Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.     

Synteny between <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and <Emphasis Type="Italic">Hordeum vulgare</Emphasis> as revealed by FISH

We investigated by fluorescence in situ hybridization (FISH) the synteny between Brachypodium distachyon with a small genome (1C = 320 Mb) and barley with a large genome (1C = 5,100 Mb) at the chromosome level. Reciprocal genomic in situ hybridization (GISH) between B. distachyon and barley labeled mainly 45S ribosomal DNA loci, indicating that most high copy DNA is weakly conserved between both grasses. Of 13 BAC clones with inserts from different B. distachyon chromosomes, only two belonging to chromosome 1 yielded hybridization signals on a barley metaphase chromosome (on 7HS and 7HL, respectively), confirming synteny between both chromosomes. FISH experiments to characterize the synteny of single-copy loci were performed. Two of four Brachypodium sylvaticum BACs spanning a 223-kb interval homologous to the region of barley that harbors a gibberellic-acid-insensitive semi-dwarfing gene, sdw3, hybridized specifically to a central position of B. distachyon chromosome 1 short arm but not to the homologous region of the barley genome. Repeat-free sequences PCR amplified from four non-overlapping barley BACs linked to the core of Sdw3 region yielded signals at distinct positions in the middle of barley chromosome arm 2HS. Together, these results (1) confirmed the synteny between B. distachyon chromosome 1 and barley chromosomes 2H and 7H at the cytological level, (2) indicated mid-arm position for the Sdw3 locus genetically mapped at the centromere of barley chromosome 2H, and (3) proved that the sdw3 core interval of <100 kb in B. distachyon corresponds to a megabase-sized syntenic region in barley.     

DNA methylation patterns of <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> chromosomes and their alteration by 5-azacytidine treatment

Sequential immunolocalisation of 5-methylcytosine (5-MeC) and fluorescence in situ hybridisation with chromosome-specific BAC clones were performed on Brachypodium distachyon mitotic metaphase chromosomes to determine specific DNA methylation patterns of each chromosome in the complement. In the majority of cells examined, chromosomes Bd4 and Bd5, which bear the loci of 5S and 35S ribosomal DNA, respectively, had characteristic 5-MeC patterns. In contrast, the distribution of 5-MeC along the metacentric chromosome pairs Bd1, Bd2 and Bd3 was more variable. There were numerous differences in distribution of methylated sites between homologous chromosomes as well as between chromosome arms. Some chromosome sites, such as pericentromeric regions, were highly methylated in all chromosomes. Additionally, the influence of a hypomethylating agent, 5-azacytidine, on B. distachyon chromosome methylation patterns was confirmed. It was found that some chromosome pairs underwent demethylation more easily than others, but there was no apparent regularity in demethylation of particular chromosome segments.     

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes

Although TaiI-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using TaiI and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of TaiI and Ty3/gypsy sequences depending on the chromosome; TaiI-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing TaiI-tandem arrays or a two-step amplification mechanism of the TaiI family where each TaiI colony was amplified to form arrays independently after the insertion of TaiI-family sequences along the entire centromere. Although sequence analysis of centromeric TaiI repeats in wheat and subtelomeric TaiI repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.     

Non-Rabl Patterns of Centromere and Telomere Distribution in the Interphase Nuclei of Plant Cells

At the anaphase of cell divisions, the divided chromosomes move to the two poles, with the centromeres as heads and telomeres as tails. Such a polarized orientation of centromeres and telomeres is believed to be preserved in the interphase and is known as Rabl model. We analyzed the distributions of centromeres and telomeres in interphase nuclei from several plant species. Although Rabl polarity was observed in wheat, rye, barley and oats, non-Rabl patterns were discovered in sorghum, rice and maize. In the non-Rabl patterns, both centromeres and telomeres were dispersed throughout the interphase nucleus, except in the area occupied by the nucleolus. Both Rabl and non-Rabl distribution patterns of centromeres and telomeres were consistent in interphase nuclei derived from meristematic root tip cells, microspore mother cells and differentiated leaf cells. Our study demonstrated that there is a diversity of interphase chromatin organization and that the classical Rabl model is not universal in plant species.     

