Direct estimation of per nucleotide and genomic deleterious mutation rates in Drosophila

Spontaneous mutations are the source of genetic variation required for evolutionary change, and are therefore important for many aspects of evolutionary biology. For example, the divergence between taxa at neutrally evolving sites in the genome is proportional to the per nucleotide mutation rate, u (ref. 1), and this can be used to date speciation events by assuming a molecular clock. The overall rate of occurrence of deleterious mutations in the genome each generation (U) appears in theories of nucleotide divergence and polymorphism, the evolution of sex and recombination, and the evolutionary consequences of inbreeding. However, estimates of U based on changes in allozymes or DNA sequences and fitness traits are discordant. Here we directly estimate u in Drosophila melanogaster by scanning 20 million bases of DNA from three sets of mutation accumulation lines by using denaturing high-performance liquid chromatography. From 37 mutation events that we detected, we obtained a mean estimate for u of 8.4 x 10(-9) per generation. Moreover, we detected significant heterogeneity in u among the three mutation-accumulation-line genotypes. By multiplying u by an estimate of the fraction of mutations that are deleterious in natural populations of Drosophila, we estimate that U is 1.2 per diploid genome. This high rate suggests that selection against deleterious mutations may have a key role in explaining patterns of genetic variation in the genome, and help to maintain recombination and sexual reproduction.     

Levels of naturally occurring DNA polymorphism correlate with recombination rates in D. melanogaster.

Two genomic regions with unusually low recombination rates in Drosophila melanogaster have normal levels of divergence but greatly reduced nucleotide diversity, apparently resulting from the fixation of advantageous mutations and the associated hitch-hiking effect. Here we show that for 20 gene regions from across the genome, the amount of nucleotide diversity in natural populations of D. melanogaster is positively correlated with the regional rate of recombination. This cannot be explained by variation in mutation rates and/or functional constraint, because we observe no correlation between recombination rates and DNA sequence divergence between D. melanogaster and its sibling species, D. simulans. We suggest that the correlation may result from genetic hitch-hiking associated with the fixation of advantageous mutants. Hitch-hiking thus seems to occur over a large fraction of the Drosophila genome and may constitute a major constraint on levels of genetic variation in nature.     

Deleterious mutations and the evolution of sexual reproduction

The origin and maintenance of sexual reproduction continues to be an important problem in evolutionary biology. If the deleterious mutation rate per genome per generation is greater than 1, then the greater efficiency of selection against these mutations in sexual populations may be responsible for the evolution of sex and related phenomena. In modern human populations detrimental mutations with small individual effects are probably accumulating faster than they are being eliminated by selection.     

Mutation and sex in a competitive world

How do deleterious mutations interact to affect fitness? The answer to this question has substantial implications for a variety of important problems in population biology, including the evolution of sex, the rate of adaptation and the conservation of small populations. Here we analyse a mathematical model of competition for food in which deleterious mutations affect competitive ability. We show that, if individuals usually compete in small groups, then competition can easily lead to a type of genetic interaction known as synergistic epistasis. This means that a deleterious mutation is most damaging in a genome that already has many other deleterious mutations. We also show that competition in small groups can produce a large advantage for sexual populations, both in mean fitness and in ability to resist invasion by asexual lineages. One implication of our findings is that experimental efforts to demonstrate synergistic epistasis may not succeed unless the experiments are redesigned to make them much more naturalistic.     

Fitness of RNA virus decreased by Muller's ratchet

Why sex exists remains an unsolved problem in biology. If mutations are on the average deleterious, a high mutation rate can account for the evolution of sex. One form of this mutational hypothesis is Muller's ratchet. If the mutation rate is high, mutation-free individuals become rare and they can be lost by genetic drift in small populations. In asexual populations, as Muller noted, the loss is irreversible and the load of deleterious mutations increases in a ratchet-like manner with the successive loss of the least-mutated individuals. Sex can be advantageous because it increases the fitness of sexual populations by re-creating mutation-free individuals from mutated individuals and stops (or slows) Muller's ratchet. Although Muller's ratchet is an appealing hypothesis, it has been investigated and documented experimentally in only one group of organisms--ciliated protozoa. I initiated a study to examine the role of Muller's ratchet on the evolution of sex in RNA viruses and report here a significant decrease in fitness due to Muller's ratchet in 20 lineages of the RNA bacteriophage phi 6. These results show that deleterious mutations are generated at a sufficiently high rate to advance Muller's ratchet in an RNA virus and that beneficial, backward and compensatory mutations cannot stop the ratchet in the observed range of fitness decrease.     

