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We use evolutionary trees of haplotypes to study phenotypic associations by exhaustively examining all possible biallelic partitions of the tree, a technique we call tree scanning. If the first scan detects significant associations, additional rounds of tree scanning are used to partition the tree into three or more allelic classes. Two worked examples are presented. The first is a reanalysis of associations between haplotypes at the Alcohol Dehydrogenase locus in Drosophila melanogaster that was previously analyzed using a nested clade analysis, a more complicated technique for using haplotype trees to detect phenotypic associations. Tree scanning and the nested clade analysis yield the same inferences when permutation testing is used with both approaches. The second example is an analysis of associations between variation in various lipid traits and genetic variation at the Apolipoprotein E (APOE) gene in three human populations. Tree scanning successfully identified phenotypic associations expected from previous analyses. Tree scanning for the most part detected more associations and provided a better biological interpretative framework than single SNP analyses. We also show how prior information can be incorporated into the tree scan by starting with the traditional three electrophoretic alleles at APOE. Tree scanning detected genetically determined phenotypic heterogeneity, within all three electrophoretic allelic classes. Overall, tree scanning is a simple, powerful, and flexible method for using haplotype trees to detect phenotype/genotype associations at candidate loci.

The 8.1 haplotype of the HLA complex has been reproducibly associated with several autoimmune diseases and traits, notably with thymus hyperplasia in patients with acquired generalized myasthenia gravis, an autoantibody-mediated disease directed at the muscle acetylcholine receptor. However, the strong linkage disequilibrium across this haplotype has prevented the identification of the causative locus, termed MYAS1. Here, we localized MYAS1 to a 1.2-Mb genome segment by reconstructing haplotypes and assessing their transmission in 73 simplex families. This segment encompasses the class III and proximal class I regions, between the BAT3 and C3-2-11 markers, therefore unambiguously excluding the class II loci. In addition, a case-control study revealed a very strong association with a core haplotype in this same region following an additive model (P = 7 x [10.sup.-11], odds ratio 6.5 for one copy and 42 for two copies of the core haplotype). Finally, we showed that this region is associated with a marked increase in serum titers of anti-acetylcholine receptor autoantibodies (P = 8 x [10.sup.-6]). Remarkably, this effect was suppressed by a second locus in cis on the 8.1 haplotype and located toward the class II region. Altogether, these data demonstrate the highly significant but complex effects of the 8.1 haplotype on the phenotype of myasthenia gravis patients and might shed light on its role in other autoimmune diseases.

The difficulty of experimental determination of haplotypes from phase-unknown genotypes has stimulated the development of nonexperimental inferral methods. One well-known approach for a group of unrelated individuals involves using the trivially deducible haplotypes (those found in individuals with zero or one heterozygous sites) and a set of rules to infer the haplotypes underlying ambiguous genotypes (those with two or more heterozygous sites). Neither the manner in which this 'rule-based' approach should be implemented nor the accuracy of this approach has been adequately assessed. We implemented eight variations of this approach that differed in how a reference list of haplotypes was derived and in the rules for the analysis of ambiguous genotypes. We assessed the accuracy of these variations by comparing predicted and experimentally determined haplotypes involving nine polymorphic sites in the human apolipoprotein E (APOE) locus. The eight variations resulted in substantial differences in the average number of correctly inferred haplotype pairs. More than one set of inferred haplotype pairs was found for each of the variations we analyzed, implying that the rule-based approach is not sufficient by itself for haplotype inferral, despite its appealing simplicity. Accordingly, we explored consensus methods in which multiple inferrals for a given ambiguous genotype are combined to generate a single inferral; we show that the set of these 'consensus' inferrals for all ambiguous genotypes is more accurate than the typical single set of inferrals chosen at random. We also use a consensus prediction to divide ambiguous genotypes into those whose algorithmic inferral is certain or almost certain and those whose less certain inferral makes molecular inferral preferable.