Total centromere size and genome size are strongly correlated in ten grass species

It has been known for decades that centromere size varies across species, but the factors involved in setting centromere boundaries are unknown. As a means to address this question, we estimated centromere sizes in ten species of the grass family including rice, maize, and wheat, which diverged 60~80 million years ago and vary by 40-fold in genome size. Measurements were made using a broadly reactive antibody to rice centromeric histone H3 (CENH3). In species-wide comparisons, we found a clear linear relationship between total centromere size and genome size. Species with large genomes and few chromosomes tend to have the largest centromeres (e.g., rye) while species with small genomes and many chromosomes have the smallest centromeres (e.g., rice). However, within a species, centromere size is surprisingly uniform. We present evidence from three oat-maize addition lines that support this claim, indicating that each of three maize centromeres propagated in oat are not measurably different from each other. In the context of previously published data, our results suggest that the apparent correlation between chromosome and centromere size is incidental to a larger trend that reflects genome size. Centromere size may be determined by a limiting component mechanism similar to that described for Caenorhabditis elegans centrosomes.     

Distinct chromosomal distributions of highly repetitive sequences in maize

The majority of genomic DNA in most plant species is made up of repetitive elements including satellites and retrotransposons. The maize genome is intermediate in size and abundance of repetitive elements between small genomes such as Arabidopsis and rice and larger genomes such as wheat. Although repetitive elements are present throughout the maize genome, individual families are non-randomly distributed along chromosomes. In this work we use fluorescence in-situ hybridization (FISH) to examine the distribution of abundant LTR retroelement families and satellites contained in heterochromatic blocks called knobs. Different retroelement families have distinct patterns of hybridization. Prem1 and Tekay, two very closely related elements, both hybridize along the length of all chromosomes but do so with greater intensity near the centromeres, although subtle differences are detectable between the hybridization patterns. Opie, Prem2/Ji, and Huck are enriched away from the centromeres and Grande is distributed uniformly along the chromosomes. Double labeling with proximally and distally enriched elements on pachytene chromosomes produces alternating blocks of element enrichment. The maize elements hybridized in the same general patterns to chromosomes of maize relatives including Zea diploperennis and Tripsacum dactyloides. Additionally, abundant Tripsacum LTR retroelements are enriched in similar chromosomal regions among the different species. The 180 bp knob satellite is present in large blocks at interstitial locations on chromosome arms. With long exposures, smaller sites of hybridization are detected at the ends of chromosomes, adjacent to the telomere tract. This distal position for knob satellites is conserved among Zea and Tripsacum species. Electronic supplementary material Supplementary material to this paper is available in electronic form at and is accessible for authorized users.     

Interstitial telomeric repeats are enriched in the centromeres of chromosomes in Solanum species

Interstitial telomeric repeats (ITRs) were reported in a number of animal and plant species. Most ITRs are organized as short tandem arrays and are likely evolutionary relics derived from chromosomal rearrangements and DNA repairs. However, megabase-sized ITR arrays were reported in Solanum species. Here, we report a fluorescence in situ hybridization (FISH) survey of ITRs in all representative diploid Solanum species, including potato, tomato, and eggplant. FISH revealed massive amplification of ITRs in the centromeric regions of chromosomes from the Solanum species containing the B and P genomes. A significant proportion of the ITR FISH signals was mapped within the primary constrictions of the pachytene chromosomes of Solanum pinnatisectum (B genome). In addition, some ITR sites overlapped with St49, a satellite repeat enriched in centromeric DNA sequences associated with CENH3 nucleosomes, in both A and B genome Solanum species. These results show that some ITR subfamilies have been amplified and invaded in the functional centromeres of chromosomes in Solanum species.     

