DNA rearrangement in orthologous orp regions of the maize, rice and sorghum genomes

The homeologous Orp1 and Orp2 regions of maize and the orthologous regions in sorghum and rice were compared by generating sequence data for >486 kb of genomic DNA. At least three genic rearrangements differentiate the maize Orp1 and Orp2 segments, including an insertion of a single gene and two deletions that removed one gene each, while no genic rearrangements were detected in the maize Orp2 region relative to sorghum. Extended comparison of the orthologous Orp regions of sorghum and japonica rice uncovered numerous genic rearrangements and the presence of a transposon-rich region in rice. Only 11 of 27 genes (40%) are arranged in the same order and orientation between sorghum and rice. Of the 8 genes that are uniquely present in the sorghum region, 4 were found to have single-copy homologs in both rice and Arabidopsis, but none of these genes are located near each other, indicating frequent gene movement. Further comparison of the Orp segments from two rice subspecies, japonica and indica, revealed that the transposon-rich region is both an ancient and current hotspot for retrotransposon accumulation and genic rearrangement. We also identify unequal gene conversion as a mechanism for maize retrotransposon rearrangement.     

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.     

Targeted analysis of orthologous phytochrome A regions of the sorghum,maize, and rice genomes using comparative gene-island sequencing

A "gene-island" sequencing strategy has been developed that expedites the targeted acquisition of orthologous gene sequences from related species for comparative genome analysis. A 152-kb bacterial artificial chromosome (BAC) clone from sorghum (Sorghum bicolor) encoding phytochrome A (PHYA) was fully sequenced, revealing 16 open reading frames with a gene density similar to many regions of the rice (Oryza sativa) genome. The sequences of genes in the orthologous region of the maize (Zea mays) and rice genomes were obtained using the gene-island sequencing method. BAC clones containing the orthologous maize and rice PHYA genes were identified, sheared, subcloned, and probed with the sorghum PHYA-containing BAC DNA. Sequence analysis revealed that approximately 75% of the cross-hybridizing subclones contained sequences orthologous to those within the sorghum PHYA BAC and less than 25% contained repetitive and/or BAC vector DNA sequences. The complete sequence of four genes, including up to 1 kb of their promoter regions, was identified in the maize PHYA BAC. Nine orthologous gene sequences were identified in the rice PHYA BAC. Sequence comparison of the orthologous sorghum and maize genes aided in the identification of exons and conserved regulatory sequences flanking each open reading frame. Within genomic regions where micro-colinearity of genes is absolutely conserved, gene-island sequencing is a particularly useful tool for comparative analysis of genomes between related species.     

Patterns of sequence divergence and evolution of the S orthologous regions between Asian and African cultivated rice species

A strong postzygotic reproductive barrier separates the recently diverged Asian and African cultivated rice species, Oryza sativa and O. glaberrima. Recently a model of genetic incompatibilities between three adjacent loci: S(1)A, S(1) and S(1)B (called together the S(1) regions) interacting epistatically, was postulated to cause the allelic elimination of female gametes in interspecific hybrids. Two candidate factors for the S(1) locus (including a putative F-box gene) were proposed, but candidates for S(1)A and S(1)B remained undetermined. Here, to better understand the basis of the evolution of regions involved in reproductive isolation, we studied the genic and structural changes accumulated in the S(1) regions between orthologous sequences. First, we established an 813 kb genomic sequence in O. glaberrima, covering completely the S(1)A, S(1) and the majority of the S(1)B regions, and compared it with the orthologous regions of O. sativa. An overall strong structural conservation was observed, with the exception of three isolated regions of disturbed collinearity: (1) a local invasion of transposable elements around a putative F-box gene within S(1), (2) the multiple duplication and subsequent divergence of the same F-box gene within S(1)A, (3) an interspecific chromosomal inversion in S(1)B, which restricts recombination in our O. sativa×O. glaberrima crosses. Beside these few structural variations, a uniform conservative pattern of coding sequence divergence was found all along the S(1) regions. Hence, the S(1) regions have undergone no drastic variation in their recent divergence and evolution between O. sativa and O. glaberrima, suggesting that a small accumulation of genic changes, following a Bateson-Dobzhansky-Muller (BDM) model, might be involved in the establishment of the sterility barrier. In this context, genetic incompatibilities involving the duplicated F-box genes as putative candidates, and a possible strengthening step involving the chromosomal inversion might participate to the reproductive barrier between Asian and African rice species.     