Molecular variation of the lagoon cockle Cerastoderma glaucum (Poiret, 1789) was examined across the species range along European coasts, from the northern Baltic to the Black Sea and the Caspian Sea. A major phylogeographic break in mitochondrial COI gene sequences (divergence 6.2%) separated a group of Ponto-Caspian and Aegean Sea haplotypes from those to the west of the Peloponnese peninsula in the Mediterranean and in the Atlantic-Baltic sector. A similar subdivision, yet not entirely geographically coincident, was found at 16 allozyme loci (Nei's distance D = 0.15). The results imply a long-term isolation of populations in parts of the Eastern Mediterranean or Black Sea basins through the Pleistocene. The subdivision does not concur with previous views on the systematics of C. glaucum complex, but the pattern is notably similar to that described earlier from some fish species. Marked phylogeographic structuring was also found at lower level within the major Mediterranean-Atlantic phylogroup of C. glaucum, which was divided into six regional or local haplotype subgroups. Divergence of these groups may date back one or several major Pleistocene climatic cycles. Local mtDNA diversity was particularly high in a sample from the Ionian Sea, whose mitochondrial identity was of the Mediterranean-Atlantic type, while its nuclear characters were more strongly associated with the Ponto-Caspian type. Patterns of shallower, starphylogeny type diversity within the Ponto-Caspian phylogroup and in the Baltic Sea area may represent more recent, post-glacial generation of variation.

Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for 'multiplex' and 1 for 'single-copy DNA molecules') that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.

Variation in DNA sequences specific to the nonrecombinant part of the human Y chromosome is particularly interesting from an anthropological point of view, because the variation relates to remote paternal […]

The association between genetic marker alleles was estimated for two regions of the bovine genome from a random sample of 50 young dairy bulls born in the United Kingdom between 1988 and 1995. Microsatellite marker genotypes were obtained for six markers on chromosome 2 and seven markers on chromosome 6, spanning 38 and 20 cM, respectively. Two different methods, which do not require family information, were used to estimate population haplotype frequencies. Haplotype frequencies were estimated for pairs of loci using the expectation-maximization algorithm and for all linked loci using a Bayesian approach via a Markov chain-Monte Carlo algorithm. Significant (P = 0.0007) linkage disequilibrium was detected between pairs of loci in syntenic groups (that is, loci in the same linkage group), extending to about 10 cM. No significant linkage disequilibrium was detected between markers in nonsyntenic regions. Given the observed level of linkage disequilibrium, mapping methods based on population-wide association might provide a better resolution than traditional quantitative trait loci mapping methods in the U.K. dairy cattle population and may reduce the required sample sizes of the experiments. Key Words: Dairy Cattle, Linkage Disequilibrium, Mapping, Markers

A novel multitrait fine-mapping method is presented. The method is implemented by a model that treats QTL effects as random variables. The covariance matrix of allelic effects is proportional to the IBD matrix, where each element is the probability that a pair of alleles is identical by descent, given marker information and QTL position. These probabilities are calculated on the basis of similarities of marker haplotypes of individuals of the first generation of genotyped individuals, using 'gene dropping' (linkage disequilibrium) and transmission of markers from genotyped parents to genotyped offspring (linkage). A small simulation study based on a granddaughter design was carried out to illustrate that the method provides accurate estimates of QTL position. Results from the simulation also indicate that it is possible to distinguish between a model postulating one pleiotropic QTL affecting two traits vs. one postulating two closely linked loci, each affecting one of the traits.

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/ SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site ([[pi].sub.S] and [[pi].sub.N]) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of [[pi].sub.N]:[[pi].sub.S] for SP11 and the hypervariable region of SRK were significantly > 1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.