Complex genomic organization of Indian muntjac centromeric DNA

A 69-kb Indian muntjac bacterial artificial chromosome (BAC) clone that screened positive for Cervid satellites I and IV was selected for complete sequence analysis and further characterization. The sequences of this BAC clone were found in the centromeres and in some interstitial sites of Indian muntjac chromosomes. Sequence analyses showed that the BAC clone contained a 14.5 kb Cervid satellite I-like DNA element and a 9 kb Cervid satellite IV-like DNA element. In addition, it contained 51 regions each organized in a complex fashion, with sequences homology to intersperse repetitive sequences such as LINEs, SINEs, LTRs, other published DNA elements, and unassigned sequences. The FISH patterns of seven non-satellite sequence elements generated from the BAC clone showed mainly specific to centromeres of the Indian muntjac representing novel centromeric DNAs of the species. Furthermore, FISH signals and Southern blot patterns of these elements suggest the existence of a not yet identified repetitive sequence with giant repeated monomers. Positive FISH signals of these elements were also detected in the centromeric regions of Formosan muntjac. This suggests that these newly identified non-Cervid satellite DNA sequences have been conserved in the centromere of the Formosan muntjac.     

Characterization of CENH3 and centromere-associated DNA sequences in sugarcane

Centromere-specific histone H3 (CENH3) has been used to detect active centromeres, and to analyse the DNA sequences closely associated with the centromere, because they localize only in active centromeres and bind directly to the DNA. In maize and rice, the centromeric retrotransposons (CR) are shown to be closely associated with their own CENH3 whereas no such association was found in Arabidopsis thaliana. In this study, this sort of association was investigated in sugarcane. Two expressed sequence tag groups encoding putative sugarcane CENH3 (SoCENH3) were found in a sugarcane-expressed sequence tag database. Their deduced amino acid sequences were similar to these of the CENH3s in rice and maize. An antibody against rice CENH3 seemed to crossreact with the SoCENH3s, and stained sugarcane centromeres. A set of immunoprecipitation tests was conducted with the antibody and chromatin from the sugarcane genome to reveal CENH3-associated DNA sequences in sugarcane. Centromeric tandem repeats (SCEN) and centromeric retrotransposons of sugarcane (CRS) were significantly precipitated with the antibody, meaning these repeats are directly interacting with CENH3 in sugarcane centromeres.     

Chromosome arrangement and behaviour of two rye homologous telosomes at the onset of meiosis in disomic wheat-5RL addition lines with and without the Ph1 locus

Fluorescence in-situ hybridization (FISH) of total genomic and repetitive DNA on microsporocytes of ditelocentric addition lines of rye 5RL in hexaploid wheat was performed to study the behaviour of the rye homologous chromosome arms in relation to centromere and telomere dynamics at premeiotic interphase and meiotic prophase I. By comparing isogenic lines with and without the Ph1 locus, we established the effect of the Ph1 gene on appearance and behaviour of the rye chromosomes. Ph1 and ph1b lines demonstrated similar premeiotic chromosome arrangement with the two rye homologues occupying separated domains despite the occurrence of centromere association. Our study confirmed that bouquet arrangement of telomeres follows the Rabl configuration. In cells displaying bouquet clustering of telomeres, centromeres of the 5RL telosomes are still at the opposite pole, suggesting anchoring of centromeres at the cytoskeleton. Once the telomeres complete clustering, the rye centromeres migrate to the telomere pole, and the rye chromosomes begin to loosen their structure. While the rye homologues in the wild-type keep separate territories in the nucleus, they become intermingled in the ph1b mutant, possibly because of their lower condensation. In a subsequent stage, the 5RL homologues appear intimately associated mainly at the distal region. Our study suggests that the lower rate of chromosome synapsis in the ph1b mutant results from abnormal chromatin decondensation and organization.     

Unstable transmission of rice chromosomes without functional centromeric repeats in asexual propagation

