After the duplication: gene loss and adaptation in Saccharomyces genomes

The ancient duplication of the Saccharomyces cerevisiae genome and subsequent massive loss of duplicated genes is apparent when it is compared to the genomes of related species that diverged before the duplication event. To learn more about the evolutionary effects of the duplication event, we compared the S. cerevisiae genome to other Saccharomyces genomes. We demonstrate that the whole genome duplication occurred before S. castellii diverged from S. cerevisiae. In addition to more accurately dating the duplication event, this finding allowed us to study the effects of the duplication on two separate lineages. Analyses of the duplication regions of the genomes indicate that most of the duplicated genes (approximately 85%) were lost before the speciation. Only a small amount of paralogous gene loss (4-6%) occurred after speciation. On the other hand, S. castellii appears to have lost several hundred genes that were not retained as duplicated paralogs. These losses could be related to genomic rearrangements that reduced the number of chromosomes from 16 to 9. In addition to S. castellii, other Saccharomyces sensu lato species likely diverged from S. cerevisiae after the duplication. A thorough analysis of these species will likely reveal other important outcomes of the whole genome duplication.     

Updated map of duplicated regions in the yeast genome

We have updated the map of duplicated chromosomal segments in the Saccharomyces cerevisiae genome originally published by Wolfe and Shields in 1997 (Nature 387, 708-713). The new analysis is based on the more sensitive Smith Waterman search method instead of BLAST. The parameters used to identify duplicated chromosomal regions were optimized such as to maximize the amount of the genome placed into paired regions, under the assumption that the hypothesis that the entire genome was duplicated in a single event is correct. The core of the new map, with 52 pairs of regions containing three or more duplicated genes, is largely unchanged from our original map. 39 tRNA gene pairs and one snRNA pair have been added. To find additional pairs of genes that may have been formed by whole genome duplication, we searched through the parts of the genome that are not covered by this core map, looking for putative duplicated chromosomal regions containing only two duplicate genes instead of three, or having lower-scoring gene pairs. This approach identified a further 32 candidate paired regions, bringing the total number of protein-coding genes on the duplication map to 905 (16% of the proteome). The updated map suggests that a second copy of the ribosomal DNA array has been deleted from chromosome IV.     

Sequences with the Potential to Form Stem-and-Loop Structures Are Associated with Coding-Region Duplications in Animal Mitochondrial DNA

Tandem duplications of gene-encoding regions occur in the mitochondrial DNA (mt DNA) of some individuals belonging to several species of whiptail lizards (genus Cnemidophorus). All or part of the duplicated regions of the mtDNAs from five different species were sequenced. In all, the duplication endpoints were within or immediately adjacent to sequences in tRNA, rRNA or protein genes that are capable of forming energetically stable stem-and-loop structures. In two of these mtDNAs, the duplication endpoints were also associated with a direct sequence repeat of 13 bp. The consistent association of stem-and-loop structures with duplication endpoints suggests that these structures may play a role in the duplication process. These data, combined with the absence of direct or palindromic repeats at three of the pairs of duplication endpoints, also suggest the existence of a mechanism for generating de novo duplications that is qualitatively different from those previously modeled.     

Yeast chromosomes have been significantly reshaped during their evolutionary history

The structure of the first eukaryotic genome, belonging to Saccharomyces cerevisiae, has been deduced; however, very little is known about its origin. In order to trace events that led to the current state of the Saccharomyces nuclear genomes, random fragments of genomic DNA from three yeasts were sequenced and compared to the S. cerevisiae database sequence. Whereas, S. cerevisiae and Saccharomyces bayanus show perfect synteny, a significant portion of the analysed fragments from Saccharomyces servazzii and Saccharomyces kluyveri show a different arrangement of genes when compared to S. cerevisiae. When the sequenced fragments were probed to the corresponding karyotype, a group of genes present on a single chromosome of S. servazzii and S. kluyveri had homologues scattered on several S. cerevisiae chromosomes. Apparently, extensive reorganisation of the chromosomes has taken place during evolution of the Saccharomyces yeasts. In addition, while one gross duplication could have taken place, at least a few genes have been duplicated independently at different time-points in the evolution.     