Previous analysis of an Australian population of D. melanogaster revealed two predominant Est6 promoter haplotypes, P1 and P7. These haplotypes, which differ at 14 sites over a 325-bp region, are associated with a 15-20% difference in male EST6 activity. Here we show that the P1/P7 sequence difference causes the male activity variation by recreating the activity difference among >60 independently transformed lines containing representative P1 or P7 promoter alleles fused to an identical Est6 coding region. Furthermore we find that the whole fly difference reflects about a twofold difference in EST6 activity in the anterior sperm ejaculatory duct. EST6 activity variation in this tissue is known to affect reproductive fitness. Using a combination of RFLP analysis and DNA sequencing, we show that P1 and P7 are predominant in six populations from America, Asia, and Australia, albeit less frequent in a population from the presumptively ancestral east African range of the species. The sequence data show significant departures from neutral expectations for the derived American and Australian populations but not the presumptively ancestral Zimbabwean population. Thus the P1/P7 difference could be a major source of adaptively significant EST6 activity variation through much of the now cosmopolitan range of D. melanogaster.

The idea of trait-based linkage analysis in half-sibs is extended by comparing the frequency of parental marker haplotypes in animals with different phenotypes. This article first presents the likelihood of observing different classes of paternal haplotypes in a half-sib family, where only family members of a certain phenotype (e.g., affected) are genotyped and are fully informative. The likelihood function is then generalized to multiple phenotypic categories. A linear predictor allows for discontinuous as well as for continuous phenotypes and other explanatory variables. Finally, how to incorporate not fully informative offspring and how to analyze super sister families are shown. Maximum-likelihood estimates of all parameters can be found by a Newton-Raphson algorithm, which mimics an iteratively weighted least-squares procedure. The method allows for any multilocus feasible mapping function and, among others, for situations with selective or nonselective genotyping, single or multiple traits, and continuous or categorical traits. No parameters are required to describe the mode of inheritance and the method copes with virtually any family size. Fields of applications are therefore mapping experiments in species with a high reproductive capacity, such as cattle, pigs, horses, honey bees, trees, and fish.

CCR5 encodes a cell surface chemokine receptor molecule that serves as the principal coreceptor, with CD4, for HIV-type 1 (HIV-1). Varied HIV-1 susceptibility and time to progression to AIDS have been associated with polymorphisms in CCR5. Many of these polymorphisms are located in the 5' cis-regulatory region of CCR5, suggesting that it may have been a target of natural selection. We characterized CCR5 sequence variation in this region in 400 chromosomes from worldwide populations and compared it to a genome-wide analysis of 100 Alu polymorphisms typed in the same populations. Variation was substantially higher than expected and characterized by an excess of intermediate-frequency alleles. A genealogy of CCR5 haplotypes had deep branch lengths despite markedly little differentiation among populations. This finding suggested a deviation from neutrality not accounted for by population structure, which was confirmed by tests for natural selection. These results are strong evidence that balancing selection has shaped the pattern of variation in CCR5 and suggest that HIV-1 resistance afforded by CCR5 5' cis-regulatory region haplotypes may be the consequence of adaptive changes to older pathogens.

A maximum-likelihood QTL mapping method that simultaneously exploits linkage and linkage disequilibrium and that is applicable in outbred half-sib pedigrees is described. The method is applied to fine map a QTL with major effect on milk fat content in a 3-cM marker interval on proximal BTA14. This proximal location is confirmed by applying a haplotype-based association method referred to as recombinant ancestral haplotype analysis. The origin of the discrepancy between the QTL position derived in this work and that of a previous analysis is examined and shown to be due to the existence of distinct marker haplotypes associated with QTL alleles having large substitution effects.

The 140-kb a1-sh2 interval of the maize genome contains at least four genes (a1, yz1, x1, and sh2). Partial sequence analysis of two haplotypes has revealed many single nucleotide polymorphisms and InDel polymorphisms, including several large structural polymorphisms. The physical positions of 101 meiotic recombination breakpoints are not distributed uniformly across the interval and are instead concentrated within three recombination hot spots. Two of these recombination hot spots are genic (a1 and yz1) and one is apparently nongenic. The x1 gene is not a recombination hot spot. Thus, these results suggest that not all hot spots are genes and indicate that not all genes are hot spots. Two of the 101 recombination events arose by means of either noncrossover events involving conversion tract lengths of at least 17 kb or double-crossover events. Only one recombination breakpoint mapped to the [approximately equal to] 80-kb distal portion of the a1-sh2 interval that contains large amounts of repetitive DNA including retrotransposons; in this region the ratio of genetic to physical distance is less than 0.5% of the genome's average. These results establish that the retrotransposon faction of the maize genome is relatively inert recombinationally.

