Assessment of linkage disequilibrium in potato genome with single nucleotide polymorphism markers

The extent of linkage disequilibrium (LD) is an important factor in designing association mapping experiments. Unlike other plant species that have been analyzed so far for the extent of LD, cultivated potato (Solanum tuberosum L.), an outcrossing species, is a highly heterozygous autotetraploid. The favored genotypes of modern cultivars are maintained by vegetative propagation through tubers. As a first step in the LD analysis, we surveyed both coding and noncoding regions of 66 DNA fragments from 47 accessions for single nucleotide polymorphism (SNP). In the process, we combined information from the potato SNP database with experimental SNP detection. The total length of all analyzed fragments was >25 kb, and the number of screened sequence bases reached almost 1.4 million. Average nucleotide polymorphism (=11.5x10(-3)) and diversity (pi=14.6x10(-3)) was high compared to the other plant species. The overall Tajima's D value (0.5) was not significant, but indicates a deficit of low-frequency alleles relative to expectation. To eliminate the possibility that an elevated D value occurs due to population subdivision, we assessed the population structure with probabilistic statistics. The analysis did not reveal any significant subdivision, indicating a relatively homogenous population structure. However, the analysis of individual fragments revealed the presence of subgroups in the fragment closely linked to the R1 resistance gene. Data pooled from all fragments show relatively fast decay of LD in the short range (r2=0.208 at 1 kb) but slow decay afterward (r2=0.137 at approximately 70 kb). The estimate from our data indicates that LD in potato declines below 0.10 at a distance of approximately 10 cM. We speculate that two conflicting factors play a vital role in shaping LD in potato: the outcrossing mating type and the very limited number of meiotic generations.     

The effect of single nucleotide polymorphism identification strategies on estimates of linkage disequilibrium

At present there is tremendous interest in characterizing the magnitude and distribution of linkage disequilibrium (LD) throughout the human genome, which will provide the necessary foundation for genome-wide LD analyses and facilitate detailed evolutionary studies. To this end, a human high-density single-nucleotide polymorphism (SNP) marker map has been constructed. Many of the SNPs on this map, however, were identified by sampling a small number of chromosomes from a single population, and inferences drawn from studies using such SNPs may be influenced by ascertainment bias (AB). Through extensive simulations, we have found that AB is a potentially significant problem in estimating and comparing LD within and between populations. Specifically, the magnitude of AB is a function of the SNP discovery strategy, number of chromosomes used for SNP discovery, population genetic characteristics of the particular genomic region considered, amount of gene flow between populations, and demographic history of the populations. We demonstrate that a balanced SNP discovery strategy (where equal numbers of chromosomes are sampled from multiple subpopulations) is the optimal study design for generating broadly applicable SNP resources. Finally, we validate our theoretical predictions by comparing our results to publicly available data from ten genes sequenced in 24 African American and 23 European American individuals.     

Statistical software for gene mapping by admixture linkage disequilibrium

Admixture mapping is a statistical methodology that detects genetic variants in recently admixed populations that are responsible for ethnic differences in disease risk. Three software packages are now available for admixture mapping and we provide a brief overview of the statistical methods and other principal features they implement.     

Optimal designs for linkage disequilibrium mapping and candidate gene association tests in livestock populations

Simulation was used to evaluate the performance of different selective genotyping strategies when using linkage disequilibrium across large half-sib families to position a QTL within a previously defined genomic region. Strategies examined included standard selective genotyping and different approaches of discordant and concordant sib selection applied to arbitrary or selected families. Strategies were compared as a function of effect and frequency of QTL alleles, heritability, and phenotypic expression of the trait. Large half-sib families were simulated for 100 generations and 2% of the population was genotyped in the final generation. Simple ANOVA was applied and the marker with the greatest F-value was considered the most likely QTL position. For traits with continuous phenotypes, genotyping the most divergent pairs of half-sibs from all families was the best strategy in general, but standard selective genotyping was somewhat more precise when heritability was low. When the phenotype was distributed in ordered categories, discordant sib selection was the optimal approach for positioning QTL for traits with high heritability and concordant sib selection was the best approach when genetic effects were small. Genotyping of a few selected sibs from many families was generally more efficient than genotyping many individuals from a few highly selected sires.     

