Identification and characterization of shared duplications between rice and wheat provide new insight into grass genome evolution

The grass family comprises the most important cereal crops and is a good system for studying, with comparative genomics, mechanisms of evolution, speciation, and domestication. Here, we identified and characterized the evolution of shared duplications in the rice (Oryza sativa) and wheat (Triticum aestivum) genomes by comparing 42,654 rice gene sequences with 6426 mapped wheat ESTs using improved sequence alignment criteria and statistical analysis. Intraspecific comparisons identified 29 interchromosomal duplications covering 72% of the rice genome and 10 duplication blocks covering 67.5% of the wheat genome. Using the same methodology, we assessed orthologous relationships between the two genomes and detected 13 blocks of colinearity that represent 83.1 and 90.4% of the rice and wheat genomes, respectively. Integration of the intraspecific duplications data with colinearity relationships revealed seven duplicated segments conserved at orthologous positions. A detailed analysis of the length, composition, and divergence time of these duplications and comparisons with sorghum (Sorghum bicolor) and maize (Zea mays) indicated common and lineage-specific patterns of conservation between the different genomes. This allowed us to propose a model in which the grass genomes have evolved from a common ancestor with a basic number of five chromosomes through a series of whole genome and segmental duplications, chromosome fusions, and translocations.     

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.     

Low X/Y divergence in four pairs of papaya sex-linked genes

Sex chromosomes in flowering plants, in contrast to those in animals, evolved relatively recently and only a few are heteromorphic. The homomorphic sex chromosomes of papaya show features of incipient sex chromosome evolution. We investigated the features of paired X- and Y-specific bacterial artificial chromosomes (BACs), and estimated the time of divergence in four pairs of sex-linked genes. We report the results of a comparative analysis of long contiguous genomic DNA sequences between the X and hermaphrodite Y (Y(h)) chromosomes. Numerous chromosomal rearrangements were detected in the male-specific region of the Y chromosome (MSY), including inversions, deletions, insertions, duplications and translocations, showing the dynamic evolutionary process on the MSY after recombination ceased. DNA sequence expansion was documented in the two regions of the MSY, demonstrating that the cytologically homomorphic sex chromosomes are heteromorphic at the molecular level. Analysis of sequence divergence between four X and Y(h) gene pairs resulted in a estimated age of divergence of between 0.5 and 2.2 million years, supporting a recent origin of the papaya sex chromosomes. Our findings indicate that sex chromosomes did not evolve at the family level in Caricaceae, and reinforce the theory that sex chromosomes evolve at the species level in some lineages.     

Microsatellite DNA markers for rice chromosomes

We found 369 complete microsatellites, of which (CGG/GCC)_n was the most frequent, in 11 798 rice sequences in the database. Of these microsatellites, 35 out of 45 could be successfully converted into microsatellite DNA markers using sequence information in their flanking regions. Thus, the time and labor used to develop new microsatellite DNA markers could be saved by using these published sequences. Twenty eight polymorphic markers between Asominori (japonica) and IR24 (indica) have been correctly mapped on the rice genome and microsatellites appear to be randomly distributed in the rice chromosomes. Integration of these markers with the published microsatellite DNA markers showed that about 35% of the rice chromosomes were covered by the 56 microsatellite DNA markers. These microsatellites were hypervariable and were easily to assay by PCR; they were distributed to all chromosomes and therefore, one can easily select plants carrying desired chromosome regions using these microsatellite DNA markers. Thus, microsatellite maps should aid the development of new breeds of rice saving time, labor, and money.     

Silene tatarica microsatellites are frequently located in repetitive DNA

The genomic distribution of microsatellites can be explained by DNA slippage, slippage like processes and base substitutions. Nevertheless, microsatellites are also frequently associated with repetitive DNA, raising the question of the relative contributions of these processes to microsatellite genesis. We show that in Silene tatarica about 50% of the microsatellites isolated by an enrichment cloning protocol are associated with repetitive DNA. Based on the flanking sequences, we distinguished seven different classes of repetitive DNA. PCR primers designed for the flanking sequences of an individual clone amplified a heterogeneous family of repetitive DNA. Despite considerable variation in the flanking sequence (pi = 0.108), the microsatellite repeats did not show any evidence for decay. Rather, we observed the emergence of a new repeat type that probably arose by mutation and was spread by replication slippage. In fact, a complete repeat type switch could be observed among the analysed clones. We propose that the analysis of microsatellite sequences embedded in repetitive DNA provides a hitherto largely unexplored tool to study microsatellite evolution.     