During sexual propagation of primary trisomic 8, chromosome 8 breaks in some rice plants, resulting in a telotrisomic (2n+·8S) line. In this study, we observed that the extra short arm of chromosome 8 (·8S) can easily be lost in the telotrisomic, and we determined by fluorescence in-situ hybridization (FISH) analysis that the centromeric region of the extra ·8S did not contain the rice centromeric satellite repeat (CentO) and centromere-specific retrotransposon (CRR); however, the extra ·8S contained part of the CentO and CRR sequences in the initially preserved telotrisomic line. We confirmed by real-time quantitative PCR (RQ-PCR) analysis that the original functional centromere of the extra ·8S was lost. Using both FISH and RQ-PCR, the breakage point of the extra ·8S was found within the BAC clone a0070J19 sequence containing the first part of the short arm near the centromere region of chromosome 8 but without any CentO or CRR sequences. However, part of the DNA sequence within the a0070J19 BAC clone played a role in the new functional centromere, contributing to the morphological variations by asexually propagated plants of rice telotrisomics in the field. We conclude that CENH3, a key element in the eukaryotic kinetochore, may not always bind properly with the new functional centromere, resulting in loss of the extra ·8S during mitosis and the chromosome numbers returning to diploid levels in subsequent generations.     

A novel, simple and rapid nondenaturing FISH (ND-FISH) technique for the detection of plant telomeres. Potential used and possible target structures detected

We report a new technique—nondenaturing FISH (ND-FISH)—for the rapid detection of plant telomeres without the need for prior denaturation of the chromosomes. In its development, two modified, synthetic oligonucleotides, 21 nt in length, fluorescently labelled at their 5′ and 3′ ends and complementary to either the cytidine-rich (C₃TA₃) or guanosine-rich (T₃AG₃) telomeric DNA strands, were used as probes. The high binding affinity of these probes and the short hybridization time required allows the visualization of plant telomeres in less than an hour. In tests, both probes gave strong signals visualized as double spots at both chromosome ends; this was true of both the mitotic and meiotic chromosomes of barley, wheat, rye, maize, Brachypodium distachyon and Rhoeo spathacea. They were also able to detect telomere motifs at certain intercalary sites in the chromosomes of R. spathacea. To investigate the nature of the target structures detected, the chromosomes were treated with RNase A and single strand-specific nuclease S1 before ND-FISH experiments. Signal formation was resistant to standard enzymatic treatment, but sensitive when much higher enzyme concentrations were used. The results are discussed in relation to current knowledge of telomere structure.     

Wheat and gluten in South African food products

Individuals with allergies and intolerances to grain products rely on accurate food labelling to prevent potentially life-threatening reactions. The aim of this study was to evaluate a number of South African food products for gluten and wheat to ascertain whether these products may pose a risk to celiac and/or wheat allergic individuals. Twenty-five products were analysed, including spelt products, flours of buckwheat, barley, rye, rice, millet, maize, semolina, triticale, oats, porridges, rice- and maize-based cereals, and rye bread. The rye and barley flours and two oat products were shown to be contaminated with wheat. Ten out of 17 naturally gluten-free products contained gluten, although in 13 of these the levels were below 20 mg/kg. The labels of four products were found to be misleading in terms of the gluten and/or wheat claims made.     

Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers

Fluorescence in-situ hybridization (FISH) is a quick and affordable approach to map DNA sequences to specific chromosomal regions. Although FISH is one of the most important physical mapping techniques, research on the resolution of FISH on different cytological targets is scarce in plants. In this study, we report the resolution of FISH mapping on mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers in rice. A majority of the FISH signals derived from bacterial artificial chromosome (BAC) clones separated by approximately 1 Mb of DNA cannot be resolved on mitotic prometaphase chromosomes. In contrast, the relative positions of closely linked or even partially overlapping BAC clones can be resolved on a euchromatic region of rice chromosome 10 at the early pachytene stage. The resolution of pachytene FISH is dependent on early or late pachytene stages and also on the location of the DNA probes in the euchromatic or heterochromatic regions. We calibrated the fiber-FISH technique in rice using seven sequenced BAC clones. The average DNA extension was 3.2 kb/μm among the seven BAC clones. Fiber-FISH results derived from a BAC contig that spanned 1 Mb DNA matched remarkably to the sequencing data, demonstrating the high resolution of this technique in cytological mapping. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mapping nonrecombining regions in barley using multicolor FISH

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.     