Comprehensive comparison between locations of orthologous genes on archaeal and bacterial genomes

MOTIVATION: Following an extensive search for orthologous genes between the complete genomes from archaea and bacteria, the spatial association of the orthologs has been investigated in terms of synteny, the conservation of the order of neighboring genes. However, the relationships between the relative locations of remote orthologs over entire genomes have not been shown. RESULTS: Comprehensive comparisons between the locations of orthologs on nineteen archaeal and bacterial genomes are presented by the location to location correspondence based on the gene-location distance. When the two genomes are rotated such that a pair of orthologs with the shortest distance is set in the same angle, a statistically significant number of orthologs maintain their relative locations between the genomes. Even by the short distances at the 5% significance level, the rotations are restricted within a narrow range, suggesting an intrinsic angle for realizing similar locations between the orthologs in each genome pair. Furthermore, the rotations in the restricted range agree with the replication origin and terminus sites for the analyzed genomes where such sites are known. The relationship between location-maintained orthologs and gene function is also discussed.     

Comparative sequence analysis reveals extensive microcolinearity in the lateral suppressor regions of the tomato, Arabidopsis, and Capsella genomes

A 57-kb region of tomato chromosome 7 harboring five different genes was compared with the sequence of the Arabidopsis genome to search for microsynteny between the genomes of these two species. For all five genes, homologous sequences could be identified in a 30-kb region located on Arabidopsis chromosome 1. Only two inversion events distinguish the arrangement of the five genes in tomato from that in Arabidopsis. Inversions were not detected when the arrangement of the five Arabidopsis genes was compared with the arrangement in the orthologous region of Capsella, a plant closely related to Arabidopsis. These results provide evidence for microcolinearity between closely and distantly related dicotyledonous species. The degree of microcolinearity found can be exploited to localize orthologous genes in Arabidopsis and tomato in an unambiguous way.     

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.     

Analysis of the barley and rice genomes by comparative RFLP linkage mapping

Comparative genetic mapping of rice and barley, both major crop species with extensive genetic resources, offers the possibility of uniting two well-established and characterized genetic systems. In the present study, we screened 229 molecular markers and utilized 110 polymorphic orthologous loci to construct comparative maps of the rice and barley genomes. While extensive chromosomal rearrangements, including inversions and intrachromosomal translocations, differentiate the rice and barley genomes, several syntenous chromosomes are evident. Indeed, several chromosomes and chromosome arms appear to share nearly identical gene content and gene order. Seventeen regions of conserved organization were detected, spanning 287 cM (24%) and 321 cM (31%) of the rice and barley genomes, respectively. The results also indicate that most (72%) of the single-copy sequences in barley are also single copy in rice, suggesting that the large barley genome arose by unequal crossing over and amplification of repetitive DNA sequences and not by the duplication of single-copy sequences. Combining these results with those previously reported for comparative analyses of rice and wheat identified nine putatively syntenous chromosomes among barley, wheat and rice. The high degree of gene-order conservation as detected by comparative mapping has astonishing implications for interpreting genetic information among species and for elucidating chromosome evolution and speciation.     

Construction of BAC libraries from two apomictic grasses to study the microcolinearity of their apospory-specific genomic regions

We have constructed bacterial artificial chromosome (BAC) libraries from two grass species that reproduce by apospory, a form of gametophytic apomixis. The library of an apomictic polyhaploid genotype (line MS228-20, with a 2C genome size of approximately 4,500 Mbp) derived from a cross between the obligate apomict, Pennisetum squamulatum, and pearl millet (P. glaucum) comprises 118,272 clones with an average insert size of 82 kb. The library of buffelgrass (Cenchrus ciliaris, apomictic line B-12-9, with a 2C genome size of approximately 3,000 Mbp) contains 68,736 clones with an average insert size of 109 kb. Based on the genome sizes of these two lines and correcting for the number for false-positive and organellar clones, library coverages were found to be 3.7 and 4.8 haploid genome equivalents for MS 228-20 and B12-9, respectively. Both libraries were screened by hybridization with six SCARs (sequence-characterized amplified regions), whose tight linkage in a single apospory-specific genomic region had been previously demonstrated in both species. Analysis of these BAC clones indicated that some of the SCAR markers are actually amplifying duplicated regions linked in coupling in both genomes and that restriction enzyme mapping will be necessary to sort out the duplications.     

