Patterns of linkage disequilibrium in mitochondrial DNA of 16 ruminant populations

Mitochondrial DNA (mtDNA) is a widely employed molecular tool in phylogeography, in the inference of human evolutionary history, in dating the domestication of livestock and in forensic science. In humans and other vertebrates the popularity of mtDNA can be partially attributed to an assumption of strict maternal inheritance, such that there is no recombination between mitochondrial lineages. The recent demonstration that linkage disequilibrium (LD) declines as a function of distance between polymorphic sites in hominid mitochondrial genomes has been interpreted as evidence of recombination between mtDNA haplotypes, and hence nonclonal inheritance. However, critics of mtDNA recombination have suggested that this association is an artefact of an inappropriate measure of LD or of sequencing error, and subsequent studies of other populations have failed to replicate the initial finding. Here we report the analysis of 16 ruminant populations and present evidence that LD significantly declines with distance in five of them. A meta-analysis of the data indicates a nonsignificant trend of LD declining with distance. Most of the earlier criticisms of patterns between LD and distance in hominid mtDNA are not applicable to this data set. Our results suggest that either ruminant mtDNA is not strictly clonal or that compensatory selection has influenced patterns of variation at closely linked sites within the mitochondrial control region. The potential impact of these processes should be considered when using mtDNA as a tool in vertebrate population genetic, phylogenetic and forensic studies.     

The distribution of long range admixture linkage disequilibrium in an African-American population

OBJECTIVES: To better understand the effect of admixture on long range linkage disequilibrium (LD), we characterized extended LD in gene-rich regions of an African-American population. METHODS: Approximately 290 cM of chromosomes 1, 3, 6, 11-17, 20 and 22 were scanned using 109 polymorphic microsatellite markers spaced an average of 3 cM apart. Disequilibrium between loci (D') was based on maximum-likelihood estimates of haplotype frequencies computed for 200 unrelated African Americans. RESULTS: Mean D' values were highest on chromosomes 6p23-p21.3 (D' = 0.33) and 15p22.2-p25.3 (D' = 0.34), and lowest on chromosome 12p11.2-q14 (D' = 0.21). Overall, the variance in LD among chromosomes accounted for approximately two-thirds of the total LD variance. Of the 434 locus pairs spaced between 0.3 and 38.7 cM apart, there was no detectable correlation between LD and recombination distance and a weak negative correlation between LD and physical distance (r(s) = -0.12; p = 0.031). For the 192 intrachromosomal locus pairs where allele frequency data were available from the Centre d'Etude du Polymorphisme humain (CEPH), we found a statistically significant positive correlation between LD and the allelic frequency differences (delta) between the African-American study population and Caucasian reference CEPH population (r(s) = 0.53; p < 0.0001). The correlation between LD and both recombination and physical distance was markedly increased for locus pairs with high delta levels. CONCLUSIONS: Our results suggest that recent Caucasian admixture maintains a high level of long range LD in African Americans on a genomic scale, and selected markers with large African American/Caucasian delta levels may be useful in association studies.     

A fine-scale linkage-disequilibrium measure based on length of haplotype sharing

High-throughput genotyping technologies for SNPs have enabled the recent completion of the International HapMap Project (phase I), which has stimulated much interest in studying genomewide linkage-disequilibrium (LD) patterns. Conventional LD measures, such as D' and r(2), are two-point measurements, and their relationship with physical distance is highly noisy. We propose a new LD measure, Delta , defined in terms of the correlation coefficient for shared haplotype lengths around two loci, thereby borrowing information from multiple loci. A U-statistic-based estimator of Delta , which takes into consideration the dependence structure of the observed data, is developed and compared with an estimator based on the usual empirical correlation coefficient. Furthermore, we propose methods for inferring LD-decay rates and recombination hotspots on the basis of Delta . The results from coalescent-simulation studies and analysis of HapMap SNP data demonstrate that the proposed estimators of Delta are superior to the two most popular conventional LD measures, in terms of their close relationship with physical distance and recombination rate, their small variability, and their strong robustness to marker-allele frequencies. These merits may offer new opportunities for mapping complex disease genes and for investigating recombination mechanisms on the basis of better-quantified LD.     