Ancestral grass karyotype reconstruction unravels new mechanisms of genome shuffling as a source of plant evolution

The comparison of the chromosome numbers of today''s species with common reconstructed paleo-ancestors has led to intense speculation of how chromosomes have been rearranged over time in mammals. However, similar studies in plants with respect to genome evolution as well as molecular mechanisms leading to mosaic synteny blocks have been lacking due to relevant examples of evolutionary zooms from genomic sequences. Such studies require genomes of species that belong to the same family but are diverged to fall into different subfamilies. Our most important crops belong to the family of the grasses, where a number of genomes have now been sequenced. Based on detailed paleogenomics, using inference from n = 5–12 grass ancestral karyotypes (AGKs) in terms of gene content and order, we delineated sequence intervals comprising a complete set of junction break points of orthologous regions from rice, maize, sorghum, and Brachypodium genomes, representing three different subfamilies and different polyploidization events. By focusing on these sequence intervals, we could show that the chromosome number variation/reduction from the n = 12 common paleo-ancestor was driven by nonrandom centric double-strand break repair events. It appeared that the centromeric/telomeric illegitimate recombination between nonhomologous chromosomes led to nested chromosome fusions (NCFs) and synteny break points (SBPs). When intervals comprising NCFs were compared in their structure, we concluded that SBPs (1) were meiotic recombination hotspots, (2) corresponded to high sequence turnover loci through repeat invasion, and (3) might be considered as hotspots of evolutionary novelty that could act as a reservoir for producing adaptive phenotypes.The Poaceae (grasses) comprise more than 10,000 species, and their basic chromosome number can widely range from two to 18 (deWet 1987; Hunziker and Stebbins 1987) both within and among subfamilies (Gaut 2002). Chromosome number can double with polyploidization, which is common in flowering plants and also referred to as whole-genome duplication (WGD). Recent comparison of map-based genomic sequences of grasses revealed shared intragenomic duplications, providing new insights into the evolution of extant genomes from an ancestral grass karyotype (AGK). Complete grass genome sequences, including three subfamilies of the grasses (Poaceae)—i.e., the Panicoideae (sorghum [Paterson et al. 2009]; maize, [Schnable et al. 2009]), Ehrhartoideae (rice [International Rice Genome Sequencing Project 2005]), and Pooideae (Brachypodium [International Brachypodium Initiative 2010])—that have diverged from a common ancestor 50–70 million yr ago (Mya) (Kellogg 2001), are available to perform paleogenomics studies, i.e., study of the ancestral genome structure of today''s species.Previous comparative analysis (i.e., establishment of orthologous and paralogous gene pair repertory) of two (rice, sorghum) whole-genome sequences and three high-density EST-based genetic maps (wheat, barley, maize) has permitted us to use synteny blocks to model an AGK based on gene order and content. We defined two new parameters for BLAST analyses (either nucleic or protein-based) that take into account not only similarity but also the relative lengths of the sequences: cumulative identity percentage (CIP) and cumulative alignment length percentage (CALP). We also systematically performed a statistical test after BLAST comparison with the CIP/CALP parameters to validate nonrandom associations between groups of sequences (Salse et al. 2009a,b). To further refine the junctions of synteny blocks from fully sequenced genomes to large mapped EST collections, we previously added two other new criteria: the density (DR) and the cluster (CR) ratios that are functions of the physical and/or genetic size, the total number of genes and/or loci, and the number of orthologous sequence pairs defined in the orthologous regions identified with the previous CIP/CALP parameters. Statistically significant collinear relationships between the two genomes are associated with the lowest DR and highest CR values, while the remaining collinear regions are considered as artificial, i.e., obtained at random (Salse et al. 2009b). Based on this approach, we recently proposed an AGK with a minimal size of 33.6 Mb structured in five protochromosomes containing at least 9138 predicted protogenes (Salse et al. 2009a). We proposed that two major evolutionary shuffling events, i.e., WGD followed by diploidization, explain the divergence of cereal genomes during their evolution from a common AGK (Bolot et al. 2009).Interestingly, both in animals and plants, similar evolutionary paths have been described with respect to the reduced numbers of protochromosomes and several rounds of WGDs, followed by lineage-specific rearrangements leading to different chromosome numbers in today''s species (Salse et al. 2009a). However, the sequence-based identification of junctions, where chromosomal rearrangements took place in grasses since their divergence from their common ancestor, was a prerequisite for discovering the molecular mechanisms that might drive these chromosome-shuffling events. The recent availability of large amounts of whole-genome sequences from diverse taxa yielded large-scale, genome-wide comparisons in mammals and provided clear identification of synteny breakpoints or junction sequences, which gave rise to a new evolutionary perspective. Synteny break points (SBPs) in mammals have then been suggested to (1) occur nonrandomly, (2) involve gene-rich intervals, (3) contain a high occurrence of segmental duplications and/or repetitive elements, (4) be involved in nonallelic homologous recombination, (5) be reusable fragile loci for chromosome rearrangement, and (6) involve adaptation traits such as disease-related ones (Trinh et al. 2004; Everts-van et al. 2004; Murphy et al. 2005; Schibler et al. 2006; Bulazel et al. 2007; Larkin et al. 2009; Lemaitre et al. 2009). However, regions of major interest, as they represent evolutionary break points caused by fusions, inversions, translocations, and other processes, were not yet investigated in detail in plants.Where, when, and how these breakpoints can and do occur are fundamental questions regarding the evolution of today''s species from their founding ancestor. Here, we undertook a new scale of plant genome comparisons because of the sets of closely related genomes that have been fully sequenced since early 2010 (rice, sorghum, maize, Brachypodium). We conducted a reconstruction of the AGK, in terms of ancestral gene order, as well as several intermediary ancestral genomes, delineating today''s species from extinct AGKs. The reconstruction of AGKs then permitted us to discover sequence intervals of several 100 kb comprising junction sequences with chromosome break and fusion points. Analysis of these intervals permitted us to propose the molecular mechanisms that have shaped today''s rice, maize, Brachypodium, and sorghum chromosomes.     