Non-protein-coding RNA genes play an important role in various biological processes. How new RNA genes originated and whether this process is controlled by similar evolutionary mechanisms for the origin of protein-coding genes remains unclear. A young chimeric RNA gene that we term sphinx (spx) provides the first insight into the early stage of evolution of RNA genes, spx originated as an insertion of a retroposed sequence of the ATP synthase chain F gene at the cytological region 60DB since the divergence of Drosophila melanogaster from its sibling species 2-3 million years ago. This retrosequence, which is located at 102F on the fourth chromosome, recruited a nearby exon and intron, thereby evolving a chimeric gene structure. This molecular process suggests that the mechanism of exon shuffling, which can generate protein-coding genes, also plays a role in the origin of RNA genes. The subsequent evolutionary process of spx has been associated with a high nucleotide substitution rate, possibly driven by a continuous positive Darwinian selection for a novel function, as is shown in its sex- and development-specific alternative splicing. To test whether spx has adapted to different environments, we investigated its population genetic structure in the unique 'Evolution Canyon' in Israel, revealing a similar haplotype structure in spx, and thus similar evolutionary forces operating on spx between environments.

Research indicates that the geographical distribution of chlorplast DNA haplotypes is structured in Fagus crenata with four haplotypes predominating the population. Phylogenetic analysis reveals that neither F.crenata nor F. japonica is monophyletic for the haplotypes, indicating either polymorphism or genetic introgression between the lineages of the two species.

We illustrate how homozygosity of haplotypes can be used to measure the level of disequilibrium between two or more markers. An excess of either homozygosity or heterozygosity signals a departure from the gametic phase equilibrium: We describe the specific form of dependence that is associated with high (low) homozygosity and derive various linkage disequilibrium measures. They feature a clear biological interpretation, can be used to construct tests, and are standardized to allow comparison across loci and populations. They are particularly advantageous to measure linkage disequilibrium between highly polymorphic markers.

Partial sequence data from laccase gene from 14 strains of Cryptococcus neoformans of serotype AD reveal two phylogenetically distinct haplotypes for each of the 14 serotypes analyzed. The haplotypes resemble either serotype A or D. Genealogical data suggest at least three independent hybridization events.

A number of alternative hypotheses seek to explain the origins of the three groups of Pacific populations--Melanesians, Micronesians, and Polynesians--who speak languages belonging to the Oceanic subfamily of Austronesian languages. To test these various hypotheses at the genetic level, we assayed diversity within the nonrecombining portion of the Y chromosome, which contains within it a relatively simple record of the human past and represents the most informative haplotypic system in the human genome. High-resolution haplotypes combining binary, microsatellite, and minisatellite markers were generated for 390 Y chromosomes from 17 Austronesian-speaking populations in southeast Asia and the Pacific. Nineteen paternal lineages were defined and a Bayesian analysis of coalescent simulations was performed upon the microsatellite diversity within lineages to provide a temporal aspect to their geographical distribution. The ages and distributions of these lineages provide little support for the dominant archeo-linguistic model of the origins of Oceanic populations that suggests that these peoples represent the Eastern fringe of an agriculturally driven expansion initiated in southeast China and Taiwan. Rather, most Micronesian and Polynesian Y chromosomes appear to originate from different source populations within Melanesia and Eastern Indonesia. The Polynesian outlier, Kapingamarangi, is demonstrated to be an admixed Micronesian/ Polynesian population. Furthermore, it is demonstrated that a geographical rather than linguistic classification of Oceanic populations best accounts for their extant Y chromosomal diversity.

********** GEORGE KIRMIZIDIS (1) Mitochondrial DNA (mtDNA) is an important tool for population geneticists because of such features as its maternal inheritance, absence of recombination, and high mutation rate. The […]

High genetic diversity during transition to resistance, high population numbers, and neutrality in the allele frequency spectra are accompanied by antibiotic resistance status and haplophytic identity of strains experiencing clonal expansion. The haplotype-antibiotic resistance indicates a genetic drift leading to selection for the resistance locus.

