Ignoring linkage disequilibrium among tightly linked markers induces false-positive evidence of linkage for affected sib pair analysis

Most multipoint linkage programs assume linkage equilibrium among the markers being studied. The assumption is appropriate for the study of sparsely spaced markers with intermarker distances exceeding a few centimorgans, because linkage equilibrium is expected over these intervals for almost all populations. However, with recent advancements in high-throughput genotyping technology, much denser markers are available, and linkage disequilibrium (LD) may exist among the markers. Applying linkage analyses that assume linkage equilibrium to dense markers may lead to bias. Here, we demonstrated that, when some or all of the parental genotypes are missing, assuming linkage equilibrium among tightly linked markers where strong LD exists can cause apparent oversharing of multipoint identity by descent (IBD) between sib pairs and false-positive evidence for multipoint model-free linkage analysis of affected sib pair data. LD can also mimic linkage between a disease locus and multiple tightly linked markers, thus causing false-positive evidence of linkage using parametric models, particularly when heterogeneity LOD score approaches are applied. Bias can be eliminated by inclusion of parental genotype data and can be reduced when additional unaffected siblings are included in the analysis.     

Lower-than-expected linkage disequilibrium between tightly linked markers in humans suggests a role for gene conversion

Understanding the pattern of linkage disequilibrium (LD) in the human genome is important both for successful implementation of disease-gene mapping approaches and for inferences about human demographic histories. Previous studies have examined LD between loci within single genes or confined genomic regions, which may not be representative of the genome; between loci separated by large distances, where little LD is seen; or in population groups that differ from one study to the next. We measured LD in a large set of locus pairs distributed throughout the genome, with loci within each pair separated by short distances (average 124 bp). Given current models of the history of the human population, nearly all pairs of loci at such short distances would be expected to show complete LD as a consequence of lack of recombination in the short interval. Contrary to this expectation, a significant fraction of pairs showed incomplete LD. A standard model of recombination applied to these data leads to an estimate of effective human population size of 110,000. This estimate is an order of magnitude higher than most estimates based on nucleotide diversity. The most likely explanation of this discrepancy is that gene conversion increases the apparent rate of recombination between nearby loci.     

Nonparametric disequilibrium mapping of functional sites using haplotypes of multiple tightly linked single-nucleotide polymorphism markers

As the speed and efficiency of genotyping single-nucleotide polymorphisms (SNPs) increase, using the SNP map, it becomes possible to evaluate the extent to which a common haplotype contributes to the risk of disease. In this study we propose a new procedure for mapping functional sites or regions of a candidate gene of interest using multiple linked SNPs. Based on a case-parent trio family design, we use expectation-maximization (EM) algorithm-derived haplotype frequency estimates of multiple tightly linked SNPs from both unambiguous and ambiguous families to construct a contingency statistic S for linkage disequilibrium (LD) analysis. In the procedure, a moving-window scan for functional SNP sites or regions can cover an unlimited number of loci except for the limitation of computer storage. Within a window, all possible widths of haplotypes are utilized to find the maximum statistic S* for each site (or locus). Furthermore, this method can be applied to regional or genome-wide scanning for determining linkage disequilibrium using SNPs. The sensitivity of the proposed procedure was examined on the simulated data set from the Genetic Analysis Workshop (GAW) 12. Compared with the conventional and generalized TDT methods, our procedure is more flexible and powerful.     

Aflp markers tightly linked to the sex locus in Asparagus officinalis L.

Nine AFLP markers linked to the sex locus in Asparagus officinalis L. have been identified by non-radioactive AFLP technique and bulked segregant analysis. A composite map of one F₂ and two F₁ populations identified three very tightly linked markers. These markers did not give recombinants in the three different populations and mapped 0.5, 0.7 and 1 cM to the sex locus. Codominant scoring of the markers in the F₂ population from a selfed andromonoecious plant could distinguish the XX, XY and YY asparagus plants. The AFLP markers were isolated from the gel and cloned into plasmid vectors. The marker E41M50, which is a low-copy sequence and did not give any recombinants in the screened populations, detected polymorphism between female and male plants when used as RFLP probe. The AFLP markers we obtained are important to plant breeding, particularly in the development of sex specific PCR primers that could be used in the screening of different asparagus plants at the seedling stage. They are likewise important in the elucidation of the mechanisms underlying sex determination and differentiation in this species.     