Neocentric activity of rye 5RL chromosome in wheat

The neocentric activity of a constriction located on the long arm of rye 5R chromosome (5RL) was analysed. It is not observed in normal rye but it is unusually stretched in bivalents involving 5RL telosomes in wheat–ditelosomic 5RL addition lines. In 20% of metaphase I cells, the 5RL bivalent presents the centromeres oriented to one pole and the constrictions oriented towards the opposite pole with a strong tension. In 5% of the cells, the constriction was able to orient the bivalent to the poles without tension in the centromeres. Sister chromatid cohesion, which is one of the distinct features of centromeric function, is persistent at the constriction in delayed 5RL chromosomes at anaphase I. Neither the elongation of the constriction nor the neocentric activity was observed at second meiotic division or mitosis. FISH studies showed that the 5RL constriction lacked detectable quantities of two repetitive DNA sequences, CCS1 and the 180-bp knob repeat, present at cereal centromeres and neocentromeres, respectively. We propose that, under special conditions, such as the wheat background, the normally non-centromeric DNA present at this region of 5RL acquires a specific chromatin structure, differentiated as an elongated constriction, which is able to function as a centromere. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hypersensitivity to Inhaled Flour Allergens Comparison between Cereals

Radioallergosorbent testing (RAST) of sera from subjects sensitized to wheat and rye flour indicated that there is significant reaction with seed extracts of 12 cereals (wheat, durum wheat, triticale, cereal rye, barley, rye grass, oats, canary grass, rice, maize, sorghum and Johnson grass). Results were evaluated in terms of taxonomic relationships and of the electrophoretically determined protein composition of the cereal extracts. RAST uptakes were uniformly low in sera from four rhinitic bakers, yet were significantly above the levels for non-allergic and cord sera. Much higher RAST uptakes were obtained with sera from four asthmatic bakers when tested with wheat and its close relatives, but there was still reasonably high reactivity with more distantly related cereals. RAST inhibition experiments indicated in a more direct way the extent of cross-reactivity between grain extracts of wheat, rye, barley and oats. One baker had a history of more severe attacks of breathlessness following inhalation of rye flour compared with wheat flour. This was confirmed by bronchial challenge testing, but the comparison was not obviously consistent with the results of prick testing or estimation of histamine released from his leucocytes. The results as a whole suggested that the bran layers of cereal grains are at least as allergenic as flour.