Mitochondrial d-loop sequence analysis from a population of 111 clingfish in 14 locations along the northeast Pacific coast and Strait of Georgia reveals significant genetic differentiation. The gene genealogy indicates two major haplotype clades, one with low diversity belonging to Strait Georgia and the other with high diversity to the Pacific coast.

Mitochondrial DNA analysis demonstrates that out of 48 Candida albicans isolates, six exhibited clonal proliferation of the DNA. Data indicate that the nuclear genotypes of the strains carrying identical mitochondrial DNA haplotypes are similar to one another than those that possess randomly derived nuclear genotypes.

The phylogenetic structure of the anemonefish Amphiprion ocellaris was established based on sequence data generated for the 5' end of the cytochrome b (cytb) gene at 11 sites throughout Southeast Asia. Considerable genetic structuring was observed throughout the range of A. ocellaris. The region has a complex geography, with the Sunda shelf having been exposed during the lower sea levels associated with periods of glaciation. The direction of subsequent flooding of the Sunda shelf, as a direct consequence of the retreating glacial sheets, can be predicted based on the major river drainage systems in the region. Much of the phylogeographic structure, including levels of intra-site genetic variation, can be explained in terms of the 'seeding' of sites on the Sunda shelf, by those on the shelf rim, as sea levels rose. We often found surface ocean currents in the region, which should influence larval dispersal, to be poorly correlated with phylogeographic structure. Several geographically close sites, which appear to be connected by surface currents, showed significant genetic stratification. We hypothesise that the phylogeographic structure of A. ocellaris is more reminiscent of the Pleistocene sea level changes than surface currents. The high contribution of sea level changes to the phylogeographic structure was also supported by several missing haplotypes in the generated spanning network. Cytb DNA sequences generated for recently recruited A. ocellaris individuals sampled from Singapore indicate that, on a local scale, there is a directional inflow of recruits, which is dependent on the monsoon seasons. The nature of dispersal and genetic structure of reef fish species found on the Sunda shelf is clearly complex, and should take into consideration past phylogeographic events.

Research has been conducted on the segregation distortion at the mouse t complex of the house mouse. The investigation of the t haplotype which has not been decomposed into its components in the course of evolution has been carried out and the results are reported.

Research is presented on the frequencies of genetic receptors in five Brazilian Indian tribes. Results of this research are compared with previous results; genetic receptors were found to be highly polymorphic while geographic dichotomy is shown between Indians of South America and those of North and Central America, with the exception of the Wai Wai of the Amazon.

The study has sequenced 361 bp of the mt DNA control region in 73 individuals from eight countries. A single dominant haplotype in 91.78% of the samples, and six more haplotypes deviating a maximum of two mutations from the dominant haplotype restricted to a single country.

Research has been conducted on South American Creole cattle. The authors discuss mitochondrial DNA characterization of this cattle, and report the existance of African and European haplotypes in cattle from South America.

The authors have investigated spotted salamander genetic diversity. They describe the use of nested cladistic and phylogenetic recontruction analyses in comparing the sequence variation among spotted salamander populations.

Research has been conducted on eukaryotes? sexual reproduction. The authors have carried out a sequence analysis of alleles of nuclear DNA markers and mitochondrial DNA haplotypes in sexual and asexual lineages of Rhopalosiphum padi, and they have concluded that ancient asexual lineages are of more recent origin than previously thought.

Research has been conducted on genetic variant distribution. The authors discuss the study of haplotype's geographical diversity in invasive Phragmites populations in the United States, and report that the invasiveness is caused by colonization and spread of distinct genotypes from Europe.

Research has been conducted on well-separated populations of the butterfly Danaus chrysippus. The authors have examined the congruence between the nuclear genotype and haplotype within the species from both allopatric and sympatric ranges in an attempt to gather evidence for sexual isolation of these populations, and they present the results.