Towards a whole‐genome sequence for rye (Secale cereale L.)

We report on a whole‐genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole‐genome shotgun sequencing of the 7.9‐Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low‐copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high‐density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high‐density genetic map and establishing a synteny‐based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome‐wide map of selection signals differentiating the divergent gene pools. This rye whole‐genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome‐based breeding in rye.     

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Comparative molecular-genetic mapping of genomes of rye (Secale cereale L.) and other cereals

The genetic map of rye consisting of 149 RFLP, 20 isozyme and 12 microsatellite markers was developed. Using the collection of cross-hybridizing probes, the presence of multiple translocations in rye genome with respect to wheat and barley genomes was shown. However, within large regions of genome a strict collinearity of marker order was observed that allow us to use the method of comparative mapping for an introduction of new genes. In the developed genetic map 18 morphological and breeding-valuable genes mapped in different rye populations were integrated. The comparative analysis of homeological loci in genomes of Triticeae species as well as in genomes of rice and maize was carried out. The genes controlling a number of morphological traits, plant height, photoperiodic response and winter/spring growth habit were shown to be conserve among cereals and to form clear homoeologous rows.     

The genomic organization and localization on the chromosomes of the HvRT family of DNA repetitive sequences in barley

HvRT family of repetitive DNA sequences from barley genome appears to have complex hierarchical organization. Tandem repetition of 118-bp monomers constitutes lower level of HvRT-family organization. Amplification units of the higher level consist of several contiguous 118-bp monomers. RFLP between different species and cultivars of barley resulted from the differences in the higher-order repeat structure. Individual chromosomes of barley contain specific HvRT subfamilies. This family also possesses separate domains differing in the restriction enzyme sites density. HvRT family is presented in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but is absent in the genomes of H. marinum, H. geniculatum and wheat.     

Major structural genomic alterations can be associated with hybrid speciation in Aegilops markgrafii (Triticeae)

During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hybridization, resulting in allopolyploidy; whereas homoploid hybrid speciation was found only in rye. Homoeologous chromosomes within the Triticeae preserved cross‐species macrocolinearity, except for a few species with rearranged genomes. Aegilops markgrafii, a diploid wild relative of wheat (2n = 2x = 14), has a highly asymmetrical karyotype that is indicative of chromosome rearrangements. Molecular cytogenetics and next‐generation sequencing were used to explore the genome organization. Fluorescence in situ hybridization with a set of wheat cDNAs allowed the macrostructure and cross‐genome homoeology of the Ae. markgrafii chromosomes to be established. Two chromosomes maintained colinearity, whereas the remaining were highly rearranged as a result of inversions and inter‐ and intrachromosomal translocations. We used sets of barley and wheat orthologous gene sequences to compare discrete parts of the Ae. markgrafii genome involved in the rearrangements. Analysis of sequence identity profiles and phylogenic relationships grouped chromosome blocks into two distinct clusters. Chromosome painting revealed the distribution of transposable elements and differentiated chromosome blocks into two groups consistent with the sequence analyses. These data suggest that introgressive hybridization accompanied by gross chromosome rearrangements might have had an impact on karyotype evolution and homoploid speciation in Ae. markgrafii.     

DNA condensation with polyamines. II. Electron microscopic studies

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long.In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to C_ot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes.Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA.Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences.Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.     

Ubiquity of the St chloroplast genome in St-containing Triticeae polyploids.

Interspecific hybridization occurs between Tritceae species in the grass family (Poaceae) giving rise to allopolyploid species. To examine bias in cytoplasmic DNA inheritance in these hybridizations, the sequence of the 3' end of the chloroplast ndhF gene was compared among 29 allopolyploid Triticeae species containing the St nuclear genome in combination with the H, I, Ns, P, W, Y, and Xm nuclear genomes. These ndhF sequences were also compared with those from diploid or allotetraploid Triticeae species having the H, I, Ns, P, W, St, and Xm genomes. The cpDNA sequences were highly similar among diploid, allotetraploid, allohexaploid, and allooctoploid Triticeae accessions containing the St nuclear genome, with 0-6-nucleotide (nt) substitutions (0-0.8%) occurring between pairs of species. Neighbor-joining analysis of the sequences showed that the ndhF DNA sequences from species containing the St nuclear genome formed a strongly supported clade. The data indicated a strong preference for cpDNA inheritance from the St nuclear genome-containing parent in hybridizations between Triticeae species. This preference was independent of the presence of the H, I, Ns, P, W, and Xm nuclear genomes, the geographic distribution of the species, and the mode of reproduction. The data suggests that hybridizations having the St-containing parent as the female may be more successful.     

