Contrasting evolutionary histories of two introns of the duchenne muscular dystrophy gene, Dmd, in humans

The Duchenne muscular dystrophy (Dmd) locus lies in a region of the X chromosome that experiences a high rate of recombination and is thus expected to be relatively unaffected by the effects of selection on nearby genes. To provide a picture of nucleotide variability at a high-recombination locus in humans, we sequenced 5. 4 kb from two introns of Dmd in a worldwide sample of 41 alleles from Africa, Asia, Europe, and the Americas. These same regions were also sequenced in one common chimpanzee and one orangutan. Dramatically different patterns of genetic variation were observed at these two introns, which are separated by >500 kb of DNA. Nucleotide diversity at intron 44 pi = 0.141% was more than four times higher than nucleotide diversity at intron 7 pi = 0.034% despite similar levels of divergence for these two regions. Intron 7 exhibited significant linkage disequilibrium extending over 10 kb and also showed a significant excess of rare polymorphisms. In contrast, intron 44 exhibited little linkage disequilibrium and no skew in the frequency distribution of segregating sites. Intron 7 was much more variable in Africa than in other continents, while intron 44 displayed similar levels of variability in different geographic regions. Comparison of intraspecific polymorphism to interspecific divergence using the HKA test revealed a significant reduction in variability at intron 7 relative to intron 44, and this effect was most pronounced in the non-African samples. These results are best explained by positive directional selection acting at or near intron 7 and demonstrate that even genes in regions of high recombination may be influenced by selection at linked sites.     

Linkage disequilibrium in the North American Holstein population

Linkage disequilibrium was estimated using 7119 single nucleotide polymorphism markers across the genome and 200 animals from the North American Holstein cattle population. The analysis of maternally inherited haplotypes revealed strong linkage disequilibrium ( r ²   >   0.8) in genomic regions of ∼50 kb or less. While linkage disequilibrium decays as a function of genomic distance, genomic regions within genes showed greater linkage disequilibrium and greater variation in linkage disequilibrium compared with intergenic regions. Identification of haplotype blocks could characterize the most common haplotypes. Although maximum haplotype block size was over 1 Mb, mean block size was 26–113 kb by various definitions, which was larger than that observed in humans (∼10 kb). Effective population size of the dairy cattle population was estimated from linkage disequilibrium between single nucleotide polymorphism marker pairs in various haplotype ranges. Rapid reduction of effective population size of dairy cattle was inferred from linkage disequilibrium in recent generations. This result implies a loss of genetic diversity because of the high rate of inbreeding and high selection intensity in dairy cattle. The pattern observed in this study indicated linkage disequilibrium in the current dairy cattle population could be exploited to refine mapping resolution. Changes in effective population size during past generations imply a necessity of plans to maintain polymorphism in the Holstein population.     

Local Patterns of Nucleotide Polymorphism Are Highly Variable in the Selfing Species Arabidopsis thaliana

Neighboring genes predictably share similar evolutionary histories to an extent delineated by recombination. This correlation should extend across multiple linked genes in a selfing species such as Arabidopsis thaliana due to its low effective recombination rate. To test this prediction, we performed a molecular population genetics analysis of nucleotide polymorphism and divergence in chromosomal regions surrounding four low-diversity loci. Three of these loci, At1g67140, At3g03700, and TERMINAL FLOWER1 (TFL1), have been previously implicated as targets of selection and we would predict stronger correlations in polymorphism between neighboring loci due to genetic hitchhiking around these loci. The remaining locus, At1g04300, was identified in a study of linkage disequilibrium surrounding the CRYPTOCHROME2 (CRY2) locus. Although we found broad valleys of reduced nucleotide variation around two of our focal genes, At1g67140 and At3g03700, all chromosomal regions exhibited extreme variation in the patterns of polymorphism and evolution between neighboring loci. Although three of our four regions contained potential targets of selection, application of the composite-likelihood-ratio test of selection in conjunction with a goodness-of-fit test supports the selection hypothesis only for the region containing At3g03700. The degree of discordance in evolutionary histories between linked loci within each region generally correlated with estimates of recombination and linkage disequilibrium for that region, with the exception of the region containing At1g04300. We discuss the implications of these data for future population genetics analyses and genomics studies in A. thaliana. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA variation in the basic chitinase locus (ChiB) region of the wild plant Arabidopsis thaliana.