Microsatellite evolution: polarity of substitutions within repeats and neutrality of flanking sequences.

Though extensively used in a variety of disciplines, the evolutionary pattern of microsatellite sequences is still unclear. We addressed several questions relating to microsatellite evolution by analysing historically accumulated mutation events in a large set of artiodactyl (CA)n repeats, through sequence analysis of orthologous bovine and ovine loci. The substitution rate in microsatellite flanking sequences was not different from that in intron sequences, suggesting that if intron sequences in general are selectively neutral, sequences close to microsatellites are similarly so. This observation thus does not support the idea that successful heterologous amplification of microsatellites across distantly related taxa would be due to flanking sequences generally being under some form of selection. Interestingly, the substitution rate at the first nucleotide positions flanking repeats was significantly higher than in sequences further away. Moreover, the substitution rate in repeat units in the very end of microsatellites was significantly higher than that in the middle of repeat regions. Together these observations suggest a relative instability close to the boundary between repetitive and unique sequences. We present three models that potentially could explain such a feature, all involving inefficiency of mismatch repair systems.     

Recent duplication of the common carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci

Genome duplications may have played a role in the early stages of vertebrate evolution, near the time of divergence of the lamprey lineage. Additional genome duplication, specifically in ray-finned fish, may have occurred before the divergence of the teleosts. The common carp (Cyprinus carpio) has been considered tetraploid because of its chromosome number (2n = 100) and its high DNA content. We studied variation using 59 microsatellite primer pairs to better understand the ploidy level of the common carp. Based on the number of PCR amplicons per individual, about 60% of these primer pairs are estimated to amplify duplicates. Segregation patterns in families suggested a partially duplicated genome structure and disomic inheritance. This could suggest that the common carp is tetraploid and that polyploidy occurred by hybridization (allotetraploidy). From sequences of microsatellite flanking regions, we estimated the difference per base between pairs of alleles and between pairs of paralogs. The distribution of differences between paralogs had two distinct modes suggesting one whole-genome duplication and a more recent wave of segmental duplications. The genome duplication was estimated to have occurred about 12 MYA, with the segmental duplications occurring between 2.3 and 6.8 MYA. At 12 MYA, this would be one of the most recent genome duplications among vertebrates. Phylogenetic analysis of several cyprinid species suggests an evolutionary model for this tetraploidization, with a role for polyploidization in speciation and diversification.     

Genealogical use of chloroplast DNA variation for intraspecific studies of Aegilops tauschii Coss.

Intraspecific patterns of chloroplast DNA variation was studied in Aegilops tauschii Coss., the D-genome progenitor of bread wheat. Nucleotide sequences of ten chloroplast microsatellite loci were analyzed for 63 accessions that cover the central part of the species distribution. As is often the case with nuclear microsatellites, those of chloroplasts of Ae. tauschii bear complex mutations. Several types of mutations other than change in the microsatellite repeat number were found, including base substitutions and length mutations in flanking regions. In total, eight mutations were present in the flanking regions of four loci. Most mutations in the flanking regions of microsatellite repeats are associated with biallelic polymorphisms. Phylogeographic analyses showed that such biallelic polymorphisms are useful to investigate intraspecific patterns of monophyletic lineage divergence. In contrast, most microsatellite repeat sites are multiallelic, variable within intraspecific lineages, and useful to compare degrees of genetic diversity between lineages. These findings show that the chloroplast genome harbors evolutionary variations informative for intraspecific studies of Ae. tauschii and can be analyzed by genealogical approaches.Electronic Supplementary Material Supplementary material is available for this article at     

Genome-wide analysis of conservation and divergence of microsatellites in rice

Studies on microsatellite distribution and divergence in related genomes contribute towards understanding of genome evolution in eukaryotes. Despite the availability of whole genome sequences of four rice genomes, occurrence and significance of microsatellites in the rice genome has remained a relatively unexplored area of research. We have aligned genomes of two rice subspecies i.e. indica and japonica to understand the trends of microsatellite conservation and divergence in the rice genome. Nearly 62% of the indica microsatellites were also found in the japonica genome. Occurrence of microsatellites showed a negative association with that of retrotransposons. Microsatellites repeat unit length and sequence showed direct influence on the microsatellite locus length. Further, microsatellite allele length was also influenced by the sequence characteristics of the neighbouring regions. CCG repeats were most conserved microsatellite sequences across the different syntenic regions in the two rice genomes and often showed association with CpG islands. Our study suggested that microsatellite distribution is not only governed by a balance between replication slippage and point mutations as proposed earlier, but also by the microsatellite motif sequence and characteristics of microsatellite neighbouring regions in the genome. Thus, this study is likely to prove an important reference for understanding the process of microsatellite evolution and dynamics in the two rice subspecies.     

