Sequence analysis of a compound coding-region microsatellite in Candida albicans resolves homoplasies and provides a high-resolution tool for genotyping

Sequence diversity at a coding-region microsatellite locus of two diploid Candida species was surveyed. Twenty-one alleles from fourteen strains of Candida albicans and three alleles from two strains of the closely related Candida dubliniensis were sequenced. Results show independent length variation in two contiguous hexanucleotide repeats, one non-contiguous hexanucleotide repeat, and two non-contiguous trinucleotide repeats within a 120 bp coding region. A neighboring, non-repetitive 120 bp region showed no variation. The information density of sequence polymorphisms in this region provides a powerful tool for genotyping microorganisms in epidemiological studies, yielding detailed resolution of closely related strains, and clearly distinguishing the two species studied here. The individual length-variable repeat regions are very short (2–8 repeats), demonstrating that even very short microsatellites can show high levels of length variability when surrounded by similarly repetitive DNA. Extensive homoplasy was discovered among the C. albicans alleles, with the majority of overall length categories consisting of alleles with more than one sequence. Our results show that microsatellite length alone should not be used to assume either sequence identity or identity by descent. Microsatellite length mutations appear to have generated the high degree of both inter- and intraspecific polymorphism seen at the ERK1 locus, and form an island of variability in an otherwise well-conserved gene.     

Multiple signatures of positive selection downstream of notch on the X chromosome in Drosophila melanogaster

To identify genomic regions affected by the rapid fixation of beneficial mutations (selective sweeps), we performed a scan of microsatellite variability across the Notch locus region of Drosophila melanogaster. Nine microsatellites spanning 60 kb of the X chromosome were surveyed for variation in one African and three non-African populations of this species. The microsatellites identified an approximately 14-kb window for which we observed relatively low levels of variability and/or a skew in the frequency spectrum toward rare alleles, patterns predicted at regions linked to a selective sweep. DNA sequence polymorphism data were subsequently collected within this 14-kb region for three of the D. melanogaster populations. The sequence data strongly support the initial microsatellite findings; in the non-African populations there is evidence of a recent selective sweep downstream of the Notch locus near or within the open reading frames CG18508 and Fcp3C. In addition, we observe a significant McDonald-Kreitman test result suggesting too many amino acid fixations species wide, presumably due to positive selection, at the unannotated open reading frame CG18508. Thus, we observe within this small genomic region evidence for both recent (skew toward rare alleles in non-African populations) and recurring (amino acid evolution at CG18508) episodes of positive selection.     

Analysis of mitochondrial DNA in Bolivian llama,alpaca and vicuna populations: a contribution to the phylogeny of the South American camelids

The objectives of this work were to assess the mtDNA diversity of Bolivian South American camelid (SAC) populations and to shed light on the evolutionary relationships between the Bolivian camelids and other populations of SACs. We have analysed two different mtDNA regions: the complete coding region of the MT‐CYB gene and 513 bp of the D‐loop region. The populations sampled included Bolivian llamas, alpacas and vicunas, and Chilean guanacos. High levels of genetic diversity were observed in the studied populations. In general, MT‐CYB was more variable than D‐loop. On a species level, the vicunas showed the lowest genetic variability, followed by the guanacos, alpacas and llamas. Phylogenetic analyses performed by including additional available mtDNA sequences from the studied species confirmed the existence of the two monophyletic clades previously described by other authors for guanacos (G) and vicunas (V). Significant levels of mtDNA hybridization were found in the domestic species. Our sequence analyses revealed significant sequence divergence within clade G, and some of the Bolivian llamas grouped with the majority of the southern guanacos. This finding supports the existence of more than the one llama domestication centre in South America previously suggested on the basis of archaeozoological evidence. Additionally, analysis of D‐loop sequences revealed two new matrilineal lineages that are distinct from the previously reported G and V clades. The results presented here represent the first report on the population structure and genetic variability of Bolivian camelids and may help to elucidate the complex and dynamic domestication process of SAC populations.     

