Development of multiple genetic markers for studies of genetic variation in arbuscular mycorrhizal fungi using AFLP™

Intraspecific and interspecific genetic variation was studied among arbuscular mycorrhizal fungi. DNA was extracted from single spores and variation was analysed by AFLP (Amplified Fragment Length Polymorphism). The patterns of amplified fragments revealed substantial genetic variation between spores from the same isolates. Possible recombination in the fungi was studied by comparing the obtained data with data generated by artificial recombination of the data sets. No evidence for recombination was found by the analysis, suggesting that the fungi reproduce clonally. The AFLP technique generated a large number of fragments, and the potential for using the technique in population genetic studies of these important unculturable biotrophic fungi is discussed.     

The influence of recombination on the population structure and evolution of the human pathogen Neisseria meningitidis.

The extent to which recombination disrupts the bifurcating treelike phylogeny and clonal structure imposed by binary fission on bacterial populations remains contentious. Here, we address this question with a study of nucleotide sequence data from 107 isolates of the human pathogen Neisseria meningitidis. Gene fragments from 12 house-keeping loci distributed around the meningococcal chromosome were analyzed, showing that (1) identical alleles are disseminated among genetically diverse isolates, with no evidence for linkage disequilibrium; (2) different loci give distinct and incongruent phylogenetic trees; and (3) allele sequences are incompatible with a bifurcating treelike phylogeny at all loci. These observations are consistent with the hypothesis that meningococcal populations comprise organisms assembled from a common gene pool, with alleles and allele fragments spreading independently, together with the occasional importation of genetic material from other species. Further, they support the view that recombination is an important genetic mechanism in the generation new meningococcal clones and alleles. Consequently, for anything other than the short-term evolution of this species, a bifurcating treelike phylogeny is not an appropriate model.     

DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees

Tandem-repetitive DNA hybridization probes based on a putative human recombination signal detect multiple polymorphic minisatellite fragments in human DNA. The genetic complexity of the resulting individual-specific DNA "fingerprints" was investigated by studying a large sibship affected by neurofibromatosis and a more extensive pedigree segregating for two different hemoglobinopathies. The segregation of up to 41 different heterozygous DNA fragments from each parent could be analyzed in a single sibship, using two different repeat probes. Most of these variable DNA fragments could not be paired as alleles, to an extent which suggests that the DNA fingerprints are together derived from approximately 60 heterozygous loci (approximately 120 variable fragments), only a proportion of which can be scored in a given individual. Two or three of the DNA fragments detected by one probe showed tight linkage and may be derived from long minisatellite(s) that are cleaved to produce more than one polymorphic DNA fragment. Excluding allelic and linked DNA fragments, almost all remaining scorable fragments segregated independently, allowing up to 34 unlinked loci to be examined simultaneously. These loci are scattered over most or all of the human autosomes. Minisatellite probes are therefore suitable for rapid marker generation and can be applied to linkage analysis in human pedigrees.     

Genetic Methods for Analysis and Manipulation of Inversion Mutations in Bacteria

A number of genetic methods for the isolation, characterization and manipulation of large chromosomal inversions in Salmonella typhimurium are described. One inversion-carrying mutant is characterized in detail and used to demonstrate a number of unique genetic properties of bacterial inversions.—Contrary to expectation, it was found that large inversion mutations can be repaired by generalized transduction. The repair results from the simultaneous introduction of two wild-type transduced fragments into a single recipient cell. Homologous recombination between the two transduced fragments and the two inversion breakpoints causes the inverted segment to be reinverted. This results in regeneration of the wild-type orientation of this chromosome segment. Similar recombination events allow a large inversion mutation to be introduced into a wild-type strain; two transduced fragments from an inversion strain cause recombination events resulting in inversion of a large chromosome segment.—Genetic methods for mapping the extent of a large inversion mutation by generalized transduction are described and tested. The methods are operationally simple and allow good resolution of the two inversion breakpoints.     

Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii

The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii . The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii , but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.     

Protoplast fusion in Bacillus species produces frequent,unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction–modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.     