Diagnosis of neurofibromatosis I by using tightly linked, flanking DNA markers.

We tested 132 individuals from 21 families segregating an allele for neurofibromatosis type 1 (NF-1), by using nine RFLPs tightly linked to the NF-1 locus. Family members had requested DNA testing either to determine whether "at risk" children were carrying the NF-1 allele or to determine whether their respective families would be informative for prenatal testing. Predictions about whether a child carries the NF-1 mutation were possible for all 32 at-risk offspring (greater than 98% accuracy based on the recombination estimates currently available for these DNA markers). At least one informative probe was available for all 23 matings in these 21 families; flanking markers were informative for 10 matings. Pairwise analysis showed that several of the polymorphisms were in tight linkage disequilibrium; few recombination events were observed with these markers in the families under study. We conclude that the DNA probes used in this study perform well for diagnostic testing of NF-1 in familial cases. A subset of five probe-enzyme systems (pHHH202/RsaI, p11-3C4.2/MspI, pTH17.19/Bg/II, p11-2C11.7/BamHI, and p11-2F9.8/TaqI) provide reliable linkage information for both clinical testing and prenatal diagnosis.     

Sequence characterized amplified region markers tightly linked to the mating factors of Lentinula edodes.

Detecting the mating types in shiitake, Lentinula edodes (Berk.) Pegler, is important for making progress in the breeding of this mushroom and determining the compatibility of the pair to cross. Shiitake is a tetrapolar fungus with two unlinking mating factors, A factor and B factor. We screened molecular markers linked to the mating factors using the randomly amplified polymorphic DNA (RAPD) method to develop the mating type identification procedure. Using 147 oligonucleotide primers, a total of 6 linkage markers for the shiitake mating factors, 4 markers for the A factor and 2 markers for the B factor, were discovered with a logarithm of the odds threshold of 3.0 for linkage. Two RAPDs that perfectly segregated with each mating factor among 72 basidiospore strains were detected. Both of these RAPDs were cloned and sequenced to convert them to the sequence characterized amplified region (SCAR) markers. Four primers, two sets of primers, were designed according to the internal sequences of two RAPDs tightly linking to the A factor or B factor. Consequently, we determined the polymerase chain reaction condition for multiplex analyses of these SCAR markers.     

Identification of co-dominant RAPD markers tightly linked to fruit skin color in apple

A simple genetic basis for the red/yellow skincolor polymorphism in apple was verified using DNA markers. Bulked segregant analysis identified one 10-base oligomer that generated different fragments in each of the bulks. After testing the primer in four populations, two fragments were found to be associated with red skin color and another two fragments associated with yellow skin color. Three of the fragments (1160, 1180, and 1230 bp) were partly sequenced and found to share high sequence homology, suggesting these were generated from the same locus. A pair of universal primers were designed to amplify the fragments. In the Rome Beauty x White Angel population, two fragments were associated with red skin color; one fragment designated as A¹ (1160 bp) was from Rome Beauty and another fragment (A², 1180 bp) was from White Angel. Progeny possessing both fragments, or either one, had red fruit. Both parents displayed an alternate fragment, a¹ (1230 bp), associated with yellowskinned fruit. In three other crosses tested, only fragment A¹ co-segregated with red skin color; two fragments, a¹ and a² (1230 bp and 1320 bp), were associated with yellow skin color. Our results are consistent with the hypothesis that the red/yellow dimorphism is controlled by a monogenic system with the presence of the red anthocyanin pigmentation being dominant. There was no indication that other modifier genes could reverse the effect of the locus (R _f ) linked to the markers. Examination of amplification products in 56 apple cultivars and advanced breeding selections demonstrated that the universal primers could be used to correctly predict fruit skin color in most cases.     

The Friedreich ataxia gene is assigned to chromosome 9q13-q21 by mapping of tightly linked markers and shows linkage disequilibrium with D9S15.