Transposable Element Distribution in the Yeast Genome Reflects A Role in Repeated Genomic Rearrangement Events on an Evolutionary Time Scale

Statistical analysis of the distribution of transposable elements (TEs) and tRNA genes in the genome of yeast Saccharomyces cerevisiae indicated that, although tRNA genes and other genes transcribed by RNA polymerase III are targets for TE insertion, the distribution of TEs was significantly more clumped than that of tRNAs. Genomic blocks putatively duplicated as the result of an ancient polyploidization event contained fewer TEs than expected by their length, and nearly two thirds of duplicated blocks lacked TEs altogether. In addition, the edges of duplicated blocks tended to be located in TE-poor genomic regions. These results can be explained by the hypotheses: (1) that transposition events have occurred well after block duplication; (2) that TEs have frequently played a role in genomic rearrangement events in yeast. According to this model, duplicated blocks identifiable as such in the present-day yeast genome are found largely in regions with low TE density because in such regions the duplicated structure has not been obscured by TE-mediated rearrangements.     

Structural diversity and evolution of the Rf-1 locus in the genus Oryza

The Rf-1 locus in rice is agriculturally important as it restores fertility in plants with BT-type cytoplasmic male sterility (CMS). The Rf-1 locus contains several duplicated copies of the gene responsible for restoration of fertility. We analyzed the genomic structure of the Rf-1 locus in the genus Oryza to clarify the structural diversity and evolution of the locus. We identified six genes (Rf-1A to Rf-1F) with homology to Rf-1 at this locus in Oryza species with an AA genome. The Rf-1 locus structures in the rice accessions examined were very complex and fell into at least six classification types. The nucleotide sequences of the duplicated genes and their flanking regions were highly conserved suggesting that the complex Rf-1 locus structures were produced by homologous recombination between the duplicated genes. The fact that complex Rf-1 locus structures were common to Oryza species that have evolved independently indicates that a duplication of the ancestral Rf-1 gene occurred early in rice evolution and that homologous recombination resulted in the diversification of Rf-1 locus structures. Additionally, the amino acid sequences of each duplicated gene were conserved between species. This suggests that the duplicated genes in the Rf-1 locus may have divergent functions and may act by controlling mitochondrial gene expression in rice as occurs in the restoration of CMS.     

Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments

There is growing evidence that duplications have played a major role in eucaryotic genome evolution. Sequencing data revealed the presence of large duplicated regions in the genomes of many eucaryotic organisms, and comparative studies have suggested that duplication of large DNA segments has been a continuing process during evolution. However, little experimental data have been produced regarding this issue. Using a gene dosage assay for growth recovery in Saccharomyces cerevisiae, we demonstrate that a majority of the revertant strains (58%) resulted from the spontaneous duplication of large DNA segments, either intra- or interchromosomally, ranging from 41 to 655 kb in size. These events result in the concomitant duplication of dozens of genes and in some cases in the formation of chimeric open reading frames at the junction of the duplicated blocks. The types of sequences at the breakpoints as well as their superposition with the replication map suggest that spontaneous large segmental duplications result from replication accidents. Aneuploidization events or suppressor mutations that do not involve large-scale rearrangements accounted for the rest of the reversion events (in 26 and 16% of the strains, respectively).     

Patterns of genome duplication within the Brassica napus genome.

The progenitor diploid genomes (A and C) of the amphidiploid Brassica napus are extensively duplicated with 73% of genomic clones detecting two or more duplicate sequences within each of the diploid genomes. This comprehensive duplication of loci is to be expected in a species that has evolved through a polyploid ancestor. The majority of the duplicate loci within each of the diploid genomes were found in distinct linkage groups as collinear blocks of linked loci, some of which had undergone a variety of rearrangements subsequent to duplication, including inversions and translocations. A number of identical rearrangements were observed in the two diploid genomes, suggesting they had occurred before the divergence of the two species. A number of linkage groups displayed an organization consistent with centric fusion and (or) fission, suggesting this mechanism may have played a role in the evolution of Brassica genomes. For almost every genetically mapped locus detected in the A genome a homologous locus was found in the C genome; the collinear arrangement of these homologous markers allowed the primary regions of homoeology between the two genomes to be identified. At least 16 gross chromosomal rearrangements differentiated the two diploid genomes during their divergence from a common ancestor.     