Nucleotide variation in a 2.2-kbp region of basic chitinase (ChiB) locus in 17 ecotypes of Arabidopsis thaliana was compared with previously investigated regions to investigate genetic mechanisms acting on DNA polymorphism. In the ChiB region, dimorphic DNA variation was detected, as in the Adh and ChiA regions. Nucleotide diversity (pi) of the entire region was 0.0091, which was similar to those of the two other regions. About half of polymorphic sites (37/87) in the ChiB region were observed in only two ecotypes. Tajima's D was negative but not significantly, while Fu and Li's D* was positive. Neither McDonald-Kreitman nor Hudson, Kreitman, Aguadé tests showed a significant result, indicating that these loci were under similar evolutionary mechanisms before and after speciation. Linkage disequilibria were observed within the three regions because of dimorphic polymorphisms. Interlocus linkage disequilibrium was not detected between the Adh and the two chitinase regions, but was observed between the ChiA and ChiB regions. This could be due to epistatic interaction between the two chitinase loci, which are located on different chromosomes.     

Selective sweeps in a 2-locus model for sex-ratio meiotic drive in Drosophila simulans

A way to identify loci subject to positive selection is to detect the signature of selective sweeps in given chromosomal regions. It is revealed by the departure of DNA polymorphism patterns from the neutral equilibrium predicted by coalescent theory. We surveyed DNA sequence variation in a region formerly identified as causing "sex-ratio" meiotic drive in Drosophila simulans. We found evidence that this system evolved by positive selection at 2 neighboring loci, which thus appear to be required simultaneously for meiotic drive to occur. The 2 regions are approximately 150-kb distant, corresponding to a genetic distance of 0.1 cM. The presumably large transmission advantage of chromosomes carrying meiotic drive alleles at both loci has not erased the individual signature of selection at each locus. This chromosome fragment combines a high level of linkage disequilibrium between the 2 critical regions with a high recombination rate. As a result, 2 characteristic traits of selective sweeps--the reduction of variation and the departure from selective neutrality in haplotype tests--show a bimodal pattern. Linkage disequilibrium level indicates that, in the natural population from Madagascar used in this study, the selective sweep may be as recent as 100 years.     

Nucleotide polymorphism and phenotypic associations within and around the phytochrome B2 Locus in European aspen (Populus tremula, Salicaceae)

We investigated the utility of association mapping to dissect the genetic basis of naturally occurring variation in bud phenology in European aspen (Populus tremula). With this aim, we surveyed nucleotide polymorphism in 13 fragments spanning an 80-kb region surrounding the phytochrome B2 (phyB2) locus. Although polymorphism varies substantially across the phyB2 region, we detected no signs for deviations from neutral expectations. We also identified a total of 41 single nucleotide polymorphisms (SNPs) that were subsequently scored in a mapping population consisting of 120 trees. We identified two nonsynonymous SNPs in the phytochrome B2 gene that were independently associated with variation in the timing of bud set and that explained between 1.5 and 5% of the observed phenotypic variation in bud set. Earlier studies have shown that the frequencies of both these SNPs vary clinally with latitude. Linkage disequilibrium across the region was low, suggesting that the SNPs we identified are strong candidates for being causally linked to variation in bud set in our mapping populations. One of the SNPs (T608N) is located in the "hinge region," close to the chromophore binding site of the phyB2 protein. The other SNP (L1078P) is located in a region supposed to mediate downstream signaling from the phyB2 locus. The lack of population structure, combined with low levels of linkage disequilibrium, suggests that association mapping is a fruitful method for dissecting naturally occurring variation in Populus tremula.     

Extent and distribution of linkage disequilibrium in three genomic regions

The positional cloning of genes underlying common complex diseases relies on the identification of linkage disequilibrium (LD) between genetic markers and disease. We have examined 127 polymorphisms in three genomic regions in a sample of 575 chromosomes from unrelated individuals of British ancestry. To establish phase, 800 individuals were genotyped in 160 families. The fine structure of LD was found to be highly irregular. Forty-five percent of the variation in disequilibrium measures could be explained by physical distance. Additional factors, such as allele frequency, type of polymorphism, and genomic location, explained <5% of the variation. Nevertheless, disequilibrium was occasionally detectable at 500 kb and was present for over one-half of marker pairs separated by <50 kb. Although these findings are encouraging for the prospects of a genomewide LD map, they suggest caution in interpreting localization due to allelic association.     