A comparison of linkage disequilibrium patterns and estimated population recombination rates across multiple populations

Large-scale studies of linkage disequilibrium (LD) have shown considerable variation in the extent and distribution of pairwise LD within and between populations. Taken at face value, these results suggest that genomewide LD maps for one population may not be generalizable to other populations. However, at least part of this diversity is due to some undesirable features of pairwise LD measures, which are well documented for the D' and r2 measures. In this report, we compare patterns of LD derived from pairwise measures with statistical estimates of population recombination rates ( rho ) along a 10-Mb stretch of chromosome 20 in four population samples, comprising East Asians, African Americans, and U.K. and U.S. individuals of western European descent. The results reveal the expected variability of D' within and between populations but show better concordance in estimates of r2 for the same markers across the population samples. Estimates of rho correlate well across populations, but there is still evidence of population-specific spikes and troughs in rho values. We conclude that it is unlikely that a single haplotype map will provide a definitive guide for association studies of many populations; rather, multiple maps will need to be constructed to provide the best-possible guides for gene mapping.     

Recombination or mutational hot spots in human mtDNA?

Awadalla, Eyre-Walker, and Maynard Smith (1999) recently argued that there might be recombination in human mitochondrial DNA (mtDNA). Their claim was based on their observation of decaying linkage disequilibrium (LD) as a function of physical distance. Their study was much criticized, and follow-up studies have failed to find any evidence for recombination. We argue that the criticisms levied, even if correct, could not possibly explain the findings of Awadalla, Eyre-Walker, and Maynard Smith (1999). Nonetheless, the test proposed by Awadalla, Eyre-Walker, and Maynard Smith (1999 ) is not robust because recombination is not the only explanation for decay of LD. We show that such a pattern can be caused by mutational hot spots as well. However, a closer look at the data suggests that the pattern observed was not caused by mutational hot spots but rather by chance. Thus, there appears to be no evidence for recombination in the mtDNA polymorphism data. In conclusion, we discuss the possibility of detecting recombination in mtDNA and the implications of its existence.     

The posterior probability of linkage allowing for linkage disequilibrium and a new estimate of disequilibrium between a trait and a marker

The posterior probability of linkage (PPL) statistic has been developed as a method for the rigorous accumulation of evidence for or against linkage allowing for both intra- and inter-sample heterogeneity. To date, the method has assumed linkage equilibrium between alleles at the trait locus and the marker locus. We now generalize the PPL to allow for linkage disequilibrium (LD), by incorporating variable phase probabilities into the underlying linkage likelihood. This enables us to recover the marginal posterior density of the recombination fraction, integrating out nuisance parameters of the trait model, including the locus heterogeneity (admixture) parameter, as well as a vector of LD parameters. The marginal posterior density can then be updated across data subsets or new data as they become available, while allowing parameters of the trait model to vary between data sets. The method applies immediately to general pedigree structures and to markers with multiple alleles. In the case of SNPs, the likelihood is parameterized in terms of the standard single LD parameter D'; and it therefore affords a mechanism for estimation of D' between the marker and the trait, again, without fixing the parameters of the trait model and allowing for updating across data sets. It is even possible to allow for a different associated allele in different populations, while accumulating information regarding the strength of LD. While a computationally efficient implementation for multi-allelic markers is still in progress, we have implemented a version of this new LD-PPL for SNPs and evaluated its performance in nuclear families. Our simulations show that LD-PPLs tend to be larger than PPLs (stronger evidence in favor of linkage/LD) with increased LD level, under a variety of generating models; while in the absence of linkage and LD, LD-PPLs tend to be smaller than PPLs (stronger evidence against linkage). The estimate of D' also behaves well even in relatively small, heterogeneous samples.     

Genome-wide linkage disequilibrium in two Japanese beef cattle breeds

There is little knowledge about the degree of linkage disequilibrium (LD) in beef cattle. This study aims to perform a genome-wide search for LD in Japanese Black and Japanese Brown beef cattle and to compare the level of LD between these two breeds. Parameter D' (the LD coefficient) was used as a measure of LD, and LD was tested for significance of allelic associations between syntenic and between non-syntenic marker pairs. Effects of breed, chromosome, genetic map distance and their interactions with D' were tested based on least squares analyses. Both breeds showed high levels of LD, which ranged over several tens of cM and declined as the marker distance increased for syntenic marker pairs. A rapid decline of the D' value was observed between markers that were spaced 5 and 20 cM apart. LD was significant in most cases for marker pairs <40 cM apart but was not significant between non-syntenic loci. The pattern of LD found in these two breeds was similar to that previously published for dairy cattle. The D' value between breeds was not significantly different (P > 0.05), but the interaction between breed and chromosome was highly significant (P < 0.001). Genetic selection seems to have caused the heterogeneity of the D' values among chromosomes within breed. These results indicate that LD mapping is a useful tool for fine-mapping quantitative trait loci of economically important traits in Japanese beef cattle.     