Identification and replication of three novel myopia common susceptibility gene loci on chromosome 3q26 using linkage and linkage disequilibrium mapping

Refractive error is a highly heritable quantitative trait responsible for considerable morbidity. Following an initial genome-wide linkage study using microsatellite markers, we confirmed evidence for linkage to chromosome 3q26 and then conducted fine-scale association mapping using high-resolution linkage disequilibrium unit (LDU) maps. We used a preliminary discovery marker set across the 30-Mb region with an average SNP density of 1 SNP/15 kb (Map 1). Map 1 was divided into 51 LDU windows and additional SNPs were genotyped for six regions (Map 2) that showed preliminary evidence of multi-marker association using composite likelihood. A total of 575 cases and controls selected from the tails of the trait distribution were genotyped for the discovery sample. Malecot model estimates indicate three loci with putative common functional variants centred on MFN1 (180,566 kb; 95% confidence interval 180,505–180, 655 kb), approximately 156 kb upstream from alternate-splicing SOX2OT (182,595 kb; 95% CI 182,533–182,688 kb) and PSARL (184,386 kb; 95% CI 184,356–184,411 kb), with the loci showing modest to strong evidence of association for the Map 2 discovery samples (p<10⁻⁷, p<10⁻¹⁰, and p=0.01, respectively). Using an unselected independent sample of 1,430 individuals, results replicated for the MFN1 (p=0.006), SOX2OT (p=0.0002), and PSARL (p=0.0005) gene regions. MFN1 and PSARL both interact with OPA1 to regulate mitochondrial fusion and the inhibition of mitochondrial-led apoptosis, respectively. That two mitochondrial regulatory processes in the retina are implicated in the aetiology of myopia is surprising and is likely to provide novel insight into the molecular genetic basis of common myopia.     

Use of a dense single nucleotide polymorphism map for in silico mapping in the mouse

Rapid expansion of available data, both phenotypic and genotypic, for multiple strains of mice has enabled the development of new methods to interrogate the mouse genome for functional genetic perturbations. In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for adenylate cyclase 7.     

Evaluation of linkage disequilibrium measures between multi-allelic markers as predictors of linkage disequilibrium between single nucleotide polymorphisms

Effectiveness of marker-assisted selection (MAS) and quantitative trait locus (QTL) mapping using population-wide linkage disequilibrium (LD) between markers and QTLs depends on the extent of LD and how it declines with distance between markers and QTLs in a population. Marker-QTL LD can be predicted from LD between markers. Our previous work evaluated LD measures between multi-allelic markers as predictors of usable LD of multi-allelic markers with QTLs. Since single nucleotide polymorphisms (SNPs) are the current marker of choice for high-density genotyping and LD-mapping of QTLs, the objective of this study was to use LD between multi-allelic markers to predict LD among biallelic SNPs or between SNPs and QTLs. Observable LD between multi-allelic markers was evaluated using nine measures. These included two pooled and standardized measures of LD between pairs of alleles at two markers based on Lewontin's LD measure, two pooled measures of squared correlations between alleles, one standardized measure using Hardy-Weinberg heterozygosities, and four measures based on the chi-square statistic for testing for association between alleles at two loci. The standardized chi-square measure that best predicted usable LD between multi-allelic markers and QTLs, based on our previous work, overestimated usable SNP-SNP or SNP-QTL LD. Instead, three other measures were found to be good predictors of usable SNP-SNP or SNP-QTL LD when LD is generated by drift. Therefore, the LD measure between multi-allelic markers that is best for predicting usable LD in a population depends on the type of markers (i.e. multi-allelic or biallelic) that will eventually be used for QTL mapping or MAS.     

Fine mapping of the congenital chloride diarrhea gene by linkage disequilibrium.

Congenital chloride diarrhea is a recessively inherited intestinal disorder affecting electrolyte transportation. The clinical presentation is a life-threatening watery diarrhea with a high chloride content. Recently, the congenital chloride diarrhea gene (CLD) was assigned to chromosome 7 by linkage in eight Finnish families. In the present study, refined mapping of CLD was performed by studying linkage and linkage disequilibrium in 24 Finnish and 4 Swedish families. Recombination mapping assigned CLD to an approximately 10-cM region flanked by D7S515 and D7S799. Linkage disequilibrium was detected over this large genetic region, with the strongest allelic association at D7S496. Application of the Luria and Delbrück-derived analysis allowed for a further narrowing of the CLD region to approximately 0.37 cM from the marker D7S496. Haplotype analysis placed CLD unequivocally between D7S501 and D7S692, very close to D7S496 and most likely on the distal side of D7S496. This combined analytical approach allowed highly accurate mapping of CLD, each component adding complementary and consistent mapping information.     

