High-resolution mapping of quantitative trait loci in outbred mice

Screening the whole genome of a cross between two inbred animal strains has proved to be a powerful method for detecting genetic loci underlying quantitative behavioural traits, but the level of resolution offered by quantitative trait loci (QTL) mapping is still too coarse to permit molecular cloning of the genetic determinants. To achieve high-resolution mapping, we used an outbred stock of mice for which the entire genealogy is known. The heterogeneous stock (HS) was established 30 years ago from an eight-way cross of C57BL/6, BALB/c, RIII, AKR, DBA/2, I, A/J and C3H inbred mouse strains. At the time of the experiment reported here, the HS mice were at generation 58, theoretically offering at least a 30-fold increase in resolution for QTL mapping compared with a backcross or an F2 intercross. Using the HS mice we have mapped a QTL influencing a psychological trait in mice to a 0.8-cM interval on chromosome 1. This method allows simultaneous fine mapping of multiple QTLs, as shown by our report of a second QTL on chromosome 12. The high resolution possible with this approach makes QTLs accessible to positional cloning.     

Genome-wide genetic association of complex traits in heterogeneous stock mice

Difficulties in fine-mapping quantitative trait loci (QTLs) are a major impediment to progress in the molecular dissection of complex traits in mice. Here we show that genome-wide high-resolution mapping of multiple phenotypes can be achieved using a stock of genetically heterogeneous mice. We developed a conservative and robust bootstrap analysis to map 843 QTLs with an average 95% confidence interval of 2.8 Mb. The QTLs contribute to variation in 97 traits, including models of human disease (asthma, type 2 diabetes mellitus, obesity and anxiety) as well as immunological, biochemical and hematological phenotypes. The genetic architecture of almost all phenotypes was complex, with many loci each contributing a small proportion to the total variance. Our data set, freely available at http://gscan.well.ox.ac.uk, provides an entry point to the functional characterization of genes involved in many complex traits.     

Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease

Integration of genome-wide expression profiling with linkage analysis is a new approach to identifying genes underlying complex traits. We applied this approach to the regulation of gene expression in the BXH/HXB panel of rat recombinant inbred strains, one of the largest available rodent recombinant inbred panels and a leading resource for genetic analysis of the highly prevalent metabolic syndrome. In two tissues important to the pathogenesis of the metabolic syndrome, we mapped cis- and trans-regulatory control elements for expression of thousands of genes across the genome. Many of the most highly linked expression quantitative trait loci are regulated in cis, are inherited essentially as monogenic traits and are good candidate genes for previously mapped physiological quantitative trait loci in the rat. By comparative mapping we generated a data set of 73 candidate genes for hypertension that merit testing in human populations. Mining of this publicly available data set is expected to lead to new insights into the genes and regulatory pathways underlying the extensive range of metabolic and cardiovascular disease phenotypes that segregate in these recombinant inbred strains.     

A phenylalanine in DGAT is a key determinant of oil content and composition in maize

Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.     

Candidate lung tumor susceptibility genes identified through whole-genome association analyses in inbred mice

We performed a whole-genome association analysis of lung tumor susceptibility using dense SNP maps ( approximately 1 SNP per 20 kb) in inbred mice. We reproduced the pulmonary adenoma susceptibility 1 (Pas1) locus identified in previous linkage studies and further narrowed this quantitative trait locus (QTL) to a region of less than 0.5 Mb in which at least two genes, Kras2 (Kirsten rat sarcoma oncogene 2) and Casc1 (cancer susceptibility candidate 1; also known as Las1), are strong candidates. Casc1 knockout mouse tumor bioassays showed that Casc1-deficient mice were susceptible to chemical induction of lung tumors. We also found three more genetic loci for lung adenoma development. Analysis of one of these candidate loci identified a previously uncharacterized gene Lasc1, bearing a nonsynonymous substitution (D102E). We found that the Lasc1 Glu102 allele preferentially promotes lung tumor cell growth. Our findings demonstrate the prospects for using dense SNP maps in laboratory mice to refine previous QTL regions and identify genetic determinants of complex traits.     