Consistent patterns of rate asymmetry and gene loss indicate widespread neofunctionalization of yeast genes after whole-genome duplication

We investigated patterns of rate asymmetry in sequence evolution among the gene pairs (ohnologs) formed by whole-genome duplication (WGD) in yeast species. By comparing three species (Saccharomyces cerevisiae, Candida glabrata, and S. castellii) that underwent WGD to a nonduplicated outgroup (Kluyveromyces lactis), and by using a synteny framework to establish orthology and paralogy relationships at each duplicated locus, we show that 56% of ohnolog pairs show significantly asymmetric protein sequence evolution. For ohnolog pairs that remain duplicated in two species there is a strong tendency for the faster-evolving copy in one species to be orthologous to the faster copy in the other species, which indicates that the evolutionary rate differences were established before speciation and hence soon after the WGD. We also present evidence that in cases where one ohnolog has been lost from the genome of a post-WGD species, the lost copy was likely to have been the faster-evolving member of the pair prior to its loss. These results suggest that a significant fraction of the retained ohnologs in yeast species underwent neofunctionalization soon after duplication.     

Conserved functions of yeast genes support the duplication, degeneration and complementation model for gene duplication

Gene duplication is often cited as a potential mechanism for the evolution of new traits, but this hypothesis has not been thoroughly tested experimentally. A classical model of gene duplication states that after gene duplication one copy of the gene preserves the ancestral function, while the other copy is free to evolve a new function. In an alternative duplication, divergence, and complementation model, duplicated genes are preserved because each copy of the gene loses some, but not all, of its functions through degenerating mutations. This results in the degenerating mutations in one gene being complemented by the other and vice versa. These two models make very different predictions about the function of the preduplication orthologs in closely related species. These predictions have been tested here for several duplicated yeast genes that appeared to be the leading candidates to fit the classical model. Surprisingly, the results show that duplicated genes are maintained because each copy carries out a subset of the conserved functions that were already present in the preduplication gene. Therefore, the results are not consistent with the classical model, but instead fit the duplication, divergence, and complementation model.     

Duplication processes in Saccharomyces cerevisiae haploid strains

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.     

Differences in the number of intrinsically disordered regions between yeast duplicated proteins, and their relationship with functional divergence

Background

Intrinsically disordered regions are enriched in short interaction motifs that play a critical role in many protein-protein interactions. Since new short interaction motifs may easily evolve, they have the potential to rapidly change protein interactions and cellular signaling. In this work we examined the dynamics of gain and loss of intrinsically disordered regions in duplicated proteins to inspect if changes after genome duplication can create functional divergence. For this purpose we used Saccharomyces cerevisiae and the outgroup species Lachancea kluyveri.

Principal Findings

We find that genes duplicated as part of a genome duplication (ohnologs) are significantly more intrinsically disordered than singletons (p<2.2^e-16, Wilcoxon), reflecting a preference for retaining intrinsically disordered proteins in duplicate. In addition, there have been marked changes in the extent of intrinsic disorder following duplication. A large number of duplicated genes have more intrinsic disorder than their L. kluyveri ortholog (29% for duplicates versus 25% for singletons) and an even greater number have less intrinsic disorder than the L. kluyveri ortholog (37% for duplicates versus 25% for singletons). Finally, we show that the number of physical interactions is significantly greater in the more intrinsically disordered ohnolog of a pair (p = 0.003, Wilcoxon).

Conclusion

This work shows that intrinsic disorder gain and loss in a protein is a mechanism by which a genome can also diverge and innovate. The higher number of interactors for proteins that have gained intrinsic disorder compared with their duplicates may reflect the acquisition of new interaction partners or new functional roles.     

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.     

Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.     