Chamberlain et al. have assigned the gene for Friedreich ataxia (FA), a recessive neurodegenerative disorder, to chromosome 9, and have proposed a regional localization in the proximal short arm (9p22-cen), on the basis of linkage to D9S15 and to interferon-beta (IFNB), the latter being localized in 9p22. We confirmed more recently the close linkage to D9S15 in another set of families but found much looser linkage to IFNB. We also reported another closely linked marker, D9S5. Additional families have now been studied, and our updated lod scores are z = 14.30 at theta = .00 for D9S15-FA linkage and z = 6.30 at theta = .00 for D9S5-FA linkage. Together with the recent data of Chamberlain et al., this shows that D9S15 is very likely within 1 cM of the FA locus. We have found very significant linkage disequilibrium (delta Std = .28, chi 2 = 9.71, P less than .01) between FA and the D9S15 MspI RFLP in French families, which further supports the very close proximity of these two loci. No recombination between D9S5 and D9S15 was found in the FA families or Centre d'Etude du Polymorphisme Humain families (z = 9.30 at theta = .00). Thus D9S5, D9S15, and FA define a cluster of tightly linked loci. We have mapped D9S5 by in situ hybridization to 9q13-q21, and, accordingly, we assign the D9S5, D9S15, and FA cluster to the proximal part of chromosome 9 long arm, close to the heterochromatic region.     

Linkage of cystic fibrosis to two tightly linked DNA markers: Joint report from a collaborative study

A collaborative study involving seven research groups provided an opportunity to investigate the linkage relationships between cystic fibrosis and two DNA marker loci, MET and pJ3.11 (D7S8), on an extended sample of 211 tested families. The maximum lod scores, recombination estimates, and confidence upper bounds (in parentheses) were 91.0 at theta = .004 (.012) for CF and MET, 71.3 at theta = .003 (.011) for CF and D7S8, and 69.3 at theta = .018 (.036) for MET and D7S8. Three-locus analyses yielded best support for the order MET-CF-D7S8, with odds against the alternate orders CF-MET-D7S8 and CF-D7S8-MET of 9:1 and 161:1, respectively. However, the number of observed recombinants was small and only one of the recombinants was jointly informative for all three markers. Significant allelic association was found between CF and both MET and D7S8. Weaker association between the latter two loci is consistent with the order MET-CF-D7S8.     

Identification of simple sequence repeat markers tightly linked to plum pox virus resistance in apricot

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit production in Europe and America. Attempts to stop the disease through the eradication of infected trees have been unsuccessful. Introgression of PPV resistance for crop improvement is therefore the most important goal in Prunus breeding programs. Due to time- and labour-consuming protocols, phenotyping for sharka is still the major bottleneck in the breeding pipeline. In this context, screening of seedlings at early stages of development and marker-assisted selection (MAS) provide the best solution for enhancing breeding efficiency. In this study, we generated 42 simple sequence repeat (SSR) markers from the peach genome assembly v1.0 and an apricot bacterial artificial chromosome clone identified in the physical map of the PPV resistance locus previously defined in apricot. Using a linkage mapping approach, we found SSR markers tightly linked to PPV resistance trait in all our progenies. Three SSR markers, PGS1.21 PGS1.23 and PGS1.24, showed allelic variants associated with PPV resistance with no recombinants in the crosses analysed. These markers unambiguously discriminated resistant from susceptible accessions in different genetic backgrounds. The results presented here are the first successful application of their use in MAS for breeding resistance in Prunus species.     

Lowe oculocerebrorenal syndrome: DNA-based linkage of the gene to Xq24-q26, using tightly linked flanking markers and the correlation to lens examination in carrier diagnosis.

The Lowe syndrome (LS), or oculocerebrorenal syndrome, has been studied using DNA-based linkage analysis, and the findings have been correlated with the result of a thorough ophthalmologic examination. It was found that the LS gene was linked to markers in the Xq24-q26 region and that the locus DXS42 was the most closely linked marker, giving a LOD score of 3.12 at zero recombination distance. Combined with earlier data, this forms the basis for carrier detection and prenatal diagnosis by using tightly linked flanking markers. A summary of our and other data suggests that the loci DXS17, DXS11, DXS87, and DXS42 are located on the proximal side, and DXS86 and DXS10 on the distal side of the Lowe locus. In isolated cases of LS the question of whether the mother is a carrier of the mutation arises. It was found that a lens examination with slit-lamp illumination and a count of the total number of lenticular opacities is a reliable method of ascertaining the carrier state.     