Orthology between genomes of Brachypodium, wheat and rice

Background

Differential expression of perforin (PRF1), a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction.

Findings

We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA) or DNA extracted from peripheral blood mononuclear cells (PBMC DNA) from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18%) to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92%) in the distal enhancer region (CpG sites 1–10), a variable methylation (range 49%–83%) in the methylation sensitive region (CpG sites 11–17), and a progressively declining methylation level (range 12%–80%) in the proximal promoter region (CpG sites 18–32) of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA.

Conclusion

This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune function.     

Further evidence supporting Morocco as a centre of origin of barley

 Thirty-five populations of H. spontaneum from nine countries, encompassing almost all the known range of distribution of the species, Afghanistan, Crete (Greece), Cyprus, Iran, Iraq, Israel, Libya, Morocco and Turkey, were studied utilizing RFLP markers (21 probes with three restriction enzymes) distributed across all seven barley chromosomes in an attempt to unveil the genetic dissimilarities existing among them. UPGMA clustering, based on the Nei and Li (1979) similarity coefficient, produced a dendrogram where three clusters could be defined: two with a clear geographical distinction (Morocco and Cyprus) and another one grouping all the Asian/Middle Eastern populations, except for an accession from Iran that clustered separately. These results confirm our previous work and suggest that barley domestication could also have taken place outside the Fertile Crescent, particularly in Morocco. Received: 10 October 1998 / Accepted: 27 October 1998     

The compositional transition between the genomes of cold- and warm-blooded vertebrates: codon frequencies in orthologous genes

The genomes of the ancestors of mammals and birds underwent a compositional change in which the gene-richest regions increased their GC levels. Here we investigated this compositional transition by analyzing the levels of G and C in third codon positions, as well as the codon frequencies of orthologous genes from human, chicken and Xenopus. The results may be summed up as follows: (i) GC-poor genes, that did not undergo the compositional transition, showed only minor differences in orthologous sets from Xenopus, human and chicken; this is remarkable in view of the very many nucleotide substitutions that occurred over the long evolutionary times separating these species; (ii) GC-rich genes, that underwent the compositional transition, showed large differences between Xenopus and warm-blooded vertebrates, but not between chicken and human. In other words, the independent changes that occurred in avian and mammalian genes, on the average, were the same.     

Dynamics of the evolution of orthologous and paralogous portions of a complex locus region in two genomes of allopolyploid wheat

Two overlapping bacterial artificial chromosome (BAC) clones from the B genome of the tetraploid wheat Triticum turgidum were identified, each of which contains one of the two high-molecular-weight (HMW) glutenin genes, comprising the complex Glu-B1 locus. The complete sequence (285 506 bp of DNA) of this chromosomal region was determined. The two paralogous x-type ( Glu-1-1 ) and y-type ( Glu-1-2 ) HMW-glutenin genes of the complex Glu-B1 locus were found to be separated by ca. 168 000 bp instead of the 51 000 bp separation previously reported for the orthologous Glu-D1 locus of Aegilops tauschii, the D-genome donor of hexaploid wheat. This difference in intergene spacing is due almost entirely to be the insertion of clusters of nested retrotransposons. Otherwise, the orientation and order of the HMW glutenins and adjacent genes were identical in the two genomes. A comparison of these orthologous regions indicates modes and patterns of sequence divergence, with implications for the overall Triticeae genome structure and evolution. A duplicate globulin gene, found 5' of each HMW-glutenin gene, assists to tentatively define the original duplication event leading to the paralogous x- and y-type HMW-glutenin genes. The intergenic regions of the two loci are composed of different patterns and classes of retrotransposons, indicating that insertion times of these retroelements were after the divergence of the two wheat genomes. In addition, a putative receptor kinase gene near the y-type HMW-glutenin gene at the Glu-B1 locus is likely active as it matches recently reported ESTs from germinating barley endosperm. The presence of four genes represented only in the Triticeae endosperm ESTs suggests an endosperm-specific chromosome domain.     

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.     