Entropy as a measure for linkage disequilibrium over multilocus haplotype blocks

OBJECTIVE: The presence of linkage disequilibrium (LD) forms the basis for a range of uses, including the fine-mapping of diseases and studies on human genealogy. Recent findings indicate that single nucleotide polymorphisms (SNP) can occur in blocks of limited haplotypic diversity with high degrees of LD. Commonly used measures for LD, such as r(2) and D', consider only two loci and might miss information to appropriately describe LD in larger haplotypic structures. METHODS: We introduce the Normalized Entropy Difference, epsilon, as a new multilocus measure for LD. A related quantity, deltaS, provides an approximate chi(2) test for the significance of LD. The ability of the measure to detect haplotype blocks is investigated using simulated data sets as well as a real data set previously analyzed by Daly et al. (2001). RESULTS: epsilon allows for arbitrary numbers of loci, describes LD with regard to the loci sequence, and can be interpreted as a multilocus extension of r(2). The application of epsilon to the data sets demonstrated the measure's ability to appropriately describe simultaneous multilocus LD and to detect haplotype blocks. CONCLUSIONS: epsilon is a reasonable multilocus LD measure and might be of potential use in the construction of the human haplotype map.     

Linkage disequilibrium for different scales and applications

Assessing the patterns of linkage disequilibrium (LD) has become an important issue in both evolutionary biology and medical genetics since the rapid accumulation of densely spaced DNA sequence variation data in several organisms. LD deals with the correlation of genetic variation at two or more loci or sites in the genome within a given population. There are a variety of LD measures which range from traditional pairwise LD measures such as D' or r2 to entropy-based multi-locus measures or haplotype-specific approaches. Understanding the evolutionary forces (in particular recombination) that generate the observed variation of LD patterns across genomic regions is addressed by model-based LD analysis. Marker type and its allelic composition also influence the observed LD pattern, microsatellites having a greater power to detect LD in population isolates than SNPs. This review aims to explain basic LD measures and their application properties.     

The effect that genotyping errors have on the robustness of common linkage-disequilibrium measures

The rapid development of a dense single-nucleotide-polymorphism marker map has stimulated numerous studies attempting to characterize the magnitude and distribution of background linkage disequilibrium (LD) within and between human populations. Although genotyping errors are an inherent problem in all LD studies, there have been few systematic investigations documenting their consequences on estimates of background LD. Therefore, we derived simple deterministic formulas to investigate the effect that genotyping errors have on four commonly used LD measures-D', r, Q, and d-in studies of background LD. We have found that genotyping error rates as small as 3% can have serious affects on these LD measures, depending on the allele frequencies and the assumed error model. Furthermore, we compared the robustness of D', r, Q, and d, in the presence of genotyping errors. In general, Q and d are more robust than D' and r, although exceptions do exist. Finally, through stochastic simulations, we illustrate how genotyping errors can lead to erroneous inferences when measures of LD between two samples are compared.     

Characterizing linkage disequilibrium in pig populations

Knowledge of the extent and range of linkage disequilibrium (LD), defined as non-random association of alleles at two or more loci, in animal populations is extremely valuable in localizing genes affecting quantitative traits, identifying chromosomal regions under selection, studying population history, and characterizing/managing genetic resources and diversity. Two commonly used LD measures, r(2) and D', and their permutation based adjustments, were evaluated using genotypes of more than 6,000 pigs from six commercial lines (two terminal sire lines and four maternal lines) at ~4,500 autosomal SNPs (single nucleotide polymorphisms). The results indicated that permutation only partially removed the dependency of D' on allele frequency and that r(2) is a considerably more robust LD measure. The maximum r(2) was derived as a function of allele frequency. Using the same genotype dataset, the extent of LD in these pig populations was estimated for all possible syntenic SNP pairs using r(2) and the ratio of r(2) over its theoretical maximum. As expected, the extent of LD highest for SNP pairs was found in tightest linkage and decreased as their map distance increased. The level of LD found in these pig populations appears to be lower than previously implied in several other studies using microsatellite genotype data. For all pairs of SNPs approximately 3 centiMorgan (cM) apart, the average r(2) was equal to 0.1. Based on the average population-wise LD found in these six commercial pig lines, we recommend a spacing of 0.1 to 1 cM for a whole genome association study in pig populations.     