Genetic linkage analysis of a dichotomous trait incorporating a tightly linked quantitative trait in affected sib pairs

Many complex diseases are usually considered as dichotomous traits but are also associated with quantitative biological markers or quantitative risk factors. For such dichotomous traits, although their associated quantitative traits may not directly underly the diagnosis of the disease status, if the associated quantitative trait is also linked to the chromosomal regions linked to the dichotomous trait, then joint analysis of dichotomous and quantitative traits should be more efficient than consideration of them separately. Previous studies have focused on the situation when a dichotomous trait can be modeled by a threshold process acting on a single underlying normal liability distribution. However, for many complex disorders, including most psychiatric disorders, diagnosis is generally based on a set of binary or discrete criteria. These traits cannot be modeled on the basis of a threshold process acting on an underlying continuous trait. We propose a likelihood-based method that efficiently combines such a discrete trait and an associated quantitative trait in the analysis, using affected-sib-pair data. Our simulation studies suggest that joint analysis increases the power to detect linkage of dichotomous traits. We also apply the proposed new method to an asthma genome-scan data set and incorporate the total serum immunoglobulin E level in the analysis.     

The use of incompletely linked markers in genetic counseling: accuracy versus linkage

The utility of incompletely linked, selectively neutral, multiallelic markers for tracing the transmission of associated genes is examined theoretically for all genetic counseling situations in which the diagnosis of deleterious progeny is in question. The analysis focuses on the fraction of progeny from each two locus mating which can be diagnosed with minimal accuracy A solely on the basis of the marker alleles transmitted, as a function of A, the recombination fraction between the loci, and the gametic frequency distribution in the population. Together the results allow a quantitative assessment of the diagnostic value of a given marker-target locus association to the total population of at-risk individuals.     

Application of comparative genomics in developing molecular markers tightly linked to the virus resistance gene Rsv4 in soybean.

The Rsv4 gene confers resistance to all the known strain groups of soybean mosaic virus in soybean (Glycine max (L.) Merr.). To construct a fine genetic map near Rsv4 in soybean, we employed a comparative genomics approach that used genome sequence information of the model legume Lotus japonicus. Sequences of the soybean expressed sequence tags (ESTs) AI856415 and BF070293 mapping to one side of the Rsv4 gene showed high similarity with gene sequences of the transformation-competent artificial chromosome (TAC) clone LjT32P24 of Lotus. LjT32P24 is tightly linked to another sequenced TAC clone, LjT26I01, in Lotus. A new marker, AW307114A, developed from soybean EST AW307114, which is homologous to a Lotus gene within LjT26I01, was mapped to the other side of the Rsv4 gene. The identification of the microsyntenic relationship facilitated the development of additional 2 EST markers between BF070293-S and AW307114A bracketing the Rsv4 gene. Several other markers developed in this study were mapped to putative homoeologous or duplicated chromosomal regions in soybean. Alignment between the soybean maps indicated that Rsv4 is located near a local chromosomal rearrangement. This targeted comparative mapping serves to provide a foundation for marker-assisted selection and cloning of the Rsv4 gene.     

Simultaneous fine mapping of multiple closely linked quantitative trait Loci using combined linkage disequilibrium and linkage with a general pedigree

