combined L+LD mapping accounting for stratification Modeling polygenic and locusspecific effects as random effects A feeling for the « individual animal model »: MVN distribution Bivariate normal distribution of the animal effects of closely related ids. Bivariate normal distribution of the animal effects of unrelated ids. Hidden Markov Models Basic principles Transition probabilities 1 2 Hidden States Emission Probabilities 1 2 3 3 Hidden Markov Modeling of haplotypes L+LD fine-mapping defines 780 Kb interval: F2 population • + 925 SNPs • LD non inbred F0 Multipoint analysis – 1 QTL/2 QTL Single-point analysis – 1 QTL 10% of phenotypic variance - Mixed model including “animal effect” - Hidden Haplotype States The grand-daughter design L+LD fine-mapping defines 780 Kb interval: outbred pop. Substitution effects of hidden haplotype states 1% of phenotypic variance Multipoint analysis – 1 QTL/2 QTL No unique haplotype associated with Q or q “q” 3% of phenotypic variance “Q” Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions HT sequencing of 780 Kb interval: =>13 candidate QTN M&M: “Progeny-tested” chromosomes of six F1 sires 103 long range PCR products Sire-specific multiplex identifiers (MIDs) Roche FLX MASA: Converting a polygenic trait in a series of monogenic entities HT sequencing of 780 Kb interval: =>13 candidate QTN Results: Average 20-fold coverage / sire 9,572 variants π: 1/300 14 candidate QTN segregation pattern compatible with QTL genotype. HT sequencing of 780 Kb interval: =>13 candidate QTN HT sequencing of 780 Kb interval: =>13 candidate QTN HT sequencing of 780 Kb interval: =>13 candidate QTN Using Hidden Haplotype States instead of sires to increase QTL detectance Exploiting “NGS” Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Across breed haplotype diversity => 8 candidate QTN Across breed haplotype diversity => 8 candidate QTN Across breed haplotype diversity => 8 candidate QTN Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Intact ORFs support regulatory pQTN Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Expression analysis: M&M 79 fetuses QRT-PCR (SYBR and/or 3’exonucl.) x/8 internal controls selected with geNorm ≤ 4 amplicons/gene The pQTN affect expression of all genes in conserved domain The pQTN affect expression of all genes in conserved domain Average: 20.86 ≈ 1.8 Allelic imbalance at (pre-)mRNA level => transcriptional effect Conservation of synteny suggests domain “regulon” Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions 3/8 candidate pQTN affect Phastcons elements Reporter assays and EMSA support 2 promotor pQTN ** Reporter assays and EMSA support 2 promotor pQTN Reporter assays and EMSA support 2 promotor pQTN Reporter assays and EMSA support 2 promotor pQTN Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 780 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Pick your favorite gene … X X Pick your favorite gene … X X Formal test for gene causality: Distribution of rare variant Σ=5% Σ=17% Formal test for gene causality: reciprocal hemizygosity Steinmetz et al. 2002 Formal test for gene causality: quantitative complementation Naturally occurring null allele excludes CHCHD7 CHCHD7 cis-eQTLwith distinct segregation vector (vs “pQTL”) Naturally occurring null allele excludes CHCHD7 “eQTN” is a donor splice site variant Naturally occurring null allele excludes CHCHD7 Naturally occurring null allele excludes CHCHD7 Splice site variant affects transcript levels in multiple (all?) tissues pQTL and eQTL have different segregation vector pQTL effect on stature is same for 4 segregating sires eQTN has no significant “residual” effect on stature No failure to quantitatively complement No failure to quantitatively complement Naturally occurring null allele excludes CHCHD7 Splice site variant affects transcript levels in multiple (all?) tissues pQTL and eQTL have different segregation vector pQTL effect on stature is same for 4 segregating sires eQTN has nosignificant “residual” effect on stature No failure to quantitatively complement => CHCHD7 can not be sole causative gene Plan Mapping the QTL Genetic identification of the QTN Intact ORFs support regulatory pQTN pQTN affects expression of PLAG1-encompassing domain Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7 bidirectional promoter Identifying the causative gene HT sequencing identifies 13 candidate pQTN Exploiting haplotype diversity to eliminate 5/13 candidate pQTN Functional analysis of the QTN Stature A QTL affecting stature maps to BTA14 L+LD fine-mapping defines a 750 Kb CI Naturally occurring null allele excludes CHCHD7 Conclusions Conclusions QTN modulating the transcription rate of a chromosome domain encompassing PLAG1 control bovine stature Domestic animal populations have unique features facilitating the genetic dissection of complex traits (line-crosses, harems, reduced effective population size, haplotype diversity) Haplotype sharing may not always be effective for the identification of old QTN The QCA can be applied in outbred populations using naturally occurring null alleles