Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and Am genomes of wheat

To study genome evolution in wheat, we have sequenced and compared two large physical contigs of 285 and 142 kb covering orthologous low molecular weight (LMW) glutenin loci on chromosome 1AS of a diploid wheat species (Triticum monococcum subsp monococcum) and a tetraploid wheat species (Triticum turgidum subsp durum). Sequence conservation between the two species was restricted to small regions containing the orthologous LMW glutenin genes, whereas >90% of the compared sequences were not conserved. Dramatic sequence rearrangements occurred in the regions rich in repetitive elements. Dating of long terminal repeat retrotransposon insertions revealed different insertion events occurring during the last 5.5 million years in both species. These insertions are partially responsible for the lack of homology between the intergenic regions. In addition, the gene space was conserved only partially, because different predicted genes were identified on both contigs. Duplications and deletions of large fragments that might be attributable to illegitimate recombination also have contributed to the differentiation of this region in both species. The striking differences in the intergenic landscape between the A and A(m) genomes that diverged 1 to 3 million years ago provide evidence for a dynamic and rapid genome evolution in wheat species.     

Conserved fragments of transposable elements in intergenic regions: evidence for widespread recruitment of MIR- and L2-derived sequences within the mouse and human genomes

We analysed the distribution of transposable elements (TEs) in 100 aligned pairs of orthologous intergenic regions from the mouse and human genomes. Within these regions, conserved segments of high similarity between the two species alternate with segments of low similarity. Identifiable TEs comprise 40-60% of segments of low similarity. Within such segments, a particular copy of a TE found in one species has no orthologue in the other. Overall, TEs comprise only approximately 20 % of conserved segments. However, TEs from two families, MIR and L2, are rather common within conserved segments. Statistical analysis of the distributions of TEs suggests that a majority of the MIR and L2 elements present in murine intergenic regions have human orthologues. These elements must have been present in the common ancestor of human and mouse and have remained under substantial negative selection that prevented their divergence beyond recognition. If so, recruitment of MIR- and L2-derived sequences to perform a function that increases host fitness is rather common, with at least two such events per host gene. The central part of the MIR consensus sequence is over-represented in conserved segments given its background frequency in the genome, suggesting that it is under the strongest selective constraint.     

Inter-species sequence comparison of Brachypodium reveals how transposon activity corrodes genome colinearity

Intergenic sequences evolve rapidly in plant genomes through a process known as genomic turnover. To investigate the influence of DNA transposons on genomic turnover, we compared 1 Mbp of orthologous genomic sequences from Brachypodium distachyon and Brachypodium sylvaticum. We found that B. distachyon and B. sylvaticum diverged approximately 1.7-2.0 million years ago. Of a total of 219 genes identified on the analyzed sequences, 211 were colinear. However, only 24 transposable elements of a total of 451 were orthologous (i.e. inserted in the common ancestor). We characterized in detail 59 insertions and 60 excisions of DNA transposons in one or other species, which altered 17% of the intergenic space. The DNA transposon excision sites showed complex and highly diagnostic sequence motifs for double-strand break (DSB) repair. DNA transposon excisions can lead to extensive deletions of hundreds of base pairs of flanking sequence if the DSB is repaired by 'single-strand annealing', or insertions of up to several hundred base pairs of 'filler DNA' if the DSB is repaired by 'synthesis-dependent strand annealing'. In some cases, DSBs were repaired by a combination of both methods. We present a model for the evolution of intergenic sequences in which repair of DSBs upon DNA transposon excision is a major factor in the rapid turnover and erosion of intergenic sequences.     