Within a small region (e.g., <10 cM), there can be multiple quantitative trait loci (QTL) underlying phenotypes of a trait. Simultaneous fine mapping of closely linked QTL needs an efficient tool to remove confounded shade effects among QTL within such a small region. We propose a variance component method using combined linkage disequilibrium (LD) and linkage information and a reversible jump Markov chain Monte Carlo (MCMC) sampling for model selection. QTL identity-by-descent (IBD) coefficients between individuals are estimated by a hybrid MCMC combining the random walk and the meiosis Gibbs sampler. These coefficients are used in a mixed linear model and an empirical Bayesian procedure combines residual maximum likelihood (REML) to estimate QTL effects and a reversible jump MCMC that samples the number of QTL and the posterior QTL intensities across the tested region. Note that two MCMC processes are used, i.e., an (internal) MCMC for IBD estimation and an (external) MCMC for model selection. In a simulation study, the use of the multiple-QTL model clearly removes the shade effects between three closely linked QTL located at 1.125, 3.875, and 7.875 cM across the region of 10 cM, using 40 markers at 0.25-cM intervals. It is shown that the use of combined LD and linkage information gives much more useful information compared to using linkage information alone for both single- and multiple-QTL analyses. When using a lower marker density (11 markers at 1-cM intervals), the signal of the second QTL can disappear. Extreme values of past effective size (resulting in extreme levels of LD) decrease the mapping accuracy.     

Characterization of a YAC and cosmid contig containing markers tightly linked to the myotonic dystrophy locus on chromosome 19.

Myotonic dystrophy (DM) is caused by a defect in an unknown gene that maps to 19q13.3, flanked by the tightly linked markers ERCC1 on the proximal side and D19S51 on the distal side. We report the isolation and characterization of overlapping YAC and cosmid clones around D19S51 for the construction of a physical map around this locus. The resulting contig contains the markers D19S51 and D19S62 (another new marker tightly linked to the DM locus) and the distal breakpoint of a radiation hybrid cell line used in the physical mapping of the DM region. We have compared the restriction maps of the YACs and cosmids with that of the genome to investigate the fidelity of these clones.     

Identification of an RFLP marker tightly linked to theHt1 gene in maize

Summary We have identified tight linkage of an RFLP marker to theHt1 gene of maize that confers resistance to the fungal pathogenHelminthosporium turcicum race 1. This was accomplished by the use of four pairs of near isogenic lines (NILs; B73, A619, W153R, and CM105), each differing by the presence or the absence of the geneHt1. SinceHt1 maps to chromosome 2, 26 clones already mapped to this chromosome were labeled and probed against Southern blots of these NILs DNA digested with three restriction enzymes:EcoRI,BamHI, andHindIII. Six markers exhibited an RFLP for at least one pair of NILs. Presumptive linkage was further tested by analyzing the segregation of five of the six markers (one was monomorphic in the cross studied) and resistance toH. turcicum race 1 on 95 F₂ individuals from the cross DF20 × LH146Ht. The results indicate a tight linkage between one of the DNA markers,UMC150B, and theHt1 gene.     

Magnitude of type I error when single-locus linkage analysis is maximized over models: a simulation study.

It is well known that maximizing the maximum LOD score over multiple parameter values or models (i.e., the method of mod scores, or MMLS), will inflate type I error, compared with assuming only one parameter value/model in the linkage analysis. On the other hand, a mod score often has greater power to detect linkage than does a LOD score (Z) calculated under a wrong genetic model. Therefore, it is of interest to determine the actual magnitude of type I error in realistic genetic situations. Simulated data sets with no linkage were generated under three dominant and three recessive single-locus models, with reduced penetrance (f = .8, .5, and .2). Data sets were analyzed for linkage by (1) maximizing over penetrance only, (2) maximizing over "dominance model" (i.e., dominant versus recessive), and (3) maximizing over both penetrance and dominance model simultaneously. In (1), the resultant significance levels were approximately doubled, compared with baseline values if one had not maximized over penetrances (i.e., compared with a one-sided chi2(1)). In (2), significance levels were increased somewhat less, and, in (3), they were increased by approximately two to three times (but not more than four times) over those of the one-sided chi2(1). This means that, for a given size of test alpha, an investigator would need to increase the Z used as a test criterion, by approximately 0.30 LOD units for analyses as in (1) or (2) and by 0.60 Z units for analyses as in (3). These guidelines, which are valid up to approximately Z = 3.0, are conservative for (1) and are very conservative for (2) and (3). By quantifying the increase in significance level (or, correspondingly, the increase in Z), our findings will enable users to rationally assess the advantages versus the disadvantages of mod scores.