Spanish population of Gremmeniella abietina is genetically unique but related to type A in Europe

Genetic structure of the European Gremmeniella abietina var. abietina was analyzed in this study. Ninety-two Spanish isolates, six Swiss isolates of Alpine biotype, 76 Finnish isolates of biotype A and 54 Finnish and seven Russian isolates of biotype B were collected. Genetic variation of different populations was analyzed using sequence analysis of specifically amplified markers GAAA1000, GAAA800 and ACA900. Variation in the GAAA1000 marker was significant, and composed of 33 alleles divided into the following four studied populations: five alleles in the Alpine type, 12 in biotype B, 16 in biotype A and two in the Spanish population. Based on variation in GAAA1000 marker, a subset of isolates were further analyzed using GAAA800 and ACA900 sequences, which showed lower overall genetic variability, and no variation among the Spanish population. Genetic differentiation analysis revealed a high genetic differentiation among populations. Finally, clustering analysis of GAAA1000 sequences showed that the Spanish isolates clearly separated from the rest of the biotypes, whereas the Alpine type was closely related to the B type. However, one of the A-type isolates had an identical GAAA1000 allele with the prevailing allele among Spanish isolates. Altogether, our data suggest that the Spanish population is genetically highly differentiated from any other G. abietina population in Europe with a probable A-type origin.     

Geographic structure of genetic variation in the widespread woodland grass Milium effusum L. A comparison between two regions with contrasting history and geomorphology.

Allozyme variation in the forest grass Milium effusum L. was studied in 21-23 populations within each of two equally sized densely sampled areas in northern and southern Sweden. In addition, 25 populations from other parts of Eurasia were studied for comparison. The structure of variation was analysed with both diversity statistics and measures based on allelic richness at a standardised sample size. The species was found to be highly variable, but no clear geographic patterns in the distribution of alleles or in overall genetic differentiation were found, either within the two regions or within the whole sample. Thus, no inferences about the direction of postglacial migration could be made. Obviously, migration and gene flow must have taken place in a manner capable of randomising the distribution of alleles. However, there were clear differences in levels and structuring of the variation between the two regions. Levels of variation, both in terms of genetic diversity and allelic richness, were lower in northern Sweden as compared with southern Sweden. In contrast, different measures of geographic structure all showed higher levels of population differentiation in the northern region. This is interpreted as due to different geomorphological conditions in the two regions, creating a relatively continuous habitat and gene flow in the southern region as compared with the northern region where the species, although common, is confined to narrow and mutually isolated corridors in the landscape.     

African sequence variation accounts for most of the sequence polymorphism in non-African Drosophila melanogaster

We compared the sequence polymorphism of 12 genomic fragments in six geographically dispersed African populations to one European Drosophila melanogaster population. On the basis of one African and one European population half of these fragments have strongly reduced levels of variability outside of Africa. Despite this striking difference in European variation, we detected no significant difference in African variation between the two fragment classes. The joint analysis of all African populations indicated that all high-frequency European alleles are of African origin. We observed a negative Tajima's D in all African populations, with three populations deviating significantly from neutral equilibrium. Low, but statistically significant, population differentiation was observed among the African populations. Our results imply that the population structure and demographic past of African D. melanogaster populations need to be considered for the inference of footprints of selection in non-African populations.     

Notes on individual sequence variation in humans: immunoglobulin kappa light chain.

Little is known concerning the magnitude of variability in the nucleic acid sequence of DNA at the individual level. We have collected a large set of sequence data from the human immunoglobulin kappa light-chain-locus constant region (10,444 bp) and subgroup IV variable region (18,580 bp). For the constant region, absolute conservation of sequence was observed, even in intron and coding-region silent sites, with the exception of one previously defined polymorphic site. For the variable region, 12 heterozygous positions were identified, giving a heterozygosity of 6 x 10(-4) per nucleotide site. The amount of nucleic acid sequence variation differs significantly (chi 2 = 4.88) between these two regions, and the observed variation is two orders of magnitude lower than that reported for two Drosophila melanogaster loci. These data suggest that, for at least some regions of the human genome, nucleic acid sequence may be less variable than previously estimated.     

Mechanisms of divergence and convergence of the human immunoglobulin alpha 1 and alpha 2 constant region gene sequences

Nucleotide sequences of the human alpha 1 and two allelic alpha 2 immunoglobulin heavy chain constant region genes are presented. The genes contain three exons, each encoding a single constant region protein domain. The protein hinge region is encoded at the 5' end of the second exon, and the rapid evolutionary changes in length of the hinge correspond to duplications or deletions within the hinge-coding region, probably facilitated by repeats in the DNA sequence. Alignment of the alpha 1 and alpha 2 gene sequences reveals an unusual coupled deletion-duplication in the 5'-flanking region, which can be explained in terms of a slipped-strand mispairing model. Comparison of nucleotide sequences of the alpha 1 gene and two alleles of the alpha 2 gene indicates a localized transfer of genetic information from the 3' end of the alpha 1 gene to one of the alpha 2 alleles, probably by a gene conversion. At one end of the region within which conversion apparently occurred, there is a 40 bp sequence of the type that can form Z-DNA.     