Further evidence of microcolinearity between barley and rice genomes at two orthologous regions

Two genetic markers, BCD135 and RZ567 were used to select clones from genomic BAC libraries of barley and rice for sequencing and subsequent sequence comparisons. A set of two orthologous BACs each from barley and rice was selected by hybridization with BCD135 and RZ567 cDNA probes. A total of 556-kb stretch including two barley BACs (773K135 and 745C13) and two orthologous rice BACs (24K23 and 49D11) was completely sequenced. Comparative sequence analysis between orthologous BACs from the two species revealed presence of two conserved genes at BCD135 region and only one gene at the RZ567 regions. The two conserved genes were in the same order and orientation in both the species however, separated by significantly larger distance in barley. The larger distance between two barley genes was mainly due to presence of different retrotransposable elements and their derivatives (78.9% of the intergenic region) that expanded the barley BCD135 region at the rate of 9.1X. An additional gene of unknown function was also inserted along with several retrotransposable elements between two conserved genes at barley BCD135 region. More genome expansion rate (10X) around barley RZ567 locus was estimated by extremely high proportion (> 70%) of retrotransposons. Among different retrotransposons, the Sabrina elements rather than BARE were more prevalent in both the regions. Contrary to it, the BCD135 region of rice was composed of only 17.1% retrotransposable elements and no significant retrotransposons except 14 miniature inverted transposable elements (MITEs) were observed in its RZ567 region. The sequence comparison between orthologous regions of rice and barley genomes was useful for gene identification and determination of individual gene structure indicating the possibility of effective utilization of rice genome sequences in understanding the large genome of barley. (The sequence data described in this paper have been submitted to the GenBank data library under the accession no. AF474072 (773K14), AF474071 (745C13), AF480497 (24K23) and AF480496 (49D11)).     

Rapid genome evolution revealed by comparative sequence analysis of orthologous regions from four triticeae genomes

Bread wheat (Triticum aestivum) is an allohexaploid species, consisting of three subgenomes (A, B, and D). To study the molecular evolution of these closely related genomes, we compared the sequence of a 307-kb physical contig covering the high molecular weight (HMW)-glutenin locus from the A genome of durum wheat (Triticum turgidum, AABB) with the orthologous regions from the B genome of the same wheat and the D genome of the diploid wheat Aegilops tauschii (Anderson et al., 2003; Kong et al., 2004). Although gene colinearity appears to be retained, four out of six genes including the two paralogous HMW-glutenin genes are disrupted in the orthologous region of the A genome. Mechanisms involved in gene disruption in the A genome include retroelement insertions, sequence deletions, and mutations causing in-frame stop codons in the coding sequences. Comparative sequence analysis also revealed that sequences in the colinear intergenic regions of these different genomes were generally not conserved. The rapid genome evolution in these regions is attributable mainly to the large number of retrotransposon insertions that occurred after the divergence of the three wheat genomes. Our comparative studies indicate that the B genome diverged prior to the separation of the A and D genomes. Furthermore, sequence comparison of two distinct types of allelic variations at the HMW-glutenin loci in the A genomes of different hexaploid wheat cultivars with the A genome locus of durum wheat indicates that hexaploid wheat may have more than one tetraploid ancestor.     

Large intraspecific haplotype variability at the Rph7 locus results from rapid and recent divergence in the barley genome

To study genome evolution and diversity in barley (Hordeum vulgare), we have sequenced and compared more than 300 kb of sequence spanning the Rph7 leaf rust disease resistance gene in two barley cultivars. Colinearity was restricted to five genic and two intergenic regions representing <35% of the two sequences. In each interval separating the seven conserved regions, the number and type of repetitive elements were completely different between the two homologous sequences, and a single gene was absent in one cultivar. In both cultivars, the nonconserved regions consisted of approximately 53% repetitive sequences mainly represented by long-terminal repeat retrotransposons that have inserted <1 million years ago. PCR-based analysis of intergenic regions at the Rph7 locus and at three other independent loci in 41 H. vulgare lines indicated large haplotype variability in the cultivated barley gene pool. Together, our data indicate rapid and recent divergence at homologous loci in the genome of H. vulgare, possibly providing the molecular mechanism for the generation of high diversity in the barley gene pool. Finally, comparative analysis of the gene composition in barley, wheat (Triticum aestivum), rice (Oryza sativa), and sorghum (Sorghum bicolor) suggested massive gene movements at the Rph7 locus in the Triticeae lineage.     