A linkage map of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.) and comparative mapping with other Poaceae species

A genetic linkage map has been constructed for meadow fescue (Festuca pratensis Huds.) (2n=2x=14) using a full-sib family of a cross between a genotype from a Norwegian population (HF2) and a genotype from a Yugoslavian cultivar (B14). The two-way pseudo-testcross procedure has been used to develop separate maps for each parent, as well as a combined map. A total number of 550 loci have been mapped using homologous and heterologous RFLPs, AFLPs, isozymes and SSRs. The combined map consists of 466 markers, has a total length of 658.8 cM with an average marker density of 1.4 cM/marker. A high degree of orthology and colinearity was observed between meadow fescue and the Triticeae genome(s) for all linkage groups, and the individual linkage groups were designated 1F–7F in accordance with the orthologous Triticeae chromosomes. As expected, the meadow fescue linkage groups were highly orthologous and co-linear with Lolium, and with oat, maize and sorghum, generally in the same manner as the Triticeae chromosomes. It was shown that the evolutionary 4AL/5AL translocation, which characterises some of the Triticeae species, is not present in the meadow fescue genome. A putative insertion of a segment orthologous to Triticeae 2 at the top of 6F, similar to the rearrangement found in the wheat B and the rye R genome, was also observed. In addition, chromosome 4F is completely orthologous to rice chromosome 3 in contrast to the Triticeae where this rice chromosome is distributed over homoeologous group 4 and 5 chromosomes. The meadow fescue genome thus has a more ancestral configuration than any of the Triticeae genomes. The extended meadow fescue map reported here provides the opportunity for beneficial cross-species transfer of genetic knowledge, particularly from the complete genome sequence of rice.Communicated by P. Langridge     

Characterization of a family of tandemly repeated DNA sequences in Triticeae

The recombinant plasmid dpTa1 has an insert of relic wheat DNA that represents a family of tandemly organized DNA sequences with a monomeric length of approximately 340 bp. This insert was used to investigate the structural organization of this element in the genomes of 58 species within the tribe Triticeae and in 7 species representing other tribes of the Poaceae. The main characteristic of the genomic organization of dpTa1 is a classical ladder-type pattern which is typical for tandemly organized sequences. The dpTa1 sequence is present in all of the genomes of the Triticeae species examined and in 1 species from a closely related tribe (Bromus inermis, Bromeae). DNA from Hordelymus europaeus (Triticeae) did not hybridize under the standard conditions used in this study. Prolonged exposure was necessary to obtain a weak signal. Our data suggest that the dpTa1 family is quite old in evolutionary terms, probably more ancient than the tribe Triticeae. The dpTa1 sequence is more abundant in the D-genome of wheat than in other genomes in Triticeae. DNA from several species also have bands in addition to the tandem repeats. The dpTa1 sequence contains short direct and inverted subrepeats and is homologous to a tandemly repeated DNA sequence from Hordeum chilense.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

CACTA transposons in Triticeae. A diverse family of high-copy repetitive elements

In comparison with retrotransposons, which comprise the majority of the Triticeae genomes, very few class 2 transposons have been described in these genomes. Based on the recent discovery of a local accumulation of CACTA elements at the Glu-A3 loci in the two wheat species Triticum monococcum and Triticum durum, we performed a database search for additional such elements in Triticeae spp. A combination of BLAST search and dot-plot analysis of publicly available Triticeae sequences led to the identification of 41 CACTA elements. Only seven of them encode a protein similar to known transposases, whereas the other 34 are considered to be deletion derivatives. A detailed characterization of the identified elements allowed a further classification into seven subgroups. The major subgroup, designated the "Caspar " family, was shown by hybridization to be present in at least 3,000 copies in the T. monococcum genome. The close association of numerous CACTA elements with genes and the identification of several similar elements in sorghum (Sorghum bicolor) and rice (Oryza sativa) led to the conclusion that CACTA elements contribute significantly to genome size and to organization and evolution of grass genomes.     

