Recodon: Coalescent simulation of coding DNA sequences with recombination, migration and demography

Background

Coalescent simulations have proven very useful in many population genetics studies. In order to arrive to meaningful conclusions, it is important that these simulations resemble the process of molecular evolution as much as possible. To date, no single coalescent program is able to simulate codon sequences sampled from populations with recombination, migration and growth.

Results

We introduce a new coalescent program, called Recodon, which is able to simulate samples of coding DNA sequences under complex scenarios in which several evolutionary forces can interact simultaneously (namely, recombination, migration and demography). The basic codon model implemented is an extension to the general time-reversible model of nucleotide substitution with a proportion of invariable sites and among-site rate variation. In addition, the program implements non-reversible processes and mixtures of different codon models.

Conclusion

Recodon is a flexible tool for the simulation of coding DNA sequences under realistic evolutionary models. These simulations can be used to build parameter distributions for testing evolutionary hypotheses using experimental data. Recodon is written in C, can run in parallel, and is freely available from http://darwin.uvigo.es/.     

High recombination rates and hotspots in a Plasmodium falciparum genetic cross

Background

The human malaria parasite Plasmodium falciparum survives pressures from the host immune system and antimalarial drugs by modifying its genome. Genetic recombination and nucleotide substitution are the two major mechanisms that the parasite employs to generate genome diversity. A better understanding of these mechanisms may provide important information for studying parasite evolution, immune evasion and drug resistance.

Results

Here, we used a high-density tiling array to estimate the genetic recombination rate among 32 progeny of a P. falciparum genetic cross (7G8 × GB4). We detected 638 recombination events and constructed a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb per centimorgan and identified 54 candidate recombination hotspots. Similar to centromeres in other organisms, the sequences of P. falciparum centromeres are found in chromosome regions largely devoid of recombination activity. Motifs enriched in hotspots were also identified, including a 12-bp G/C-rich motif with 3-bp periodicity that may interact with a protein containing 11 predicted zinc finger arrays.

Conclusions

These results show that the P. falciparum genome has a high recombination rate, although it also follows the overall rule of meiosis in eukaryotes with an average of approximately one crossover per chromosome per meiosis. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination rate observed. The lack of recombination activity in centromeric regions is consistent with the observations of reduced recombination near the centromeres of other organisms.     

A coalescent model of recombination hotspots

Recent experimental findings suggest that the assumption of a homogeneous recombination rate along the human genome is too naive. These findings point to block-structured recombination rates; certain regions (called hotspots) are more prone than other regions to recombination. In this report a coalescent model incorporating hotspot or block-structured recombination is developed and investigated analytically as well as by simulation. Our main results can be summarized as follows: (1) The expected number of recombination events is much lower in a model with pure hotspot recombination than in a model with pure homogeneous recombination, (2) hotspots give rise to large variation in recombination rates along the genome as well as in the number of historical recombination events, and (3) the size of a (nonrecombining) block in the hotspot model is likely to be overestimated grossly when estimated from SNP data. The results are discussed with reference to the current debate about block-structured recombination and, in addition, the results are compared to genome-wide variation in recombination rates. A number of new analytical results about the model are derived.     

Refinement of primate copy number variation hotspots identifies candidate genomic regions evolving under positive selection

Background

Copy number variants (CNVs), defined as losses and gains of segments of genomic DNA, are a major source of genomic variation.

Results

In this study, we identified over 2,000 human CNVs that overlap with orthologous chimpanzee or orthologous macaque CNVs. Of these, 170 CNVs overlap with both chimpanzee and macaque CNVs, and these were collapsed into 34 hotspot regions of CNV formation. Many of these hotspot regions of CNV formation are functionally relevant, with a bias toward genes involved in immune function, some of which were previously shown to evolve under balancing selection in humans. The genes in these primate CNV formation hotspots have significant differential expression levels between species and show evidence for positive selection, indicating that they have evolved under species-specific, directional selection.

Conclusions

These hotspots of primate CNV formation provide a novel perspective on divergence and selective pressures acting on these genomic regions.     