Development of a single nucleotide polymorphism map of porcine chromosome 2

Single nucleotide polymorphism markers are developed on SSC2, predominantly on the p-arm. Several studies reported a quantitative trait loci (QTL) for backfat thickness in this region. Single nucleotide polymorphisms were identified by comparative re-sequencing of polymerase chain reaction (PCR) products from a panel of eight individuals. The panel consisted of five Large Whites (each from a different Dutch breeding company), a Meishan, a Pietrain and a Wild Boar. In total, 67 different PCR products were sequenced and 301 SNPs were identified in 32,429 bp of consensus sequence, an average of one SNP in every 108 bp. After correction for sample size, this polymorphism rate corresponds to a heterozygosity value of one SNP in every 357 bp. For 63% of the SNPs, there was variation among the five Large Whites, and these SNPs are relevant for linkage and association studies in commercial populations. Comparing the Whites with other breeds revealed higher variation rates with: (i) Meishan, 89%; (ii) Pietrain, 69%; (iii) Wild Boar, 70%. Because many of the experimental populations to identify QTL are based on crosses between these breeds, these SNPs are relevant for the fine mapping of the QTL identified within these crosses.     

Estimation of linkage disequilibrium and interspecific gene flow in Ficedula flycatchers by a newly developed 50k single‐nucleotide polymorphism array

With the access to draft genome sequence assemblies and whole‐genome resequencing data from population samples, molecular ecology studies will be able to take truly genome‐wide approaches. This now applies to an avian model system in ecological and evolutionary research: Old World flycatchers of the genus Ficedula, for which we recently obtained a 1.1 Gb collared flycatcher genome assembly and identified 13 million single‐nucleotide polymorphism (SNP)s in population resequencing of this species and its sister species, pied flycatcher. Here, we developed a custom 50K Illumina iSelect flycatcher SNP array with markers covering 30 autosomes and the Z chromosome. Using a number of selection criteria for inclusion in the array, both genotyping success rate and polymorphism information content (mean marker heterozygosity = 0.41) were high. We used the array to assess linkage disequilibrium (LD) and hybridization in flycatchers. Linkage disequilibrium declined quickly to the background level at an average distance of 17 kb, but the extent of LD varied markedly within the genome and was more than 10‐fold higher in ‘genomic islands’ of differentiation than in the rest of the genome. Genetic ancestry analysis identified 33 F₁ hybrids but no later‐generation hybrids from sympatric populations of collared flycatchers and pied flycatchers, contradicting earlier reports of backcrosses identified from much fewer number of markers. With an estimated divergence time as recently as <1 Ma, this suggests strong selection against F₁ hybrids and unusually rapid evolution of reproductive incompatibility in an avian system.     

Haplotype frequency distribution and linkage disequilibrium analysis of single nucleotide polymorphisms at the human FMO3 gene locus

We analyzed flavin-containing monooxygenase 3 (FMO3) polymorphisms, haplotype structure, and linkage disequilibrium (LD) in 256 Han Chinese and 50 African-American individuals to compare their haplotype frequencies and LD with other world populations. For the Han Chinese, genotyping of three haplotype tag single nucleotide polymorphisms (E158K, V257M, and E308G) was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For the African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism. Haplotype frequencies, LD, and evolutionary rates were inferred and estimated computationally. There were significant differences in haplotype frequency distribution and LD pattern among Han Chinese, African-Americans, and other world populations. Four major haplotypes of Han Chinese were EVE, KVE, EME, and EVG. Two major haplotypes of African-Americans were EVE and KVE. We found that sites 158 and 257 are in significant LD in both populations. This is the first report comparing FMO haplotypes and LD of Han Chinese with African-Americans. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.     

High nucleotide polymorphism and rapid decay of linkage disequilibrium in wild populations of Caenorhabditis remanei

The common ancestor of the self-fertilizing nematodes Caenorhabditis elegans and C. briggsae must have reproduced by obligate outcrossing, like most species in this genus. However, we have only a limited understanding about how genetic variation is patterned in such male-female (gonochoristic) Caenorhabditis species. Here, we report results from surveying nucleotide variation of six nuclear loci in a broad geographic sample of wild isolates of the gonochoristic C. remanei. We find high levels of diversity in this species, with silent-site diversity averaging 4.7%, implying an effective population size close to 1 million. Additionally, the pattern of polymorphisms reveals little evidence for population structure or deviation from neutral expectations, suggesting that the sampled C. remanei populations approximate panmixis and demographic equilibrium. Combined with the observation that linkage disequilibrium between pairs of polymorphic sites decays rapidly with distance, this suggests that C. remanei will provide an excellent system for identifying the genetic targets of natural selection from deviant patterns of polymorphism and linkage disequilibrium. The patterns revealed in this obligately outcrossing species may provide a useful model of the evolutionary circumstances in C. elegans' gonochoristic progenitor. This will be especially important if self-fertilization evolved recently in C. elegans history, because most of the evolutionary time separating C. elegans from its known relatives would have occurred in a state of obligate outcrossing.     