Epstein-Barr virus intrastrain recombination in oral hairy leukoplakia.

In laboratory lymphoblastoid cell lines and in natural human infections, Epstein-Barr virus (EBV) strains have been identified by DNA restriction fragment length polymorphisms of the BamHI H fragment. Multiple, heterogeneous BamHI H fragments have been detected in oral hairy leukoplakia (HLP), raising the question of EBV coinfection with multiple strains. To investigate whether the heterogeneous BamHI H fragments represent different EBV strains or recombinant variants of the same strain, EBV DNA from HLP lesions was analyzed to characterize the viral strains and determine the source of possible recombinant variants. Clones of heterogeneous BamHI H fragments from a single HLP lesion were determined to have strain identity on the basis of sequence identity of the EBNA-2 genes. Intrastrain homologous recombination within the IR2 internal repeat region and nonhomologous recombination of other sequences accounted for the heterogeneity of the BamHI H fragments. PCR amplification from additional HLP specimens detected similar recombinant variants. A possible example of site-specific recombination joining the BamHI Y portion of the EBNA-2 gene to sequences within the BamHI S fragment was also detected in multiple HLP specimens. These data indicate that intrastrain recombination during productive replication confounds the use of restriction fragment length polymorphism analysis of the BamHI Y and H fragments to identify EBV strains in HLP. In patients with permissive epithelial EBV infections, EBV strains could be more accurately distinguished by sequence identity or divergence within known regions of genetic strain variation.     

Cre reconstitution allows for DNA recombination selectively in dual-marker-expressing cells in transgenic mice

Cre/LoxP-based DNA recombination has been used to introduce desired DNA rearrangements in various organisms, having for example, greatly assisted genetic analyses in mice. For most applications, single gene promoters are used to drive Cre production for conditional gene activation/inactivation or lineage-tracing experiments. Such a manipulation introduces Cre in all cells in which the utilized promoter is active. To overcome the limited selectivity of single promoters for cell-type-specific recombination, we have explored the ‘dual promoter combinatorial control’ of Cre activity, so that Cre activity could be restricted to cells that express dual protein markers. We efficiently reconstituted Cre activity from two modified, inactive Cre fragments. Cre re-association was greatly enhanced by fusing the Cre fragments separately to peptides that can form a tight antiparallel leucine zipper. The co-expressed Cre fusion fragments showed substantial activity in cultured cells. As proof of principle of the utility of this technique in vivo for manipulating genes specifically in dual-marker-positive cells, we expressed each inactive Cre fragments in transgenic mice via individual promoters. Result showed the effective reconstitution of Cre activates LoxP recombination in the co-expressing cells.     

Variation in recombination rate may bias human genetic disease mapping studies

The availability of the human genome sequence and variability information (as from the International HapMap project) will enhance our ability to map genetic disorders and choose targets for therapeutic intervention. However, several factors, such as regional variation in recombination rate, can bias conclusions from genetic mapping studies. Here, we examine the impact of regional variation in recombination rate across the human genome. Through computer simulations and literature surveys, we conclude that genetic disorders have been mapped to regions of low recombination more often than expected if such diseases were randomly distributed across the genome. This concentration in low recombination regions may be an artifact, and disorders appearing to be caused by a few genes of large effect may be polygenic. Future genetic mapping studies should be conscious of this potential complication by noting the regional recombination rate of regions implicated in diseases.     

Genetic diversity, recombination and cryptic clades in Pseudomonas viridiflava infecting natural populations of Arabidopsis thaliana

Species-level genetic diversity and recombination in bacterial pathogens of wild plant populations have been nearly unexplored. Pseudomonas viridiflava is a common natural bacterial pathogen of Arabidopsis thaliana, for which pathogen defense genes and mechanisms are becoming increasing well known. The genetic variation contained within a worldwide sample of P. viridiflava collected from wild populations of A. thaliana was investigated using five genomic sequence fragments totaling 2.3 kb. Two distinct and deeply diverged clades were found within the P. viridiflava sample and in close proximity in multiple populations, each genetically diverse with synonymous variation as high as 9.3% in one of these clades. Within clades, there is evidence of frequent recombination within and between each sequenced locus and little geographic differentiation. Isolates from both clades were also found in a small sample of other herbaceous species in Midwest populations, indicating a possibly broad host range for P. viridiflava. The high levels of genetic variation and recombination together with a lack of geographic differentiation in this pathogen distinguish it from other bacterial plant pathogens for which intraspecific variation has been examined.     