Insights into cereal genomes from two draft genome sequences of rice

Draft genome sequences have been reported for two subspecies of rice. The drafts include the sequences of an estimated 99% of all rice genes and provide major advances in our understanding of the content and complexity of cereal genomes in general and the rice genome in particular.     

Shifts in the evolutionary rate and intensity of purifying selection between two Brassica genomes revealed by analyses of orthologous transposons and relics of a whole genome triplication

Recent sequencing of the Brassica rapa and Brassica oleracea genomes revealed extremely contrasting genomic features such as the abundance and distribution of transposable elements between the two genomes. However, whether and how these structural differentiations may have influenced the evolutionary rates of the two genomes since their split from a common ancestor are unknown. Here, we investigated and compared the rates of nucleotide substitution between two long terminal repeats (LTRs) of individual orthologous LTR‐retrotransposons, the rates of synonymous and non‐synonymous substitution among triplicated genes retained in both genomes from a shared whole genome triplication event, and the rates of genetic recombination estimated/deduced by the comparison of physical and genetic distances along chromosomes and ratios of solo LTRs to intact elements. Overall, LTR sequences and genic sequences showed more rapid nucleotide substitution in B. rapa than in B. oleracea. Synonymous substitution of triplicated genes retained from a shared whole genome triplication was detected at higher rates in B. rapa than in B. oleracea. Interestingly, non‐synonymous substitution was observed at lower rates in the former than in the latter, indicating shifted densities of purifying selection between the two genomes. In addition to evolutionary asymmetry, orthologous genes differentially regulated and/or disrupted by transposable elements between the two genomes were also characterized. Our analyses suggest that local genomic and epigenomic features, such as recombination rates and chromatin dynamics reshaped by independent proliferation of transposable elements and elimination between the two genomes, are perhaps partially the causes and partially the outcomes of the observed inter‐specific asymmetric evolution.     

Macro- and microcolinearity between the genomic region of wheat chromosome 5B containing the Tsn1 gene and the rice genome

The Tsn1 gene in wheat confers sensitivity to a proteinaceous host-selective toxin (Ptr ToxA) produced by the tan spot fungus (Pyrenophora tritici-repentis) and lies within a gene-rich region of chromosome 5B. To use the rice genome sequence information for the map-based cloning of Tsn1, colinearity between the wheat genomic region containing Tsn1 and the rice genome was determined at the macro- and microlevels. Macrocolinearity was determined by testing 28 expressed sequence markers (ESMs) spanning a 25.5-cM segment and encompassing Tsn1 for similarity to rice sequences. Twelve ESMs had no similarity to rice sequences, and 16 had similarity to sequences on seven different rice chromosomes. Segments of colinearity with rice chromosomes 3 and 9 were identified, but frequent rearrangements and disruptions occurred. Microcolinearity was determined by testing the sequences of 26 putative genes identified from BAC contigs of 205 and 548 kb in length and flanking Tsn1 for similarity to rice genomic sequences. Fourteen of the predicted genes detected orthologous sequences on six different rice chromosomes, whereas the remaining 12 had no similarity with rice sequences. Four genes were colinear on rice chromosome 9, but multiple disruptions, rearrangements, and duplications were observed in wheat relative to rice. The data reported provide a detailed analysis of a region of wheat chromosome 5B that is highly rearranged relative to rice.     

Matrix attachment regions and structural colinearity in the genomes of two grass species.

In order to gain insights into the relationship between spatial organization of the genome and genome function we have initiated studies of the co-linear Sh2/A1- homologous regions of rice (30 kb) and sorghum (50 kb). We have identified the locations of matrix attachment regions (MARs) in these homologous chromosome segments, which could serve as anchors for individual structural units or loops. Despite the fact that the nucleotide sequences serving as MARs were not detectably conserved, the general organizational patterns of MARs relative to the neighboring genes were preserved. All identified genes were placed in individual loops that were of comparable size for homologous genes. Hence, gene composition, gene orientation, gene order and the placement of genes into structural units has been evolutionarily conserved in this region. Our analysis demonstrated that the occurrence of various 'MAR motifs' is not indicative of MAR location. However, most of the MARs discovered in the two genomic regions were found to co-localize with miniature inverted repeat transposable elements (MITEs), suggesting that MITEs preferentially insert near MARs and/or that they can serve as MARs.