Global Analysis of ATM Polymorphism Reveals Significant Functional Constraint

ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.     

Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.     

Molecular evolution of the Est-6 gene in Drosophila melanogaster: contrasting patterns of DNA variability in adjacent functional regions

We have investigated nucleotide polymorphism at the esterase 6 gene (Est-6) gene, including the complete coding region (1686 bp), as well as the 5'-flanking (1183 bp) and 3'-flanking (193 bp) regions of the gene, in 30 strains of Drosophila melanogaster and in one strain of Drosophila simulans. The level of silent variation is similar in the coding and in the 3'-flanking region, but smaller in the 5'-flanking region. Strong linkage disequilibrium occurs within each region; and also, although less pronounced, between the 5'-flanking region and the rest of the gene, including the 3'-flanking region. We suggest that the pattern of nucleotide polymorphism of Est-6 may be shaped by: (1) directional and balancing selection acting on the promoter and the coding region; and (2) interactions between the two regions that involve variable degrees of hitchhiking. The patterns of linkage disequilibrium, as well as the statistics Z(nS) (Genetics 146 (1997)1197) and B and Q (Genet. Res. 74 (1999) 65), may be interpreted as there being multiple targets of selection within the gene. The previously reported Est-6 allozyme latitudinal clines may be accounted for by the interaction between selective processes in the promoter and coding regions.     

Identification of a locus under complex positive selection in Drosophila simulans by haplotype mapping and composite-likelihood estimation

The recent action of positive selection is expected to influence patterns of intraspecific DNA sequence variation in chromosomal regions linked to the selected locus. These effects include decreased polymorphism, increased linkage disequilibrium, and an increased frequency of derived variants. These effects are all expected to dissipate with distance from the selected locus due to recombination. Therefore, in regions of high recombination, it should be possible to localize a target of selection to a relatively small interval. Previously described patterns of intraspecific variation in three tandemly arranged, testes-expressed genes (janusA, janusB, and ocnus) in Drosophila simulans included all three of these features. Here we expand the original sample and also survey nucleotide polymorphism at three neighboring loci. On the basis of recombination events between derived and ancestral alleles, we localize the target of selection to a 1.5-kb region surrounding janusB. A composite-likelihood-ratio test based on the spatial distribution and frequency of derived polymorphic variants corroborates this result and provides an estimate of the strength of selection. However, the data are difficult to reconcile with the simplest model of positive selection, whereas a new composite-likelihood method suggests that the data are better described by a model in which the selected allele has not yet gone to fixation.     

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.     

Genetic exchange versus genetic differentiation in a medium-sized inversion of Drosophila: the A2/Ast arrangements of Drosophila subobscura

Chromosomal inversion polymorphism affects nucleotide variation at loci associated with inversions. In Drosophila subobscura, a species with a rich chromosomal inversion polymorphism and the largest recombinational map so far reported in the Drosophila genus, extensive genetic structure of nucleotide variation was detected in the segment affected by the O(3) inversion, a moderately sized inversion at Muller's element E. Indeed, a strong genetic differentiation all over O(3) and no evidence of a higher genetic exchange in the center of the inversion than at breakpoints were detected. In order to ascertain, whether other polymorphic and differently sized inversions of D. subobscura also exhibited a strong genetic structure, nucleotide variation in 5 gene regions (P236, P275, P150, Sxl, and P125) located along the A(2) inversion was analyzed in A(st) and A(2) chromosomes of D. subobscura. A(2) is a medium-sized inversion at Muller's element A and forms a single inversion loop in heterokaryotypes. The lower level of variation in A(2) relative to A(st) and the significant excess of low-frequency variants at polymorphic sites indicate that nucleotide variation at A(2) is not at mutation-drift equilibrium. The closest region to an inversion breakpoint, P236, exhibits the highest level of genetic differentiation (F(ST)) and of linkage disequilibrium (LD) between arrangements and variants at nucleotide polymorphic sites. The remaining 4 regions show a higher level of genetic exchange between A(2) and A(st) chromosomes than P236, as revealed by F(ST) and LD estimates. However, significant genetic differentiation between the A(st) and A(2) arrangements was detected not only at P236 but also in the other 4 regions separated from the nearest breakpoint by 1.2-2.9 Mb. Therefore, the extent of genetic exchange between arrangements has not been high enough to homogenize nucleotide variation in the center of the A(2) inversion. A(2) can be considered a typical successful inversion of D. subobscura according to its relative length. Chromosomal inversion polymorphism of D. subobscura might thus cause the genome of this species to be highly structured and to harbor different gene pools that might contribute to maintain adaptations to particular environments.     