Microsatellites in Zea - variability,patterns of mutations,and use for evolutionary studies

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees. Received: 10 February 2001 / Accepted: 21 May 2001     

Genetic differentiation of water buffalo (Bubalus bubalis) populations in China, Nepal and south-east Asia: inferences on the region of domestication of the swamp buffalo

Data from three published studies of genetic variation at 18 microsatellite loci in water buffalo populations in China (18 swamp type, two river type), Nepal (one wild, one domestic river, one hybrid) and south-east Asia (eight swamp, three river) were combined so as to gain a broader understanding of genetic relationships among the populations and their demographic history. Mean numbers of alleles and expected heterozygosities were significantly different among populations. Estimates of θ (a measure of population differentiation) were significant among the swamp populations for all loci and among the river populations for most loci. Differentiation among the Chinese swamp populations (which was due primarily to just one population) was much less than among the south-east Asian. The Nepal wild animals, phenotypically swamp type but genetically like river type, are significantly different from all the domestic river populations and presumably represent the ancestral Bubalus arnee (possibly with some river-type introgression). Relationships among the swamp populations (D(A) genetic distances, principal component analysis and structure analyses) show the south-east Asian populations separated into two groups by the Chinese populations. Given these relationships and the patterns of genetic variability, we postulate that the swamp buffalo was domesticated in the region of the far south of China, northern Thailand and Indochina. Following domestication, it spread south through peninsular Malaysia to Sumatra, Java and Sulawesi, and north through China, and then to Taiwan, the Philippines and Borneo.     

Population structure of rice (Oryza sativa) landraces from high altitude area of Indian Himalayas

This study examined the genetic diversity in 20 rice landrace populations from parts of traditional farming areas of the Indian Himalayas using 11 mapped simple sequence repeats (SSR) loci. Twenty‐four individuals sampled from each of the 20 landraces (480 individuals), which were collected from farmers from Northwest to Northeast Himalaya, showed that all landraces showed within population variation and none were homogeneous. The number of polymorphic loci in a landrace population ranged from 5 to 11. A total of 71 alleles were recorded of which 58 were common and 13 were rare. Of the 71 alleles, 46 were common to both Northwest and Northeast regions, whereas 9 were unique to the former and 16 were unique to the latter. The mean number of alleles per locus was 6.45 and for landrace populations from Northwest and Northeast regions were 5.0 and 5.64, respectively. Population differentiation, as shown by a high F_ST value (0.61), was greater for Northeast populations. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram classified the populations into three major clusters: cluster I comprised seven populations from the Northwest region, cluster II comprised seven populations from the Northeast region and cluster III comprised populations from both regions. Investigating the population genetic structure can help monitor change in diversity over time and space, and also help devise a rational plan for management of crop landraces on‐farm under farmer management.     

Comparative study of the validity of three regions of the 18S‐rRNA gene for massively parallel sequencing‐based monitoring of the planktonic eukaryote community

The nuclear 18S‐rRNA gene has been used as a metabarcoding marker in massively parallel sequencing (MPS)‐based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations into 18S‐rRNA were carried out; we investigated the effective number of sequences deposited in international nucleotide sequence databases (INSDs), the amplification bias, and the amplicon sequence variability among the three variable regions, V1–3, V4–5 and V7–9, using in silico polymerase chain reaction (PCR) amplification based on INSDs. We also examined the primer universality and the taxonomic identification power, using MPS‐based environmental surveys in the Sea of Okhotsk, to determine which region is more useful for MPS‐based monitoring. The primer universality was not significantly different among the three regions, but the number of sequences deposited in INSDs was markedly larger for the V4–5 region than for the other two regions. The sequence variability was significantly different, with the highest variability in the V1–3 region, followed by the V7–9 region, and the lowest variability in the V4–5 region. The results of the MPS‐based environmental surveys showed significantly higher identification power in the V1–3 and V7–9 regions than in the V4–5 region, but no significant difference was detected between the V1–3 and V7–9 regions. We therefore conclude that the V1–3 region will be the most suitable for future MPS‐based monitoring of natural eukaryote communities, as the number of sequences deposited in INSDs increases.     