Conservation and purifying selection of transcribed genes located in a rice centromere

Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8 genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements.     

Evolution of three Pyrenophora cereal pathogens: Recent divergence, speciation and evolution of non-coding DNA

Three of the most important fungal pathogens of cereals are Pyrenophora tritici-repentis, the cause of tan spot on wheat, and Pyrenophora teres f. teres and Pyrenophora teres f. maculata, the cause of spot form and net form of net blotch on barley, respectively. Orthologous intergenic regions were used to examine the genetic relationships and divergence times between these pathogens. Mean divergence times were calculated at 519kya (±30) between P. teresf. teres and P. teresf. maculata, while P. tritici-repentis diverged from both Pyrenophora teresforms 8.04Mya (±138ky). Individual intergenic regions showed a consistent pattern of co-divergence of the P. teresforms from P. tritici-repentis, with the pattern supported by phylogenetic analysis of conserved genes. Differences in calculated divergence times between individual intergenic regions suggested that they are not entirely under neutral selection, a phenomenon shared with higher Eukaryotes. P. tritici-repentis regions varied in divergence time approximately 5-12Mya from the P. teres lineage, compared to the separation of wheat and barley some 12Mya, while the P. teresf. teres and P. teresf. maculata intergenic region divergences correspond to the middle Pleistocene. The data suggest there is no correlation between the divergence of these pathogens the domestication of wheat and barley, and show P. teresf. teres and P. teresf. maculata are closely related but autonomous. The results are discussed in the context of speciation and the evolution of intergenic regions.     

Effect of Divergence Time and Recombination Rate on Molecular Evolution of <Emphasis Type="Italic">Drosophila</Emphasis> INE-1 Transposable Elements and Other Candidates for Neutrally Evolving Sites

Interspecies divergence of orthologous transposable element remnants is often assumed to be simply due to genetic drift of neutral mutations that occurred after the divergence of the species. However, divergence may also be affected by other factors, such as variation in the mutation rate, ancestral polymorphisms, or selection. Here we attempt to determine the impact of these forces on divergence of three classes of sites that are often assumed to be selectively unconstrained (INE-1 TE remnants, sites within short introns, and fourfold degenerate sites) in two different pairwise comparisons of Drosophila (D. melanogaster vs. D. simulans and D. simulans vs. D. sechellia). We find that divergence of these three classes of sites is strongly influenced by the recombination environment in which they are located, and this is especially true for the closer D. simulans vs. D. sechellia comparison. We suggest that this is mainly a result of the contribution of ancestral polymorphisms in different recombination regions. We also find that intergenic INE-1 elements are significantly more diverged than intronic INE-1 in both pairwise comparisons, implying the presence of either negative selection or lower mutation rates in introns. Furthermore, we show that substitution rates in INE-1 elements are not associated with the length of the noncoding sequence in which they are located, suggesting that reduced divergence in long noncoding sequences is not due to reduced mutation rates in these regions. Finally, we show that GC content for each site within INE-1 sequences has evolved toward an equilibrium value (approximately 33%) since insertion.     

Molecular Analysis of the Hot Spot Region Related to Length Mutations in Wheat Chloroplast Dnas. I. Nucleotide Divergence of Genes and Intergenic Spacer Regions Located in the Hot Spot Region