Reciprocal recombination products of VK-JK joining reactions in human lymphoid cell lines.

The recombination process that joins a VK to a JK segment of an immunoglobulin gene generates a second, reciprocal recombination product called f fragment. In this second product the regions flanking the VK and JK segments in the germline are joined in a head to head fashion. We now analysed f fragments in the human lymphoid cell lines Daudi, JI and IARC/BL41. All three f fragments contain JK1 flanks; the VK derived moiety of f Daudi and f41 could be traced back to known germline VK genes. There is a precise head to head joining of the heptanucleotide signal sequences in f Daudi and fJI while in f41 six nucleotides are present between the signal sequences. In contrast to the VK-JK recombination products, the f fragments were found to lack somatic mutations. The structures of the f fragments are discussed in the context of the VK-JK rearrangement mechanism.     

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.     

RNA structures facilitate recombination-mediated gene swapping in HIV-1

Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.     

Individual variation in recombination among human males.

Studies of recombination between the markers D6S291 and D6S109 in individuals by sperm typing provide direct evidence for significant variation in recombination among humans. A statistically significant difference in the recombination fraction (range 5.1%-11.2%) was detected among five donors. This variation could reflect polymorphisms in genes affecting recombination or in chromosome structure. Ignoring this variability in studies designed to examine the relationship between physical and genetic distances could lead to incorrect inferences. Individual variation in recombination makes it difficult to predict the recombination fraction for an interval in any particular individual. This could be important in certain genetic counseling situations.     

Low species barriers in halophilic archaea and the formation of recombinant hybrids

Speciation of sexually reproducing organisms requires reproductive barriers. Prokaryotes reproduce asexually but?often exchange DNA by lateral gene transfer mechanisms and recombination [1], yet distinct lineages are still observed. Thus, barriers to gene flow such as geographic isolation, genetic incompatibility or a physiological inability to transfer DNA represent potential underlying mechanisms behind preferred exchange groups observed in prokaryotes [2-6]. In Bacteria, experimental evidence showed that sequence divergence impedes homologous recombination between bacterial species [7-11]. Here we study interspecies gene exchange in halophilic archaea that possess a parasexual mechanism of genetic exchange that is functional between species [12, 13]. In this process, cells fuse forming a diploid state containing the full genetic repertoire of both parental cells, which facilitates genetic exchange and recombination. Later, cells separate, occasionally resulting in hybrids of the parental strains [14]. We show high recombination frequencies between Haloferax volcanii and Haloferax mediterranei, two species that have an average nucleotide sequence identity of 86.6%. Whole genome sequencing of Haloferax interspecies hybrids revealed the exchange of chromosomal fragments ranging from 310Kb to 530Kb. These results show that recombination barriers may be more permissive in halophilic archaea than they are in bacteria.     

Microsatellite variation and recombination rate in the human genome

Background (purifying) selection on deleterious mutations is expected to remove linked neutral mutations from a population, resulting in a positive correlation between recombination rate and levels of neutral genetic variation, even for markers with high mutation rates. We tested this prediction of the background selection model by comparing recombination rate and levels of microsatellite polymorphism in humans. Published data for 28 unrelated Europeans were used to estimate microsatellite polymorphism (number of alleles, heterozygosity, and variance in allele size) for loci throughout the genome. Recombination rates were estimated from comparisons of genetic and physical maps. First, we analyzed 61 loci from chromosome 22, using the complete sequence of this chromosome to provide exact physical locations. These 61 microsatellites showed no correlation between levels of variation and recombination rate. We then used radiation-hybrid and cytogenetic maps to calculate recombination rates throughout the genome. Recombination rates varied by more than one order of magnitude, and most chromosomes showed significant suppression of recombination near the centromere. Genome-wide analyses provided no evidence for a strong positive correlation between recombination rate and polymorphism, although analyses of loci with at least 20 repeats suggested a weak positive correlation. Comparisons of microsatellites in lowest-recombination and highest-recombination regions also revealed no difference in levels of polymorphism. Together, these results indicate that background selection is not a major determinant of microsatellite variation in humans.     