Evidence for widespread convergent evolution around human microsatellites

Microsatellites are a major component of the human genome, and their evolution has been much studied. However, the evolution of microsatellite flanking sequences has received less attention, with reports of both high and low mutation rates and of a tendency for microsatellites to cluster. From the human genome we generated a database of many thousands of (AC)_n flanking sequences within which we searched for common characteristics. Sequences flanking microsatellites of similar length show remarkable levels of convergent evolution, indicating shared mutational biases. These biases extend 25–50 bases either side of the microsatellite and may therefore affect more than 30% of the entire genome. To explore the extent and absolute strength of these effects, we quantified the observed convergence. We also compared homologous human and chimpanzee loci to look for evidence of changes in mutation rate around microsatellites. Most models of DNA sequence evolution assume that mutations are independent and occur randomly. Allowances may be made for sites mutating at different rates and for general mutation biases such as the faster rate of transitions over transversions. Our analysis suggests that these models may be inadequate, in that proximity to even very short microsatellites may alter the rate and distribution of mutations that occur. The elevated local mutation rate combined with sequence convergence, both of which we find evidence for, also provide a possible resolution for the apparently contradictory inferences of mutation rates in microsatellite flanking sequences.     

Mutation and evolution of microsatellite loci in Neurospora

The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora. First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora. To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data. This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies. In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions. By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species. In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses. Frequency distributions of alleles within species were generally consistent with the stepwise mutational model. By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies. A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles. To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Sequence-tagged site markers for microsatellites: Simplified technique for rapidly obtaining flanking sequences

A sequencing strategy is described for the rapid recovery of DNA sequences flanking repeat sequences of microsatellites in plant nuclear genomes. Primers that represent a perfect microsatellite repeat and end in a 3′ degenerate base have been used to sequence directly from microsatellite repeats in one direction. The procedure allows the design of one flanking primer that is then used to sequence back through the repeat to define the microsatellite site precisely and also provides for the design of the second flanking primer. The strategy is versatile with various repeat sizes and different categories of microsatellites; perfect, imperfect, and compound were found to be suitable templates for analysis.     

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.     

Evolutionary constraint in flanking regions of avian genes

An important comprehension from comparative genomic analysis is that sequence conservation beyond neutral expectations is frequently found outside protein-coding regions, indicating important functional roles of noncoding DNA. Understanding the causes of constraint on noncoding sequence evolution forms an important area of research, not least in light of the importance for understanding the evolution of gene expression. We aligned all orthologous genes of chicken and zebra finch together with 5 kb of their upstream and downstream noncoding sequences, to study the evolution of gene flanking sequences in the avian genome. Using ancestral repeats as a neutral reference, we detected significant evolutionary constraint in the 3' flanking region, highest directly after termination (60%) and then gradually decreasing to about 20% 5 kb downstream. Constraint was higher in annotated 3' untranslated regions (UTRs) than in non-UTRs at the same distance from the stop codon and higher in sequences annotated as microRNA (miRNA)-binding sites than in non-miRNA-binding sites within 3' UTRs. Constraint was also higher when estimated for a smaller data set of genes from more closely related songbird species, indicating turnover of functional elements during avian evolution. On the 5' flanking side constraint was readily seen within the first 125 bp immediately upstream of the start codon (34%) and was about 10% for remaining sequence within 5 kb upstream. Analysis of chicken polymorphism data gave further support for the highest constraint directly before and after the translated region. Finally, we found that genes evolving under the highest constraint measured by d(N)/d(S) also had the highest level of constraint in the 3' flanking region. This study broadens the insights into gene flanking sequence evolution by adding new findings from a vertebrate lineage other than mammals.     