Linkage disequilibrium in the domesticated pig

This study investigated the extent of linkage disequilibrium (LD) in two genomic regions (on chromosomes 4 and 7) in five populations of domesticated pigs. LD was measured with D' and tested for significance with the Fisher exact test. Effects of genetic (linkage) distance, chromosome, population, and their interactions on D' were tested both through a linear model analysis of covariance and by a theoretical nonlinear model. The overall result was that (1) the distance explained most of the variability of D', (2) the effect of chromosome was significant, and (3) the effect of population was significant. The significance of the chromosome effect may have resulted from selection and the significance of the population effect illustrates the effects of population structures and effective population sizes on LD. These results suggest that mapping methods based on LD may be valuable even with only moderately dense marker spacing in pigs.     

Linkage disequilibrium in domestic sheep

The last decade has seen a dramatic increase in the number of livestock QTL mapping studies. The next challenge awaiting livestock geneticists is to determine the actual genes responsible for variation of economically important traits. With the advent of high density single nucleotide polymorphism (SNP) maps, it may be possible to fine map genes by exploiting linkage disequilibrium between genes of interest and adjacent markers. However, the extent of linkage disequilibrium (LD) is generally unknown for livestock populations. In this article microsatellite genotype data are used to assess the extent of LD in two populations of domestic sheep. High levels of LD were found to extend for tens of centimorgans and declined as a function of marker distance. However, LD was also frequently observed between unlinked markers. The prospects for LD mapping in livestock appear encouraging provided that type I error can be minimized. Properties of the multiallelic LD coefficient D' were also explored. D' was found to be significantly related to marker heterozygosity, although the relationship did not appear to unduly influence the overall conclusions. Of potentially greater concern was the observation that D' may be skewed when rare alleles are present. It is recommended that the statistical significance of LD is used in conjunction with coefficients such as D' to determine the true extent of LD.     

"Patchy-tachy" leads to false positives for recombination

Indirect tests have detected recombination in mitochondrial DNA (mtDNA) from many animal lineages, including mammals. However, it is possible that features of the molecular evolutionary process without recombination could be incorrectly inferred by indirect tests as being due to recombination. We have identified one such example, which we call "patchy-tachy" (PT), where different partitions of sequences evolve at different rates, that leads to an excess of false positives for recombination inferred by indirect tests. To explore this phenomena, we characterized the false positive rates of six widely used indirect tests for recombination using simulations of general models for mtDNA evolution with PT but without recombination. All tests produced 30-99% false positives for recombination, although the conditions that produced the maximal level of false positives differed between the tests. To evaluate the degree to which conditions that exacerbate false positives are found in published sequence data, we turned to 20 animal mtDNA data sets in which recombination is suggested by indirect tests. Using a model where different regions of the sequences were free to evolve at different rates in different lineages, we demonstrated that PT is prevalent in many data sets in which recombination was previously inferred using indirect tests. Taken together, our results argue that PT without recombination is a viable alternative explanation for detection of widespread recombination in animal mtDNA using indirect tests.     

Linkage disequilibrium between STRPs and SNPs across the human genome

Patterns of linkage disequilibrium (LD) reveal the action of evolutionary processes and provide crucial information for association mapping of disease genes. Although recent studies have described the landscape of LD among single nucleotide polymorphisms (SNPs) from across the human genome, associations involving other classes of molecular variation remain poorly understood. In addition to recombination and population history, mutation rate and process are expected to shape LD. To test this idea, we measured associations between short-tandem-repeat polymorphisms (STRPs), which can mutate rapidly and recurrently, and SNPs in 721 regions across the human genome. We directly compared STRP-SNP LD with SNP-SNP LD from the same genomic regions in the human HapMap populations. The intensity of STRP-SNP LD, measured by the average of D', was reduced, consistent with the action of recurrent mutation. Nevertheless, a higher fraction of STRP-SNP pairs than SNP-SNP pairs showed significant LD, on both short (up to 50 kb) and long (cM) scales. These results reveal the substantial effects of mutational processes on LD at STRPs and provide important measures of the potential of STRPs for association mapping of disease genes.     

A simple and robust statistical test for detecting the presence of recombination

Recombination is a powerful evolutionary force that merges historically distinct genotypes. But the extent of recombination within many organisms is unknown, and even determining its presence within a set of homologous sequences is a difficult question. Here we develop a new statistic, phi(w), that can be used to test for recombination. We show through simulation that our test can discriminate effectively between the presence and absence of recombination, even in diverse situations such as exponential growth (star-like topologies) and patterns of substitution rate correlation. A number of other tests, Max chi2, NSS, a coalescent-based likelihood permutation test (from LDHat), and correlation of linkage disequilibrium (both r2 and /D'/) with distance, all tend to underestimate the presence of recombination under strong population growth. Moreover, both Max chi2 and NSS falsely infer the presence of recombination under a simple model of mutation rate correlation. Results on empirical data show that our test can be used to detect recombination between closely as well as distantly related samples, regardless of the suspected rate of recombination. The results suggest that phi(w) is one of the best approaches to distinguish recurrent mutation from recombination in a wide variety of circumstances.     