High mutation rate and predominance of insertions in the Caenorhabditis elegans nuclear genome

Mutations have pivotal functions in the onset of genetic diseases and are the fundamental substrate for evolution. However, present estimates of the spontaneous mutation rate and spectrum are derived from indirect and biased measurements. For instance, mutation rate estimates for Caenorhabditis elegans are extrapolated from observations on a few genetic loci with visible phenotypes and vary over an order of magnitude. Alternative approaches in mammals, relying on phylogenetic comparisons of pseudogene loci and fourfold degenerate codon positions, suffer from uncertainties in the actual number of generations separating the compared species and the inability to exclude biases associated with natural selection. Here we provide a direct and unbiased estimate of the nuclear mutation rate and its molecular spectrum with a set of C. elegans mutation-accumulation lines that reveal a mutation rate about tenfold higher than previous indirect estimates and an excess of insertions over deletions. Because deletions dominate patterns of C. elegans pseudogene variation, our observations indicate that natural selection might be significant in promoting small genome size, and challenge the prevalent assumption that pseudogene divergence accurately reflects the spontaneous mutation spectrum.     

The DNA sequence and biological annotation of human chromosome 1

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.     

The Drosophila melanogaster Genetic Reference Panel

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.     

Genome reshuffling of the copia element in an inbred line of Drosophila melanogaster

Mobile genetic elements are found in the genomes of many organisms, and because of their effects on genes and their ability to induce chromosomal rearrangements they are an important source of genetic variability. Transposition rates are usually found to be low, estimated at around 10(-3) per generation. Higher rates of transposition are observed, however, in crosses between certain strains of Drosophila melanogaster ('hybrid dysgenesis'), which can lead to a dramatic rearrangement of many mobile elements ('transposition bursts'). We have studied the chromosomal distribution of mdg-1 and copia mobile elements in 17 highly inbred lines of D. melanogaster, after 69 generations of sib-mating. Most lines show no changes, but one showed a complete reshuffling of the copia element. We conclude that the transpositions of the copia element in this line occurred rapidly in a few generations. This phenomenon, distinct from 'transposition bursts' in that only copia elements are involved, may account for the instability sometimes observed in inbred lines and may be important in creating genetic variability in highly homozygous populations.     

Rate of de novo mutations and the importance of father's age to disease risk

Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 Icelandic parent-offspring trios at high coverage. We show that in our samples, with an average father's age of 29.7, the average de novo mutation rate is 1.20?×?10(-8) per nucleotide per generation. Most notably, the diversity in mutation rate of single nucleotide polymorphisms is dominated by the age of the father at conception of the child. The effect is an increase of about two mutations per year. An exponential model estimates paternal mutations doubling every 16.5?years. After accounting for random Poisson variation, father's age is estimated to explain nearly all of the remaining variation in the de novo mutation counts. These observations shed light on the importance of the father's age on the risk of diseases such as schizophrenia and autism.     

Sequence and analysis of chromosome 2 of Dictyostelium discoideum

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.     

Demasculinization of X chromosomes in the Drosophila genus

X chromosomes evolve differently from autosomes, but general governing principles have not emerged. For example, genes with male-biased expression are under-represented on the X chromosome of D. melanogaster, but are randomly distributed in the genome of Anopheles gambiae. In direct global profiling experiments using species-specific microarrays, we find a nearly identical paucity of genes with male-biased expression on D. melanogaster, D. simulans, D. yakuba, D. ananassae, D. virilis and D. mojavensis X chromosomes. We observe the same under-representation on the neo-X of D. pseudoobscura. It has been suggested that precocious meiotic silencing of the X chromosome accounts for reduced X chromosome male-biased expression in nematodes, mammals and Drosophila. We show that X chromosome genes with male-biased expression are under-represented in somatic cells and in mitotic male germ cells. These data are incompatible with simple X chromosome inactivation models. Using expression profiling and comparative sequence analysis, we show that selective gene extinction on the X chromosome, creation of new genes on autosomes and changed genomic location of existing genes contribute to the unusual X chromosome gene content.     

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.     

DNA sequence and analysis of human chromosome 8

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.     

Haploidy or diploidy: which is better?