The nucleotide divergence of chloroplast DNAs around the hot spot region related to length mutation in Triticum (wheat) and Aegilops was analyzed. DNA sequences (ca. 4.5 kbp) of three chloroplast genome types of wheat complex were compared with one another and with the corresponding region of other grasses. The sequences region contained rbcL and psaI, two open reading frames, and a pseudogene, rpl23' (pseudogene for ribosomal protein L23) disrupted by AT-rich intergic spacer regions. The evolution of these genes in the closely related wheat complex is characterized by nonbiased nucleotide substitutions in terms of being synonymous/nonsynonymous, having A-T pressure transitions over transversions, and frequent changes at the third codon position, in contrast with the gene evolution among more distant plant groups where biased nucleotide substitutions have frequently occurred. The sequences of these genes had diverged almost in proportion to taxonomic distance. The sequence of the pseudogene rpl23' changed approximately two times faster than that of the coding region. Sequence comparison between the pseudogene and its protein-coding counterpart revealed different degrees of nucleotide homology in wheat, rice and maize, suggesting that the transposition timing of the pseudogene differed and/or that different rates of gene conversion operated on the pseudogene in the cpDNA of the three plant groups in Gramineae. The intergenic spacer regions diverged approximately ten times faster than the genes. The divergence of wheat from barley, and that from rice are estimated based on the nucleotide similarity to be 1.5, 10 and 40 million years, respectively.     

Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat.

We used PCR to obtain phylogenetically informative sequences from the high molecular weight glutenin genes of wheat. The validity of partial sequence comparisons as a means of studying glutenin phylogenetics was established by constructing neighbour-joining trees from partial alignments of 12 published glutenin allele sequences. PCR was then used to obtain 20 novel glutenin allele sequences from various Triticum and Aegilops species, including a 3000 year old preserved wheat. A neighbour-joining tree derived from all known glutenin allele sequences had eight clades, representing the eight loci from which the allele sequences were derived, and was split into two halves, one comprising alleles from the Glu-1-1 loci and the other comprising Glu-1-2 alleles. The topology was compatible with the postulated relationships between the A, B, D, and G genomes. The Glu gene duplication event was tentatively dated at 7.2-10.0 million years ago (MYA), the origin of the four genomes at 5.0-6.9 MYA, and the split between the B and G genomes at 2.5-3.5 MYA. The Glu-B1-1 alleles in cultivated wheats fell into two subgroups that diverged 1.4-2.0 MYA, suggesting that emmer was domesticated twice. The D allele sequences were relatively diverse, indicating that the hybridization event that resulted in the hexaploid bread wheats might have occurred more than once.     

Nucleotide sequence of the rat gamma-crystallin gene region and comparison with an orthologous human region

The sequences of a 51-kb region containing the cluster of five rat gamma-crystallin-coding genes (CRYG) and of a 7-kb region surrounding the sixth rat CRYG gene were determined. Approximately 78% of the total sequence represents intergenic DNA. We also sequenced 22 kb of DNA from the human CRYG gene cluster. All CRYG genes are associated with CpG-rich regions. The sequence similarity between the human and rat gene regions drops sharply (to 65%) in intronic and 3'-flanking regions but decreases only gradually in the 5'-flanking region. Highly conserved regions (greater than 80%) are found as far upstream as 1.5 kb. Overall intergenic distances are conserved. The human region contains much more repetitive DNA (24% vs. 10%) but less simple-sequence (sps) DNA (0.7% vs. 4%) than the rat region. Almost all repeats and spsDNA elements are located in the intergenic region. The location of repetitive and spsDNA differs between the orthologous regions and these elements were probably inserted after the evolutionary separation of rat and man. The Alu repeats in man and the B3 repeats in the rat are close copies of their respective consensus sequences and bordered by virtually perfect repeats. In contrast, the B1 and B2 repeats in the rat have diverged considerably from the consensus sequence and the surrounding direct repeats are usually imperfect. Thus the dispersion of the B1 and B2 repeats in the rat probably preceded that of the B3 repeats. Within the rat genomic region the spacing of Z-DNA elements is surprisingly regular, they are located about 12 kb apart. A search for putative matrix-associated regions suggests that the rat CRYG gene cluster is organized into two chromosomal domains.     