Genetic variation of mitochondrial DNA in Chinese populations of Pectinophora gossypiella (Lepidoptera: Gelechiidae)

The pink bollworm Pectinophora gossypiella is an invasive pest insect that has successfully established populations in many cotton growing regions around the world. In this study, the genetic diversity and population structure of Chinese populations of P. gossypiella were evaluated using mitochondrial DNA sequence data (COII and Nad4). For comparison, individuals of Pakistan and America were also sequenced at the same two mtDNA regions. Extremely low genetic variation was observed in the two mitochondrial regions among all populations examined. Most of the populations harbored only one to two haplotypes. Although the Nad4 region showed relatively high haplotype diversity and nucleotide variation, ranging from 0.363 to 0.591 and from 0.00078 to 0.00140, respectively, there were only three haplotypes observed in this region. COII and Nad4 haplotype networks shaved one or two common haplotype(s) forming the center of a star-shaped phylogeny. Pairwise tests showed that most of the populations were not significantly differentiated from each other. The Chinese populations were differentiated from the Pakistani and American populations in the Nad4 region. The low level of population genetic variation of P. gossypiella is attributed to invasion bottlenecks, which may have been subsequently strengthened by its nonmigratory biology and the mosaic pattern of agricultural activities.     

Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting

Background

Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial.

Results

Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles.

Conclusion

The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.     

Molecular evolution near a two-locus balanced polymorphism

Balancing selection at one locus can increase the amount of selectively neutral variation within neighboring genomic regions. Discrete phenotypic polymorphisms studied in natural populations are frequently determined by sets of interacting genes instead of alternative alleles at single loci. We extend coalescent theory to investigate balancing selection on combinations of linked genes. We find that variation at neutral sites is increased across a much larger genomic region relative to the single-locus models: the entire region lying between the two loci in balanced combination is affected to some degree. Epistatic selection maintains these high levels of neutral variation because it directly opposes the homogenizing effect of recombination. The results of the theory are discussed in relation to published gene sequence data, primarily from Drosophila.     

Sequence Variation within the KIV-2 Copy Number Polymorphism of the Human LPA Gene in African,Asian, and European Populations

Amazingly little sequence variation is reported for the kringle IV 2 copy number variation (KIV 2 CNV) in the human LPA gene. Apart from whole genome sequencing projects, this region has only been analyzed in some detail in samples of European populations. We have performed a systematic resequencing study of the exonic and flanking intron regions within the KIV 2 CNV in 90 alleles from Asian, European, and four different African populations. Alleles have been separated according to their CNV length by pulsed field gel electrophoresis prior to unbiased specific PCR amplification of the target regions. These amplicons covered all KIV 2 copies of an individual allele simultaneously. In addition, cloned amplicons from genomic DNA of an African individual were sequenced. Our data suggest that sequence variation in this genomic region may be higher than previously appreciated. Detection probability of variants appeared to depend on the KIV 2 copy number of the analyzed DNA and on the proportion of copies carrying the variant. Asians had a high frequency of so-called KIV 2 type B and type C (together 70% of alleles), which differ by three or two synonymous substitutions respectively from the reference type A. This is most likely explained by the strong bottleneck suggested to have occurred when modern humans migrated to East Asia. A higher frequency of variable sites was detected in the Africans. In particular, two previously unreported splice site variants were found. One was associated with non-detectable Lp(a). The other was observed at high population frequencies (10% to 40%). Like the KIV 2 type B and C variants, this latter variant was also found in a high proportion of KIV 2 repeats in the affected alleles and in alleles differing in copy numbers. Our findings may have implications for the interpretation of SNP analyses in other repetitive loci of the human genome.     

Nine polymorphic microsatellite markers in Crepis sancta (Asteraceae)

We describe nine new variable di‐ and trinucleotide microsatellites in the plant Crepis sancta, as well as conditions for multiplexing and simultaneous genotyping sets of loci. Their variability was assessed in four populations. Four to 33 alleles were detected per locus; two to 23 alleles per population. Significant deviations from Hardy–Weinberg equilibrium could be attributed either to a mating system involving partial selfing or to null alleles. Genetic differentiation over all loci was significant among the four populations studied. These markers will constitute an efficient tool for investigating dispersal patterns in fragmented landscapes. Cross‐species amplification provided few loci variability in three other Crepis species (C. pulcra, C. foetida and C. rubra).     

云南4个地区高山姬鼠线粒体控制区种群遗传分化研究

对云南4个种群38个个体的线粒体控制区（D-loop）905 bp的核苷酸序列遗传变异进行分析,探讨了高山姬鼠种群遗传结构和分化。在905 bp D-loop基因的碱基序列中,共发现了57个变异位点（全变异的6.30%）,共定义了23个单倍型,其中有一个单倍型（Hap1）为横断山3个种群（中甸、丽江和剑川）所共享,其余22个单倍型均为各个种群所特有。分子变异分析（AMOVA）表明,种群间的遗传变异占33.7%,种群内的遗传变异占66.3%。FST统计结果表明,除昆明种群和横断山种群之间差异显著（P〈0.05）,其它地理种群间的差异均不显著（P〉0.05）,说明昆明种群与横断山种群之间出现了明显的遗传分化。     

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod–res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.