Nuclear genetic control of mitochondrial DNA segregation

Mammalian mitochondrial DNA (mtDNA) is a high copy-number, maternally inherited genome that codes for a small number of essential proteins involved in oxidative phosphorylation. Mutations in mtDNA are responsible for a broad spectrum of clinical disorders. The segregation pattern of pathogenic mtDNA mutants is an important determinant of the nature and severity of mitochondrial disease, but it varies with the specific mutation, cell type and nuclear background and generally does not correlate well with mitochondrial dysfunction. To identify nuclear genes that modify the segregation behavior of mtDNA, we used a heteroplasmic mouse model derived from two inbred strains (BALB/c and NZB; ref. 12), in which we had previously demonstrated tissue-specific and age-dependent directional selection for different mtDNA genotypes in the same mouse. Here we show that this phenotype segregates in F2 mice from a genetic cross (BALB/c x CAST/Ei) and that it maps to at least three quantitative-trait loci (QTLs). Genome-wide scans showed linkage of the trait to loci on Chromosomes 2, 5 and 6, accounting for 16-35% of the variance in the trait, depending on the tissue and age of the mouse. This is the first genetic evidence for nuclear control of mammalian mtDNA segregation.     

Genetic dissection of a behavioral quantitative trait locus shows that Rgs2 modulates anxiety in mice

Here we present a strategy to determine the genetic basis of variance in complex phenotypes that arise from natural, as opposed to induced, genetic variation in mice. We show that a commercially available strain of outbred mice, MF1, can be treated as an ultrafine mosaic of standard inbred strains and accordingly used to dissect a known quantitative trait locus influencing anxiety. We also show that this locus can be subdivided into three regions, one of which contains Rgs2, which encodes a regulator of G protein signaling. We then use quantitative complementation to show that Rgs2 is a quantitative trait gene. This combined genetic and functional approach should be applicable to the analysis of any quantitative trait.     

Dopa decarboxylase (Ddc) affects variation in Drosophila longevity

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.     

Control of rice grain-filling and yield by a gene with a potential signature of domestication

Grain-filling, an important trait that contributes greatly to grain weight, is regulated by quantitative trait loci and is associated with crop domestication syndrome. However, the genes and underlying molecular mechanisms controlling crop grain-filling remain elusive. Here we report the isolation and functional analysis of the rice GIF1 (GRAIN INCOMPLETE FILLING 1) gene that encodes a cell-wall invertase required for carbon partitioning during early grain-filling. The cultivated GIF1 gene shows a restricted expression pattern during grain-filling compared to the wild rice allele, probably a result of accumulated mutations in the gene's regulatory sequence through domestication. Fine mapping with introgression lines revealed that the wild rice GIF1 is responsible for grain weight reduction. Ectopic expression of the cultivated GIF1 gene with the 35S or rice Waxy promoter resulted in smaller grains, whereas overexpression of GIF1 driven by its native promoter increased grain production. These findings, together with the domestication signature that we identified by comparing nucleotide diversity of the GIF1 loci between cultivated and wild rice, strongly suggest that GIF1 is a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement.     

Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome

Meckel-Gruber syndrome (MKS) is a genetically heterogeneous, neonatally lethal malformation and the most common form of syndromic neural tube defect (NTD). To date, several MKS-associated genes have been identified whose protein products affect ciliary function. Here we show that mutations in MKS1, MKS3 and CEP290 (also known as NPHP6) either can cause Bardet-Biedl syndrome (BBS) or may have a potential epistatic effect on mutations in known BBS-associated loci. Five of six families with both MKS1 and BBS mutations manifested seizures, a feature that is not a typical component of either syndrome. Functional studies in zebrafish showed that mks1 is necessary for gastrulation movements and that it interacts genetically with known bbs genes. Similarly, we found two families with missense or splice mutations in MKS3, in one of which the affected individual also bears a homozygous nonsense mutation in CEP290 that is likely to truncate the C terminus of the protein. These data extend the genetic stratification of ciliopathies and suggest that BBS and MKS, although distinct clinically, are allelic forms of the same molecular spectrum.     