Genome evolution in diploid and tetraploid Coffea species as revealed by comparative analysis of orthologous genome segments

Sequence comparison of orthologous regions enables estimation of the divergence between genomes, analysis of their evolution and detection of particular features of the genomes, such as sequence rearrangements and transposable elements. Despite the economic importance of Coffea species, little genomic information is currently available. Coffea is a relatively young genus that includes more than one hundred diploid species and a single tetraploid species. Three Coffea orthologous regions of 470-900 kb were analyzed and compared: both subgenomes of allotetraploid Coffea arabica (contributed by the diploid species Coffea eugenioides and Coffea canephora) and the genome of diploid C. canephora. Sequence divergence was calculated on global alignments or on coding and non-coding sequences separately. A search for transposable elements detected 43 retrotransposons and 198 transposons in the sequences analyzed. Comparative insertion analysis made it possible to locate 165 TE insertions in the phylogenetic tree of the three genomes/subgenomes. In the tetraploid C. arabica, a homoeologous non-reciprocal transposition (HNRT) was detected and characterized: a 50 kb region of the C. eugenioides derived subgenome replaced the C. canephora derived counterpart. Comparative sequence analysis on three Coffea genomes/subgenomes revealed almost perfect gene synteny, low sequence divergence and a high number of shared transposable elements. Compared to the results of similar analysis in other genera (Aegilops/Triticum and Oryza), Coffea genomes/subgenomes appeared to be dramatically less diverged, which is consistent with the relatively recent radiation of the Coffea genus. Based on nucleotide substitution frequency, the HNRT was dated at 10,000-50,000 years BP, which is also the most recent estimation of the origin of C. arabica.     

Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.     

Different types and rates of genome evolution detected by comparative sequence analysis of orthologous segments from four cereal genomes

Orthologous regions in barley, rice, sorghum, and wheat were studied by bacterial artificial chromosome sequence analysis. General microcolinearity was observed for the four shared genes in this region. However, three genic rearrangements were observed. First, the rice region contains a cluster of 48 predicted small nucleolar RNA genes, but the comparable region from sorghum contains no homologous loci. Second, gene 2 was inverted in the barley lineage by an apparent unequal recombination after the ancestors of barley and wheat diverged, 11-15 million years ago (mya). Third, gene 4 underwent direct tandem duplication in a common ancestor of barley and wheat 29-41 mya. All four of the shared genes show the same synonymous substitution rate, but nonsynonymous substitution rates show significant variations between genes 4a and 4b, suggesting that gene 4b was largely released from the strong purifying selection that acts on gene 4a in both barley and wheat. Intergenic retrotransposon blocks, many of them organized as nested insertions, mostly account for the lower gene density of the barley and wheat regions. All but two of the retrotransposons were found in the regions between genes, while all but 2 of the 51 inverted repeat transposable elements were found as insertions in genic regions and outside the retrotransposon blocks.     

Sequence-level analysis of the diploidization process in the triplicated FLOWERING LOCUS C region of Brassica rapa

Strong evidence exists for polyploidy having occurred during the evolution of the tribe Brassiceae. We show evidence for the dynamic and ongoing diploidization process by comparative analysis of the sequences of four paralogous Brassica rapa BAC clones and the homologous 124-kb segment of Arabidopsis thaliana chromosome 5. We estimated the times since divergence of the paralogous and homologous lineages. The three paralogous subgenomes of B. rapa triplicated 13 to 17 million years ago (MYA), very soon after the Arabidopsis and Brassica divergence occurred at 17 to 18 MYA. In addition, a pair of BACs represents a more recent segmental duplication, which occurred approximately 0.8 MYA, and provides an exception to the general expectation of three paralogous segments within the B. rapa genome. The Brassica genome segments show extensive interspersed gene loss relative to the inferred structure of the ancestral genome, whereas the Arabidopsis genome segment appears little changed. Representatives of all 32 genes in the Arabidopsis genome segment are represented in Brassica, but the hexaploid complement of 96 has been reduced to 54 in the three subgenomes, with compression of the genomic region lengths they occupy to between 52 and 110 kb. The gene content of the recently duplicated B. rapa genome segments is identical, but intergenic sequences differ.