Bayesian Inference of Local Trees Along Chromosomes by the Sequential Markov Coalescent

We propose a genealogy-sampling algorithm, Sequential Markov Ancestral Recombination Tree (SMARTree), that provides an approach to estimation from SNP haplotype data of the patterns of coancestry across a genome segment among a set of homologous chromosomes. To enable analysis across longer segments of genome, the sequence of coalescent trees is modeled via the modified sequential Markov coalescent (Marjoram and Wall, Genetics 7:16, 2006). To assess performance in estimating these local trees, our SMARTree implementation is tested on simulated data. Our base data set is of the SNPs in 10 DNA sequences over 50 kb. We examine the effects of longer sequences and of more sequences, and of a recombination and/or mutational hotspot. The model underlying SMARTree is an approximation to the full recombinant-coalescent distribution. However, in a small trial on simulated data, recovery of local trees was similar to that of LAMARC (Kuhner et al. Genetics 156:1393-1401, 2000a), a sampler which uses the full model.     

Dengue virus serotype infection specifies the activation of the unfolded protein response

Background

Tomato-infecting begomoviruses are widely distributed across the world and cause diseases of high economic impact on wide range of agriculturally important crops. Though recombination plays a pivotal role in diversification and evolution of these viruses, it is currently unknown whether there are differences in the number and quality of recombination events amongst different tomato-infecting begomovirus species. To examine this we sought to characterize the recombination events, estimate the frequency of recombination, and map recombination hotspots in tomato-infecting begomoviruses of South and Southeast Asia.

Results

Different methods used for recombination breakpoint analysis provided strong evidence for presence of recombination events in majority of the sequences analyzed. However, there was a clear evidence for absence or low Recombination events in viruses reported from North India. In addition, we provide evidence for non-random distribution of recombination events with the highest frequency of recombination being mapped in the portion of the N-terminal portion of Rep.

Conclusion

The variable recombination observed in these viruses signified that all begomoviruses are not equally prone to recombination. Distribution of recombination hotspots was found to be reliant on the relatedness of the genomic region involved in the exchange. Overall the frequency of phylogenetic violations and number of recombination events decreased with increasing parental sequence diversity. These findings provide valuable new information for understanding the diversity and evolution of tomato-infecting begomoviruses in Asia.     

Enrichment of meiotic recombination hotspot sequences by avidin capture technology

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5′-CCNCCNTNNCCNC-3′. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (> 3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.     

Phosphorylation-Independent Regulation of Atf1-Promoted Meiotic Recombination by Stress-Activated,p38 Kinase Spc1 of Fission Yeast

Background

Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.

Methodology/Principal Findings

We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.

Conclusions/Significance

The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.     

Suppression of gamma ray-induced illegitimate recombination in Escherichia coli by the DNA-binding protein H-NS

To study the mechanism of gamma-ray-induced illegitimate recombination, we examined the formation of lambdabio transducing phage in Escherichia coli after gamma-ray irradiation. We show that gamma-ray irradiation enhances the formation of lambdabio transducing phage during prophage induction. Moreover, an hns mutation synergistically enhanced the incidence of lambda-ray-induced illegitimate recombination. Next we determined the sequences at the recombination junctions of the lambdabio transducing phages induced by gamma-ray irradiation. Most of the recombination sites coincided with known hotspots. Among them, hotspot I accounted for 67% and 77% of gamma-ray-induced lambdabio transducing phages in the wild type and the hns mutant, respectively. Therefore, the recombination sites appear to occur mostly at hotspot I or at other hotspots, but rarely at non-hotspot sites. These results suggest that types of DNA damage other than the double-strand breaks induced at random sites are mainly responsible for the introduction of the site-specific or region-specific DNA double strand breaks that lead to recombination at the hotspots. The results also showed that the recombination events took place between DNA sequences possessing short stretches of homology. H-NS protein, which binds to curved DNA, suppresses illegitimate recombination in the presence and absence of gamma-ray irradiation. Models for gamma-ray-induced illegitimate recombination are discussed.     

Inference of demographic history from genealogical trees using reversible jump Markov chain Monte Carlo

Background  

Coalescent theory is a general framework to model genetic variation in a population. Specifically, it allows inference about population parameters from sampled DNA sequences. However, most currently employed variants of coalescent theory only consider very simple demographic scenarios of population size changes, such as exponential growth.     

Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots

Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3′ side of 5′ GCTGGTGG 3′, the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD. We found strongly enhanced cutting at Chi with certain preferred sequences, such as A or G at nucleotides 4–7, on the 3′ flank of the Chi octamer. These sequences also strongly increased Chi hotspot activity in E. coli cells. Our combined enzymatic and genetic results redefine the Chi hotspot sequence, implicate the nuclease domain in Chi recognition, indicate that nicking of one strand at Chi is RecBCD''s biologically important reaction in living cells, and enable more precise analysis of Chi''s role in recombination and genome evolution.     

Hotspots of homologous recombination in mouse meiosis

The molecular mapping of recombinational breakpoints in the proximal region of the mouse MHC has revealed four hotspots at which breakpoints are clustered. A direct comparison of the nucleotide sequences of two independent hotspots revealed common molecular elements: a consensus sequence of the middle-repetitive MT-family, a repeat of tetramer sequences and a sequence homologous to a solitary LTR of mouse retroviruses. Extremely high frequency of recombination is observed at these hotspots when particular MHC haplotypes are used in genetic crosses. Wild mouse-derived wm7 haplotype instigates recombination at the hotspot located at the 3′-end of the Lmp-2 gene only during female meiosis. Fine genetic analysis demonstrated that the wm7 haplotype carries a genetic factor to instigate recombination and another factor to suppress recombination specifically during male meiosis. In addition, there is no dose effect of the hotspot on frequency of recombination. Finally, we described an attempt to establish an efficient in vitro assay system for monitoring recombination using plasmid DNAs that contain the Lmp-2 hotspot and nuclear extracts prepared from mouse testis.     

A combination of cis and trans control can solve the hotspot conversion paradox

There is growing evidence that in a variety of organisms the majority of meiotic recombination events occur at a relatively small fraction of loci, known as recombination hotspots. If hotspot activity results from the DNA sequence at or near the hotspot itself (in cis), these hotspots are expected to be rapidly lost due to biased gene conversion, unless there is strong selection in favor of the hotspot itself. This phenomenon makes it very difficult to maintain existing hotspots and even more difficult for new hotspots to evolve; it has therefore come to be known as the "hotspot conversion paradox." I develop an analytical framework for exploring the evolution of recombination hotspots under the forces of selection, mutation, and conversion. I derive the general conditions under which cis- and trans-controlled hotspots can be maintained, as well as those under which new hotspots controlled by both a cis and a trans locus can invade a population. I show that the conditions for maintenance of and invasion by trans- or cis-plus-trans-controlled hotspots are broader than for those controlled entirely in cis. Finally, I show that a combination of cis and trans control may allow for long-lived polymorphisms in hotspot activity, the patterns of which may explain some recently observed features of recombination hotspots.     

A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.     

Recombination hotspots as a point process

The variation of the recombination rate along chromosomal DNA is one of the important determinants of the patterns of linkage disequilibrium. A number of inferential methods have been developed which estimate the recombination rate and its variation from population genetic data. The majority of these methods are based on modelling the genealogical process underlying a sample of DNA sequences and thus explicitly include a model of the demographic process. Here we propose a different inferential procedure based on a previously introduced framework where recombination is modelled as a point process along a DNA sequence. The approach infers regions containing putative hotspots based on the inferred minimum number of recombination events; it thus depends only indirectly on the underlying population demography. A Poisson point process model with local rates is then used to infer patterns of recombination rate estimation in a fully Bayesian framework. We illustrate this new approach by applying it to several population genetic datasets, including a region with an experimentally confirmed recombination hotspot.     

Origins of chromosomal rearrangement hotspots in the human genome: evidence from the <Emphasis Type="Italic">AZFa</Emphasis>deletion hotspots

Background  

The origins of the recombination hotspots that are a common feature of both allelic and non-allelic homologous recombination in the human genome are poorly understood. We have investigated, by comparative sequencing, the evolution of two hotspots of non-allelic homologous recombination on the Y chromosome that lie within paralogous sequences known to sponsor deletions resulting in male infertility.     

The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.     

Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.