Nucleotide Diversity and Haplotype Structure of the Human Angiotensinogen Gene in Two Populations

Variation in the angiotensinogen gene, AGT, has been associated with variation in plasma angiotensinogen levels. In addition, the T235M polymorphism in the AGT product is associated with an increased risk of essential hypertension in multiple populations, making AGT a good example of a quantitative-trait locus underlying susceptibility to a common disease. To better understand genetic variation in AGT, we sequenced a 14.4-kb genomic region spanning the entire AGT and identified 44 single-nucleotide polymorphisms (SNPs). Forty-two SNPs were observed both in 88 white and in 77 Japanese unselected subjects. Six major haplotypes accounted for most of the variation in this region, indicating less allelic complexity than in many other genomic regions. Although the two populations were found to share all of the major AGT haplotypes, there were substantial differences in haplotype frequencies. Pairwise linkage disequilibrium (LD), measured by the D', r(2), and d(2) statistics, demonstrated a general pattern of decline with increasing distance, but, as expected in a small genomic region, individual LD values were highly variable. LD between T235M and each of the other 39 SNPs was assessed in order to model the usefulness of LD to detect a disease-associated mutation. Among the Japanese subjects, 13 (33%) of the SNPs had r(2) values >0.1, whereas this statistic was substantially higher for the white subjects (occurring in 35/39 [90%]). LD between a hypertension-associated promoter mutation, A-6G, and 39 SNPs was also measured. Similar results were obtained, with 33% of the SNPs showing r(2)>0.1 in the Japanese subjects and 92% of the SNPs showing r(2)>0.1 in the white subjects. This difference, which occurs despite an overall similarity in LD patterns in the two populations, reflects a much higher frequency of the M235-associated haplotype in the white sample. These results have important implications for the usefulness of LD approaches in the mapping of genes underlying susceptibility to complex diseases.     

An isochore transition in the NF1 gene region coincides with a switch in the extent of linkage disequilibrium

Whole-genome association studies will be a powerful tool to identify genes responsible for common human diseases. A crucial task for association-mapping studies is the evaluation of the relationship between linkage disequilibrium (LD) and physical distance for the genomic region under study. Since it is known that the extent of LD is nonuniformly distributed throughout the human genome, the required marker density has to be determined specifically for the region under study. These regions may be related to isochores and chromosomal bands, as indicated by earlier cytogenetic findings concerning chiasma distribution in meiosis. Therefore we analyzed the neurofibromatosis type 1 (NF1) gene region on chromosome 17q11.2, which is characterized by a nonuniform LD pattern and an L1-to-H2 isochore transition. Long-range LD within the NF1 gene was found to extend over 200 kb (D' = 0.937) in the L1 isochore, whereas, in the neighboring H2 isochore, no LD is apparent between markers spaced by 26 kb (D' = 0.144). Recombination frequencies derived from the LD are at.00019 (high LD) and.01659 (low LD) per megabase, the latter identical to the average value from segregation analysis. The boundary between these regions coincides precisely with a transition in the GC content of the sequences, with low values (37.2%) in the region with long-range LD and high values (51%) in the other. Our results suggest a correlation between the LD pattern and the isochores, at least in the NF1 region. If this correlation can be generalized, the marker densities required for association studies have to be adjusted to the regional GC content and may be chosen according to the isochores.     

Bayesian Inference of Shared Recombination Hotspots Between Humans and Chimpanzees

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies.     

A broad survey of recombination in animal mitochondria

Recombination in mitochondrial DNA (mtDNA) remains a controversial topic. Here we present a survey of 279 animal mtDNA data sets, of which 12 were from asexual species. Using four separate tests, we show that there is widespread evidence of recombination; for one test as many as 14.2% of the data sets reject a model of clonal inheritance and in several data sets, including primates, the recombinants can be identified visually. We show that none of the tests give significant results for obligate clonal species (apomictic pathogens) and that the sexual species show significantly greater evidence of recombination than asexual species. For some data sets, such as Macaca nemestrina, additional data sets suggest that the recombinants are not artifacts. For others, it cannot be determined whether the recombinants are real or produced by laboratory error. Either way, the results have important implications for how mtDNA is sequenced and used.