Sequential Quantitative Trait Locus Mapping in Experimental Crosses

The etiology of complex diseases is heterogeneous. The presence of risk alleles in one or more genetic loci affects the function of a variety of intermediate biological pathways, resulting in the overt expression of disease. Hence, there is an increasing focus on identifying the genetic basis of disease by sytematically studying phenotypic traits pertaining to the underlying biological functions. In this paper we focus on identifying genetic loci linked to quantitative phenotypic traits in experimental crosses. Such genetic mapping methods often use a one stage design by genotyping all the markers of interest on the available subjects. A genome scan based on single locus or multi-locus models is used to identify the putative loci. Since the number of quantitative trait loci (QTLs) is very likely to be small relative to the number of markers genotyped, a one-stage selective genotyping approach is commonly used to reduce the genotyping burden, whereby markers are genotyped solely on individuals with extreme trait values. This approach is powerful in the presence of a single quantitative trait locus (QTL) but may result in substantial loss of information in the presence of multiple QTLs. Here we investigate the efficiency of sequential two stage designs to identify QTLs in experimental populations. Our investigations for backcross and F2 crosses suggest that genotyping all the markers on 60% of the subjects in Stage 1 and genotyping the chromosomes significant at 20% level using additional subjects in Stage 2 and testing using all the subjects provides an efficient approach to identify the QTLs and utilizes only 70% of the genotyping burden relative to a one stage design, regardless of the heritability and genotyping density. Complex traits are a consequence of multiple QTLs conferring main effects as well as epistatic interactions. We propose a two-stage analytic approach where a single-locus genome scan is conducted in Stage 1 to identify promising chromosomes, and interactions are examined using the loci on these chromosomes in Stage 2. We examine settings under which the two-stage analytic approach provides sufficient power to detect the putative QTLs.

"Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death"

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (,30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (similar to 30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2 nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) × Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

A strong quantitative trait locus for wing length on chromosome 2 in a wild population of great reed warblers

Wing length is a key character for essential behaviours related to bird flight such as migration and foraging. In the present study, we initiate the search for the genes underlying wing length in birds by studying a long-distance migrant, the great reed warbler (Acrocephalus arundinaceus). In this species wing length is an evolutionary interesting trait with pronounced latitudinal gradient and sex-specific selection regimes in local populations. We performed a quantitative trait locus (QTL) scan for wing length in great reed warblers using phenotypic, genotypic, pedigree and linkage map data from our long-term study population in Sweden. We applied the linkage analysis mapping method implemented in GRIDQTL (a new web-based software) and detected a genome-wide significant QTL for wing length on chromosome 2, to our knowledge, the first detected QTL in wild birds. The QTL extended over 25 cM and accounted for a substantial part (37%) of the phenotypic variance of the trait. A genome scan for tarsus length (a bodysize-related trait) did not show any signal, implying that the wing-length QTL on chromosome 2 was not associated with body size. Our results provide a first important step into understanding the genetic architecture of avian wing length, and give opportunities to study the evolutionary dynamics of wing length at the locus level. This journal is© 2010 The Royal Society.

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype.

A major quantitative trait locus for CD4-CD8 ratio is located on chromosome 11

CD4-CD8 ratio is an important diagnostic measure of immune system functioning. In particular, CD4-CD8 ratio predicts the time taken for progression of HIV infection to acquired immune deficiency syndrome (AIDS) and the long-term survival of AIDS patients. To map genes that regulate differences between healthy individuals in CD4-CD8 ratio, we typed 757 highly polymorphic microsatellite markers at an average spacing of similar to5 cM across the genome in 405 pairs of dizygotic twins at ages 12, 14 and 16. We used multipoint variance components linkage analysis to test for linkage between marker loci and CD4-CD8 ratio at each age. We found suggestive evidence of linkage on chromosome 11p in 12-year-old twins (LOD=2.55, P=0.00031) and even stronger evidence of linkage in the same region at age 14 (LOD 3.51, P=0.00003). Possible candidate genes include CD5 and CD6, which encode cell membrane proteins involved in the positive selection of thymocytes. We also found suggestive evidence of linkage at other areas of the genome including regions on chromosomes 1, 3, 4, 5, 6, 12, 13, 15, 17 and 22.