Heterogeneous Nature and Distribution of Interruptions in Dinucleotides May Indicate the Existence of Biased Substitutions Underlying Microsatellite Evolution

Some aspects of microsatellite evolution, such as the role of base substitutions, are far from being fully understood. To examine the significance of base substitutions underlying the evolution of microsatellites we explored the nature and the distribution of interruptions in dinucleotide repeats from the human genome. The frequencies that we inferred in the repetitive sequences were statistically different from the frequencies observed in other noncoding sequences. Additionally, we detected that the interruptions tended to be towards the ends of the microsatellites and 5'-3' asymmetry. In all the estimates nucleotides forming the same repetitive motif seem to be affected by different base substitution rates in AC and AG. This tendency itself could generate patterning and similarity in flanking sequences and reconcile these phenomena with the high mutation rate found in flanking sequences without invoking convergent evolution. Nevertheless, our data suggest that there is a regional bias in the substitution pattern of microsatellites. The accumulation of random substitutions alone cannot explain the heterogeneity and the asymmetry of interruptions found in this study or the relative frequency of different compound microsatellites in the human genome. Therefore, we cannot rule out the possibility of a mutational bias leading to convergent or parallel evolution in flanking sequences.     

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites. Across 36,229 base pairs of noncoding sequence representing 41 genetic loci, a total of 307 polymorphisms including 248 polymorphic microsatellites and 39 SNPs were identified. We found a significant excess of microsatellite polymorphisms having a repeat unit length of one or two, compared to those with longer repeat lengths, as well as a nonrandom distribution of SNP polymorphisms. Almost half of the SNPs localized to only three of the 41 genetic loci sampled. Furthermore, we find significant differences in the frequency of polymorphisms across the two chromosomes (2 and 3) examined most extensively, with an excess of SNPs and a surplus of polymorphic microsatellites on chromosome 3 as compared to chromosome 2 (P=0.0001). Furthermore, at some individual genetic loci we also find a nonrandom distribution of polymorphisms between coding and flanking noncoding sequences, where completely monomorphic regions may flank highly polymorphic genes. These data, combined with our previous findings of nonrandom distribution of SNPs across chromosome 2, suggest that the Plasmodium falciparum genome may be a mosaic with regard to genetic diversity, containing chromosomal regions that are highly polymorphic interspersed with regions that are much less polymorphic.     

Extensive concerted evolution of rice paralogs and the road to regaining independence

Many genes duplicated by whole-genome duplications (WGDs) are more similar to one another than expected. We investigated whether concerted evolution through conversion and crossing over, well-known to affect tandem gene clusters, also affects dispersed paralogs. Genome sequences for two Oryza subspecies reveal appreciable gene conversion in the approximately 0.4 MY since their divergence, with a gradual progression toward independent evolution of older paralogs. Since divergence from subspecies indica, approximately 8% of japonica paralogs produced 5-7 MYA on chromosomes 11 and 12 have been affected by gene conversion and several reciprocal exchanges of chromosomal segments, while approximately 70-MY-old "paleologs" resulting from a genome duplication (GD) show much less conversion. Sequence similarity analysis in proximal gene clusters also suggests more conversion between younger paralogs. About 8% of paleologs may have been converted since rice-sorghum divergence approximately 41 MYA. Domain-encoding sequences are more frequently converted than nondomain sequences, suggesting a sort of circularity--that sequences conserved by selection may be further conserved by relatively frequent conversion. The higher level of concerted evolution in the 5-7 MY-old segmental duplication may reflect the behavior of many genomes within the first few million years after duplication or polyploidization.     

Contrasted patterns of molecular evolution in dominant and recessive self-incompatibility haplotypes in Arabidopsis

Self-incompatibility has been considered by geneticists a model system for reproductive biology and balancing selection, but our understanding of the genetic basis and evolution of this molecular lock-and-key system has remained limited by the extreme level of sequence divergence among haplotypes, resulting in a lack of appropriate genomic sequences. In this study, we report and analyze the full sequence of eleven distinct haplotypes of the self-incompatibility locus (S-locus) in two closely related Arabidopsis species, obtained from individual BAC libraries. We use this extensive dataset to highlight sharply contrasted patterns of molecular evolution of each of the two genes controlling self-incompatibility themselves, as well as of the genomic region surrounding them. We find strong collinearity of the flanking regions among haplotypes on each side of the S-locus together with high levels of sequence similarity. In contrast, the S-locus region itself shows spectacularly deep gene genealogies, high variability in size and gene organization, as well as complete absence of sequence similarity in intergenic sequences and striking accumulation of transposable elements. Of particular interest, we demonstrate that dominant and recessive S-haplotypes experience sharply contrasted patterns of molecular evolution. Indeed, dominant haplotypes exhibit larger size and a much higher density of transposable elements, being matched only by that in the centromere. Overall, these properties highlight that the S-locus presents many striking similarities with other regions involved in the determination of mating-types, such as sex chromosomes in animals or in plants, or the mating-type locus in fungi and green algae.