Genome-wide association analysis identifies 20 loci that influence adult height

Adult height is a model polygenic trait, but there has been limited success in identifying the genes underlying its normal variation. To identify genetic variants influencing adult human height, we used genome-wide association data from 13,665 individuals and genotyped 39 variants in an additional 16,482 samples. We identified 20 variants associated with adult height (P < 5 x 10(-7), with 10 reaching P < 1 x 10(-10)). Combined, the 20 SNPs explain approximately 3% of height variation, with a approximately 5 cm difference between the 6.2% of people with 17 or fewer 'tall' alleles compared to the 5.5% with 27 or more 'tall' alleles. The loci we identified implicate genes in Hedgehog signaling (IHH, HHIP, PTCH1), extracellular matrix (EFEMP1, ADAMTSL3, ACAN) and cancer (CDK6, HMGA2, DLEU7) pathways, and provide new insights into human growth and developmental processes. Finally, our results provide insights into the genetic architecture of a classic quantitative trait.     

SNP and haplotype mapping for genetic analysis in the rat

The laboratory rat is one of the most extensively studied model organisms. Inbred laboratory rat strains originated from limited Rattus norvegicus founder populations, and the inherited genetic variation provides an excellent resource for the correlation of genotype to phenotype. Here, we report a survey of genetic variation based on almost 3 million newly identified SNPs. We obtained accurate and complete genotypes for a subset of 20,238 SNPs across 167 distinct inbred rat strains, two rat recombinant inbred panels and an F2 intercross. Using 81% of these SNPs, we constructed high-density genetic maps, creating a large dataset of fully characterized SNPs for disease gene mapping. Our data characterize the population structure and illustrate the degree of linkage disequilibrium. We provide a detailed SNP map and demonstrate its utility for mapping of quantitative trait loci. This community resource is openly available and augments the genetic tools for this workhorse of physiological studies.     

The roads from phenotypic variation to gene discovery: mutagenesis versus QTLs

In model organisms, chemical mutagenesis provides a powerful alternative to natural, polygenic variation (for example, quantitative trait loci (QTLs)) for identifying functional pathways and complex disease genes. Despite recent progress in QTLs, we expect that mutagenesis is will ultimately prove more effective because the prospects of gene identification are high and every gene affecting a trait is potentially a target.     

A rice quantitative trait locus for salt tolerance encodes a sodium transporter

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.     

The sex-specific genetic architecture of quantitative traits in humans

Mapping genetically complex traits remains one of the greatest challenges in human genetics today. In particular, gene-environment and gene-gene interactions, genetic heterogeneity and incomplete penetrance make thorough genetic dissection of complex traits difficult, if not impossible. Sex could be considered an environmental factor that can modify both penetrance and expressivity of a wide variety of traits. Sex is easily determined and has measurable effects on recognizable morphology; neurobiological circuits; susceptibility to autoimmune disease, diabetes, asthma, cardiovascular and psychiatric disease; and quantitative traits like blood pressure, obesity and lipid levels, among others. In this study, we evaluated sex-specific heritability and genome-wide linkages for 17 quantitative traits in the Hutterites. The results of this study could have important implications for mapping complex trait genes.     

A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2.

Variation of flowering time is found in the natural populations of many plant species. The underlying genetic variation, mostly of a quantitative nature, is presumed to reflect adaptations to different environments contributing to reproductive success. Analysis of natural variation for flowering time in Arabidopsis thaliana has identified several quantitative trait loci (QTL), which have yet to be characterized at the molecular level. A major environmental factor that determines flowering time is photoperiod or day length, the length of the light period, which changes across the year differently with geographical latitude. We identified the EDI locus as a QTL partly accounting for the difference in flowering response to the photoperiod between two Arabidopsis accessions: the laboratory strain Landsberg erecta (Ler), originating in Northern Europe, and Cvi, collected in the tropical Cape Verde Islands. Positional cloning of the EDI QTL showed it to be a novel allele of CRY2, encoding the blue-light photoreceptor cryptochrome-2 that has previously been shown to promote flowering in long-day (LD) photoperiods. We show that the unique EDI flowering phenotype results from a single amino-acid substitution that reduces the light-induced downregulation of CRY2 in plants grown under short photoperiods, leading to early flowering.