Major quantitative trait locus for eosinophil count is located on chromosome 2q

Background: Eosinophils are granulocytic white blood cells implicated in asthma and atopic disease. The degree of eosinophilia in the blood of patients with asthma correlates with the severity of asthmatic symptoms. Quantitative trait loci (QTL) linkage analysis of eosinophil count may be a more powerful strategy of mapping genes involved in asthma than linkage analysis using affected relative pairs. 1 Objective: To identify QTLs responsible for variation in eosinophil count in adolescent twins. Methods: We measured eosinophil count longitudinally in 738 pairs of twins at 12, 14, and 16 years of age. We typed 757 highly polymorphic microsatellite markers at an average spacing of similar to5 centimorgans across the genome. We then used multipoint variance components linkage analysis to test for linkage between marker loci and eosinophil concentrations at each age across the genome. Results: We found highly significant linkage on chromosome 2q33 in 12-year-old twins (logarithm of the odds = 4.6; P = .000002) and suggestive evidence of linkage in the same region in 14-year-olds (logarithm of the odds = 1.0; P = .016). We also found suggestive evidence of linkage at other areas of the genome, including regions on chromosomes 2, 3, 4, 8, 9, 11, 12, 17, 20, and 22. Conclusion: A QTL for eosinophil count is present on chromosome 2q33. This QTL might represent a gene involved in asthma pathophysiology.

Novel Statistical Methods in Quantitative Genetics : Modeling Genetic Variance for Quantitative Trait Loci Mapping and Genomic Evaluation

This thesis develops and evaluates statistical methods for different types of genetic analyses, including quantitative trait loci (QTL) analysis, genome-wide association study (GWAS), and genomic evaluation. The main contribution of the thesis is to provide novel insights in modeling genetic variance, especially via random effects models. In variance component QTL analysis, a full likelihood model accounting for uncertainty in the identity-by-descent (IBD) matrix was developed. It was found to be able to correctly adjust the bias in genetic variance component estimation and gain power in QTL mapping in terms of precision.  Double hierarchical generalized linear models, and a non-iterative simplified version, were implemented and applied to fit data of an entire genome. These whole genome models were shown to have good performance in both QTL mapping and genomic prediction. A re-analysis of a publicly available GWAS data set identified significant loci in Arabidopsis that control phenotypic variance instead of mean, which validated the idea of variance-controlling genes.  The works in the thesis are accompanied by R packages available online, including a general statistical tool for fitting random effects models (hglm), an efficient generalized ridge regression for high-dimensional data (bigRR), a double-layer mixed model for genomic data analysis (iQTL), a stochastic IBD matrix calculator (MCIBD), a computational interface for QTL mapping (qtl.outbred), and a GWAS analysis tool for mapping variance-controlling loci (vGWAS).

MAPfastR : quantitative trait loci mapping in outbred line crosses

MAPfastR is a software package developed to analyze QTL data from inbred and outbred line-crosses. The package includes a number of modules for fast and accurate QTL analyses. It has been developed in the R language for fast and comprehensive analyses of large datasets. MAPfastR is freely available at: http://www.computationalgenetics.se/?page_id=7.

Dissection of a quantitative trait locus for genetic hypertension on rat chromosome 10.