A high-resolution single nucleotide polymorphism genetic map of the mouse genome

High-resolution genetic maps are required for mapping complex traits and for the study of recombination. We report the highest density genetic map yet created for any organism, except humans. Using more than 10,000 single nucleotide polymorphisms evenly spaced across the mouse genome, we have constructed genetic maps for both outbred and inbred mice, and separately for males and females. Recombination rates are highly correlated in outbred and inbred mice, but show relatively low correlation between males and females. Differences between male and female recombination maps and the sequence features associated with recombination are strikingly similar to those observed in humans. Genetic maps are available from http://gscan.well.ox.ac.uk/#genetic_map and as supporting information to this publication.     

Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an F1 (p50T x C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.     

Improved power by use of a weighted score test for linkage disequilibrium mapping

Association studies offer an exciting approach to finding underlying genetic variants of complex human diseases. However, identification of genetic variants still includes difficult challenges, and it is important to develop powerful new statistical methods. Currently, association methods may depend on single-locus analysis--that is, analysis of the association of one locus, which is typically a single-nucleotide polymorphism (SNP), at a time--or on multilocus analysis, in which multiple SNPs are used to allow extraction of maximum information about linkage disequilibrium (LD). It has been shown that single-locus analysis may have low power because a single SNP often has limited LD information. Multilocus analysis, which is more informative, can be performed on the basis of either haplotypes or genotypes. It may lose power because of the often large number of degrees of freedom involved. The ideal method must make full use of important information from multiple loci but avoid increasing the degrees of freedom. Therefore, we propose a method to capture information from multiple SNPs but with the use of fewer degrees of freedom. When a set of SNPs in a block are correlated because of LD, we might expect that the genotype variation among the different phenotypic groups would extend across all the SNPs, and this information could be compressed into the low-frequency components of a Fourier transform. Therefore, we develop a test based on weighted Fourier transformation coefficients, with more weight given to the low-frequency components. Our simulation results demonstrate the validity and substantially higher power of the proposed method compared with other common methods. This method provides an additional tool to existing methods for identification of causative genetic variants underlying complex diseases.     

A localized linkage map of the citrus tristeza virus resistance gene region

A localized genetic linkage map was developed of the region surrounding the citrus tristeza virus (CTV) resistance gene (designated Ctv) from Poncirus trifoliate L., a sexually compatible Citrus relative. Bulked segregant analysis (BSA) was used to identify potential resistance-associated RAPD fragment markers in four intergeneric backcross families that were segregating for CTV resistance. Eight RAPD fragments were found that were consistently linked to Ctv in the four families. Map distances and locus order were determined with MAPMAKER 3.0, using the results obtained from 59 individuals in the largest family. Also, a consensus map was constructed with JOINMAP 1.3, using pooled results from the four backcross families. Marker orders were identical, except for 1 marker, on these independently developed maps. Family-specific resistance-associated markers were also identified, as were numerous susceptibility-associated markers. The identification of markers tightly linked to Ctv will enable citrus breeders to identify plants likely to be CTV-resistant by indirect, marker-assisted selection, rather than by labor-intensive direct challenge with the pathogen. These markers also provide a basis for future efforts to isolate Ctv for subsequent genetic manipulation.Florida Agricultural Experimental Station Journal Series No. R-04491     

Fine mapping of a quantitative trait locus for twinning rate using combined linkage and linkage disequilibrium mapping

A novel and robust method for the fine-scale mapping of genes affecting complex traits, which combines linkage and linkage-disequilibrium information, is proposed. Linkage information refers to recombinations within the marker-genotyped generations and linkage disequilibrium to historical recombinations before genotyping started. The identity-by-descent (IBD) probabilities at the quantitative trait locus (QTL) between first generation haplotypes were obtained from the similarity of the marker alleles surrounding the QTL, whereas IBD probabilities at the QTL between later generation haplotypes were obtained by using the markers to trace the inheritance of the QTL. The variance explained by the QTL is estimated by residual maximum likelihood using the correlation structure defined by the IBD probabilities. Unlinked background genes were accounted for by fitting a polygenic variance component. The method was used to fine map a QTL for twinning rate in cattle, previously mapped on chromosome 5 by linkage analysis. The data consisted of large half-sib families, but the method could also handle more complex pedigrees. The likelihood of the putative QTL was very small along most of the chromosome, except for a sharp likelihood peak in the ninth marker bracket, which positioned the QTL within a region <1 cM in the middle part of bovine chromosome 5. The method was expected to be robust against multiple genes affecting the trait, multiple mutations at the QTL, and relatively low marker density.