The implications of a low rate of horizontal transfer in Borrelia.

The nature and rate of recombination can be studied by comparing the sequences of multiple genes across a set of strains. When this approach is applied to Borrelia burgdorferi, four results emerge: (1) chromosomal genes are clonal; (2) there is little or no plasmid exchange; (3) the major mode of horizontal transfer of genetic material inserts a small fragment of DNA, typically <1 kb, during recombination; and (4) the level of horizontal transfer in Borrelia is so low that there is evidence for horizontal transfer only in genes where there is positive selection for diversity, that is, positive selection for the recombinant. Thus, Borrelia can serve as a model of a low recombination taxon. The implications of these results lead us to postulate that an unknown agent that is part of the Borrelia genome mediates the horizontal transfer of small fragments of DNA; the rare transfer of small fragments of DNA excludes both DNA parasites and virulence factors from the genome.     

Polymorphic variation in human meiotic recombination

In this study, our phenotype of interest is meiotic recombination. Using genotypes of approximately 6,000 SNP markers in members of the Centre d'Etude du Polymorphisme Humain Utah pedigrees, we found extensive individual variation in the number of female and male recombination events. The locations and frequencies of these recombination events vary along the genome. In both female and male meiosis, the regions with the most recombination events are found at the ends of the chromosomes. Our analysis also shows that there are polymorphic differences among individuals in the activity of the recombination "jungles"; these preferred sites of meiotic recombination differ greatly among individuals. These findings have important implications for understanding genetic disorders that result from improper chromosome segregation.     

Circularity of the Caulobacter crescentus chromosome determined by pulsed-field gel electrophoresis.

Previous genetic analyses of the Caulobacter crescentus chromosome have resulted in the construction of a linear genetic map. To establish the circularity of the C. crescentus chromosome, restriction fragments generated by digestion with AseI and SpeI were analyzed by pulsed-field gel electrophoresis and Southern hybridization. The size of each fragment was calculated and used to demonstrate that C. crescentus has a genome size of approximately 4,000 kilobases. In addition, both enzymes gave rise to large DNA fragments which contained genes from both ends of the genetic map. Thus, there is physical linkage between the genes at the ends of the genetic map and the chromosome is circular. Since this region of the chromosome appears to contain the replication terminus, we propose that recombination occurs at a high frequency in the vicinity of the terminus. This high frequency of recombination would prevent genetic linkage from being observed between genes on opposite sides of the terminus. Additional experiments using insertions which introduced new AseI and DraI restriction sites into the genome allowed us to calculate the physical distance between genes located in the vicinity of the replication terminus.     

The breakdown of genomic ancestry blocks in hybrid lineages given a finite number of recombination sites

When a lineage originates from hybridization genomic blocks of contiguous ancestry from different ancestors are fragmented through genetic recombination. The resulting blocks are delineated by so called junctions, which accumulate with every generation that passes. Modeling the accumulation of ancestry block junctions can elucidate processes and timeframes of genomic admixture. Previous models have not addressed ancestry block dynamics for chromosomes that consist of a finite number of recombination sites. However, genomic data typically consist of informative markers that are interspersed with fragments for which no ancestry information is available. Hence, repeated recombination events may occur between markers, effectively removing existing junctions. Here, we present an analytical treatment of the dynamics of the mean number of junctions over time, taking into account the number of recombination sites per chromosome, population size, genetic map length, and the frequency of the ancestral species in the founding hybrid swarm. We describe the expected number of junctions using equidistant molecular markers and estimate the number of junctions using random markers. This extended theory of junctions thus reflects properties of empirical data and can serve to study the genomic patterns following admixture.