We have previously identified a locus on rat chromosome 10 as carrying a major hypertension gene, BP/SP-1. The 100:1 odds support interval for this gene extended over a 35-centimorgan (cM) region of the chromosome that included the angiotensin I-converting enzyme (ACE) locus as demonstrated in a cross between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and the normotensive Wistar-Kyoto (WKY-0HD) rat. Here we report on the further characterization of BP/SP-1, using a congenic strain, WKY-1HD. WKY-1HD animals carry a 6-cM chromosomal fragment genotypically identical with SHRSPHD on chromosome 10, 26 cM away from the ACE locus. Higher blood pressures in the WKY-1HD strain compared with the WKY-0HD strain, as well as absence of linkage of the chromosome 10 region to blood pressure in an F2 (WKY-1HD x SHRSPHD) population suggested the existence of a quantitative trait locus, termed BP/SP-1a, that lies within the SHRSP-congenic region in WKY-1HD. Linkage analysis in the F2 (WKY-0HD x SHRSPHD) cross revealed that BP/SP-1a is linked to basal blood pressure, whereas a second locus on chromosome 10, termed BP/SP-1b, that maps closer to the ACE locus cosegregates predominantly with blood pressure after exposure to excess dietary NaCl. Thus, we hypothesize that the previously reported effect of BP/SP-1 represents a composite phenotype that can be dissected into at least two specific components on the basis of linkage data and congenic experimentation. One of the loci identified, BP/SP-1a, represents the most precisely mapped locus affecting blood pressure that has so far been characterized by random-marker genome screening.

Genome-wide scan of IQ finds significant linkage to a quantitative trait locus on 2q

A genome-wide linkage scan of 795 microsatellite markers (761 autosomal, 34 X chromosome) was performed on Multidimensional Aptitude Battery subtests and verbal, performance and full scale scores, the WAIS-R Digit Symbol subtest, and two word-recognition tests (Schonell Graded Word Reading Test, Cambridge Contextual Reading Test) highly predictive of IQ. The sample included 361 families comprising 2-5 siblings who ranged in age from 15.7 to 22.2 years; genotype, but not phenotype, data were available for 81% of parents. A variance components analysis which controlled for age and sex effects showed significant linkage for the Cambridge reading test and performance IQ to the same region on chromosome 2, with respective LOD scores of 4.15 and 3.68. Suggestive linkage (LOD score > 2.2) for various measures was further supported on chromosomes 6, 7, 11, 14, 21 and 22. Where location of linkage peaks converged for IQ subtests within the same scale, the overall scale score provided increased evidence for linkage to that region over any individual subtest. Association studies of candidate genes, particularly those involved in neural transmission and development, will be directed to genes located under the linkage peaks identified in this study.

Mapping quantitative trait loci in a wild population using linkage and linkage disequilibrium analyses

Historical information can be used, in addition to pedigree, traits and genotypes, to map quantitative trait locus (QTL) in general populations via maximum likelihood estimation of variance components. This analysis is known as linkage disequilibrium (LD) and linkage mapping, because it exploits both linkage in families and LD at the population level. The search for QTL in the wild population of Soay sheep on St. Kilda is a proof of principle. We analysed the data from a previous study and confirmed some of the QTLs reported. The most striking result was the confirmation of a QTL affecting birth weight that had been reported using association tests but not when using linkage-based analyses. Copyright © Cambridge University Press 2010.

Mapping quantitative trait loci for resistance to Pratylenchus thornei from synthetic hexaploid wheat in the International Triticeae Mapping Initiative (ITMI) population

Root-lesion nematode (Pratylenchus thornei) is a serious pathogen of wheat in many countries. The International Triticeae Mapping Initiative (ITMI) population of recombinant inbred lines (RILs) was assessed for resistance to P. thornei to determine the chromosome locations of the resistance genes. The ITMI population is derived from a cross between the resistant synthetic hexaploid wheat W-7984 and a susceptible bread wheat cultivar Opata 85. Two years of phenotypic data for resistance to P. thornei were obtained in replicated glasshouse trials. Quantitative trait locus (QTL) analysis was performed using available segregation and map data for 114 RILs. A QTL on chromosome 6DS showed consistent effects for reduced nematode numbers (partial resistance) across years and accounted for 11% and 23% of the phenotypic variation. A second QTL for P. thornei resistance on chromosome 2BS accounted for an additional 19% and 5%. Restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers associated with the QTLs are physically located in regions rich in major genes at the distal ends of the short chromosome arms of 6D and 2B. SSR markers with potential for marker-assisted selection of P. thornei resistance effective in different genetic backgrounds have been identified.