Chromosomal inversion polymorphism leads to extensive genetic structure: a multilocus survey in Drosophila subobscura

The adaptive character of inversion polymorphism in Drosophila subobscura is well established. The O(ST) and O(3+4) chromosomal arrangements of this species differ by two overlapping inversions that arose independently on O(3) chromosomes. Nucleotide variation in eight gene regions distributed along inversion O(3) was analyzed in 14 O(ST) and 14 O(3+4) lines. Levels of variation within arrangements were quite similar along the inversion. In addition, we detected (i) extensive genetic differentiation between arrangements in all regions, regardless of their distance to the inversion breakpoints; (ii) strong association between nucleotide variants and chromosomal arrangements; and (iii) high levels of linkage disequilibrium in intralocus and also in interlocus comparisons, extending over distances as great as approximately 4 Mb. These results are not consistent with the higher genetic exchange between chromosomal arrangements expected in the central part of an inversion from double-crossover events. Hence, double crossovers were not produced or, alternatively, recombinant chromosomes were eliminated by natural selection to maintain coadapted gene complexes. If the strong genetic differentiation detected along O(3) extends to other inversions, nucleotide variation would be highly structured not only in D. subobscura, but also in the genome of other species with a rich chromosomal polymorphism.     

Polymorphism and differentiation of rainbow trout Y chromosomes.

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.     

Detection of a NotI polymorphism with the pmetH probe by pulsed-field gel electrophoresis.

The pmetH probe, tightly linked to the locus for cystic fibrosis (CF), detected a NotI polymorphism with allele sizes of 1,000 and 550 kb that could be separated by pulsed-field gel electrophoresis. Both alleles were found on chromosomes bearing either the normal or the CF allele. Preliminary data showed that this polymorphism was not in strong linkage disequilibrium with the CF locus. Preliminary large-scale restriction mapping of the fragment showed that the NotI polymorphic site is 200-370 kb distant from the MET locus; thus it defines a new polymorphic locus in the CF region. This locus is of potential interest for studying CF families that show recombinants between CF and its tightly linked probes, in order to increase the precision of the genetic map of the region.     

Use of tyramide-fluorescence in situ hybridization and chromosome microdissection for ascertaining homology relationships and chromosome linkage group associations in oats

The physical mapping of single locus sequences by tyramide-fluorescence in situ hybridization (Tyr-FISH) and the analysis of sequences obtained from microdissected chromosomes were assayed as potential tools for (1) determining homology and homoeology among chromosome regions of Avena species, and (2) establishing associations between linkage groups and specific chromosomes. Low copy number probes, derived from resistance gene analogues (RGAs) and 2.8-4.5 kb long, successfully produced hybridization signals on specific chromosomes. Four sets of homoeologous chromosome regions were identified in the hexaploids using 3 probes that produced 4 single locus markers in A. strigosa and 2 in A. eriantha. Laser capture microdissection of metaphase I cells of A. sativa monosomic lines allowed the isolation of critical univalents. Sequences derived from 2 RGAs were successfully amplified in DNA extracted from univalents. In one instance, it was possible to map a nucleotide polymorphism specific for 1 chromosome. An association was established between this chromosome and its linkage groups in 2 hexaploid genetic maps. The results indicate that Tyr-FISH is useful in the characterization of homoeologous chromosome segments in hexaploids, whereas chromosome microdissection, as employed in this work, needs to be improved before it can routinely be used with meiotic chromosomes.