Duplication and Gene Conversion in the Drosophila melanogaster Genome

Using the genomic sequences of Drosophila melanogaster subgroup, the pattern of gene duplications was investigated with special attention to interlocus gene conversion. Our fine-scale analysis with careful visual inspections enabled accurate identification of a number of duplicated blocks (genomic regions). The orthologous parts of those duplicated blocks were also identified in the D. simulans and D. sechellia genomes, by which we were able to clearly classify the duplicated blocks into post- and pre-speciation blocks. We found 31 post-speciation duplicated genes, from which the rate of gene duplication (from one copy to two copies) is estimated to be 1.0×10⁻⁹ per single-copy gene per year. The role of interlocus gene conversion was observed in several respects in our data: (1) synonymous divergence between a duplicated pair is overall very low. Consequently, the gene duplication rate would be seriously overestimated by counting duplicated genes with low divergence; (2) the sizes of young duplicated blocks are generally large. We postulate that the degeneration of gene conversion around the edges could explain the shrinkage of “identifiable” duplicated regions; and (3) elevated paralogous divergence is observed around the edges in many duplicated blocks, supporting our gene conversion–degeneration model. Our analysis demonstrated that gene conversion between duplicated regions is a common and genome-wide phenomenon in the Drosophila genomes, and that its role should be especially significant in the early stages of duplicated genes. Based on a population genetic prediction, we applied a new genome-scan method to test for signatures of selection for neofunctionalization and found a strong signature in a pair of transporter genes.     

Genome Duplication and Gene Loss Affect the Evolution of Heat Shock Transcription Factor Genes in Legumes

Whole-genome duplication events (polyploidy events) and gene loss events have played important roles in the evolution of legumes. Here we show that the vast majority of Hsf gene duplications resulted from whole genome duplication events rather than tandem duplication, and significant differences in gene retention exist between species. By searching for intraspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found that genome duplications accounted for 42 of 46 Hsf-containing segments in Glycine max, while paired segments were rarely identified in Lotus japonicas, Medicago truncatula and Cajanus cajan. However, by comparing interspecies microsynteny, we determined that the great majority of Hsf-containing segments in Lotus japonicas, Medicago truncatula and Cajanus cajan show extensive conservation with the duplicated regions of Glycine max. These segments formed 17 groups of orthologous segments. These results suggest that these regions shared ancient genome duplication with Hsf genes in Glycine max, but more than half of the copies of these genes were lost. On the other hand, the Glycine max Hsf gene family retained approximately 75% and 84% of duplicated genes produced from the ancient genome duplication and recent Glycine-specific genome duplication, respectively. Continuous purifying selection has played a key role in the maintenance of Hsf genes in Glycine max. Expression analysis of the Hsf genes in Lotus japonicus revealed their putative involvement in multiple tissue-/developmental stages and responses to various abiotic stimuli. This study traces the evolution of Hsf genes in legume species and demonstrates that the rates of gene gain and loss are far from equilibrium in different species.     

Stability of large segmental duplications in the yeast genome

The high level of gene redundancy that characterizes eukaryotic genomes results in part from segmental duplications. Spontaneous duplications of large chromosomal segments have been experimentally demonstrated in yeast. However, the dynamics of inheritance of such structures and their eventual fixation in populations remain largely unsolved. We analyzed the stability of a vast panel of large segmental duplications in Saccharomyces cerevisiae (from 41 kb for the smallest to 268 kb for the largest). We monitored the stability of three different types of interchromosomal duplications as well as that of three intrachromosomal direct tandem duplications. In the absence of any selective advantage associated with the presence of the duplication, we show that a duplicated segment internally translocated within a natural chromosome is stably inherited both mitotically and meiotically. By contrast, large duplications carried by a supernumerary chromosome are highly unstable. Duplications translocated into subtelomeric regions are lost at variable rates depending on the location of the insertion sites. Direct tandem duplications are lost by unequal crossing over, both mitotically and meiotically, at a frequency proportional to their sizes. These results show that most of the duplicated structures present an intrinsic level of instability. However, translocation within another chromosome significantly stabilizes a duplicated segment, increasing its chance to get fixed in a population even in the absence of any immediate selective advantage conferred by the duplicated genes.     

Molecular Evolution of the Duplicated Amy Locus in the Drosophila Melanogaster Species Subgroup: Concerted Evolution Only in the Coding Region and an Excess of Nonsynonymous Substitutions in Speciation

From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a ``specialist' species for host plants and probably also occurs in the process of speciation in general.     

Pervasive and persistent redundancy among duplicated genes in yeast

The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication (~100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.