Although the evolutionary advantages of sexual reproduction have been extensively discussed, much less attention has been paid to haploid and diploid phases of the sexual life cycle. The relative lengths of these phases differ greatly in various taxa, including as extremes those with one or the other phase reduced to a single cell. Here we consider the efficiency of elimination of deleterious mutations as an evolutionary force and compare the mutation loads under haploid and diploid selection, Ln and L2n. With truncation-like selection, partial dominance, and heterozygous effect of a mutation less than about 1/4 its hemizygous effect, L2n less than Ln; otherwise L2n greater than Ln. The difference becomes important when the genomic deleterious mutation rate exceeds about 1 per genome. This suggests that the mutation rate, degree of dominance and mode of selection can be important in life-cycle evolution.     

Fitness reduction associated with the deletion of a satellite DNA array

Satellite DNA refers to a class of tandem repeats of very simple sequences, usually A + T or G + C rich, which form a satellite band on a CsCl gradient. Their ubiquity and abundance in higher eukaryotes have led to speculation about their functions. It has often been suggested that satellite DNAs are merely innocuous genetic parasites or comprise 'junk' DNA. The recent identification of an array of satellite DNA repeats as the Responder (Rsp) locus of Drosophila melanogaster provides a new perspective on these elements. Rsp is in the centromeric heterochromatin of most natural second chromosomes. It causes spermatids bearing it to degenerate after meiosis when the homologous second chromosome is a Segregation Distorter (SD) chromosome. That is, SD targets the Rsp locus on its homologue for destruction during spermatogenesis, causing meiotic drive. Why then does the Rsp locus, a large array of satellite repeats, exist at all? One plausible explanation is that its existence contributes to the fitness of flies bearing it, compensating for the loss through meiotic drive. A direct demonstration of the usefulness of any family of satellite DNA is to compare the fitnesses of individuals with and without it. Previously, such an experiment has been difficult because the absence of a characteristic phenotype has precluded an efficient selection of deletion mutations. In this report we attempt to demonstrate a fitness reduction associated with the deletion of Rsp satellite DNA as well as the life stages at which such a reduction occurs.     

Reduced adaptation of a non-recombining neo-Y chromosome

Sex chromosomes are generally believed to have descended from a pair of homologous autosomes. Suppression of recombination between the ancestral sex chromosomes led to the genetic degeneration of the Y chromosome. In response, the X chromosome may become dosage-compensated. Most proposed mechanisms for the degeneration of Y chromosomes involve the rapid fixation of deleterious mutations on the Y. Alternatively, Y-chromosome degeneration might be a response to a slower rate of adaptive evolution, caused by its lack of recombination. Here we report patterns of DNA polymorphism and divergence at four genes located on the neo-sex chromosomes of Drosophila miranda. We show that a higher rate of protein sequence evolution of the neo-X-linked copy of Cyclin B relative to the neo-Y copy is driven by positive selection, which is consistent with the adaptive hypothesis for the evolution of the Y chromosome. In contrast, the neo-Y-linked copies of even-skipped and roundabout show an elevated rate of protein evolution relative to their neo-X homologues, probably reflecting the reduced effectiveness of selection against deleterious mutations in a non-recombining genome. Our results provide evidence for the importance of sexual recombination for increasing and maintaining the level of adaptation of a population.     

Genome sequence and analysis of the tuber crop potato

Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop.     

Evolution of digital organisms at high mutation rates leads to survival of the flattest.

Darwinian evolution favours genotypes with high replication rates, a process called 'survival of the fittest'. However, knowing the replication rate of each individual genotype may not suffice to predict the eventual survivor, even in an asexual population. According to quasi-species theory, selection favours the cloud of genotypes, interconnected by mutation, whose average replication rate is highest. Here we confirm this prediction using digital organisms that self-replicate, mutate and evolve. Forty pairs of populations were derived from 40 different ancestors in identical selective environments, except that one of each pair experienced a 4-fold higher mutation rate. In 12 cases, the dominant genotype that evolved at the lower mutation rate achieved a replication rate >1.5-fold faster than its counterpart. We allowed each of these disparate pairs to compete across a range of mutation rates. In each case, as mutation rate was increased, the outcome of competition switched to favour the genotype with the lower replication rate. These genotypes, although they occupied lower fitness peaks, were located in flatter regions of the fitness surface and were therefore more robust with respect to mutations.