Genome merger: from sequence rearrangements in triticale to their elimination in wheat–rye addition lines

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat–rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat–rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale × wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat–rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat–rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.     

Dynamics of Tandem Repetitive Afa-Family Sequences in Triticeae, Wheat-Related Species

The Afa-family sequences in wheat-related species, Triticeae, are tandem repetitive sequences of 340 bp. All the analyzed Triticeae species carried the sequences in their genomes, though the copy numbers varied about 100-fold among the species. The nucleotide fragments amplified by PCR were cloned and sequenced, and their behavior in the evolution of Triticeae was analyzed by the neighbor-joining (NJ) method. The sequences in genomes with many copies of this family clustered at independent branches of the phylogenic tree, whereas the sequences in genomes with a few copies did not. This may suggest that Afa-family sequences had amplified several times in the evolution of Triticeae, each using a limited number of different master copies. In addition, the sequences of the A and B genomes of hexaploid common wheat indicated that the Afa-family sequences had not evolved in a concerted manner between the genomes. Furthermore, the sequences of each chromosome of the D genome of this species indicated that the sequences had amplified on all over the D-genome chromosomes in a short period. Received: 1 September 1997 / Accepted: 19 January 1998     

Comparison of Acetyl-CoA carboxylase 1 (Acc-1) gene diversity among different Triticeae genomes

It has widely been documented that life form and mating system have significant influences on genetic diversity. In the tribe Triticeae, several genera contain both annual and perennial species, whereas other genera comprise strictly annual or perennial species. It was suggested that Triticeae annuals have originated from Triticeae perennials. The present study aims to analyze nucleotide diversity of Acc-1 gene among different Triticeae genomes, and attempts to link effects of life history (annuals and perennials) and mating systems. The nucleotide diversity of 364 Acc-1 sequences in Triticeae species was characterized. The highest estimates of nucleotide diversity values (π = 0.01919, θ = 0.03515) were found for the Ns genome among the genomes analyzed. Nucleotide diversities in the D genome and Ns genome of polyploids are higher than those in respective genomes of diploids, while in the St genome of polyploids, it is lower than that in the St genome of diploids. The averaged π value (0.013705) in the genomes of perennials is more than twice of the value (0.00508) in the genomes of annuals. The averaged π value (0.01323) in the genomes of outcrossing species is two-fold of the value (0.005664) in the genomes of selfer. Our results suggested that the evolutionary history and mating system may play an important role in determining nucleotide diversity of Acc-1 gene in each genome.     

Molecular phylogeny of RPB2 gene reveals multiple origin, geographic differentiation of H genome, and the relationship of the Y genome to other genomes in Elymus species

It has been hypothesized from isozymic and cytological studies of Elymus species that the Old and New World taxa may be of separate origin of the H genome in the StH genome species. To test this hypothesis, and estimate the phylogenetic relationships of polyploid Elymus species within the Triticeae, the second largest subunit of RNA polymerase II (RPB2) sequence of 36 Elymus accessions containing StH or StY genomes was analyzed with those of Pseudoroegneria (St), Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum(Ee), Thinopyrum(Eb) and Dasypyrum (V). Our data indicated that the H genome in Elymus species is differentiated in accordance with geographical origin, and that the Eurasian and American StH genome species have independent alloploid origins with different H-genome donors. Phylogenetic analysis of Y genome sequences with other genome donors (St, H, P, W) of Elymus revealed that W and P genomes are sister to Y genome with a 87% bootstrap support, and that StY and StH species group might have acquired their RPB2 St sequences from distinct Pseudoroegneria gene pools. Our data did not support the suggestion that the St and Y genomes have the same origin as put forward in a previous study using ITS data. Our result provides some insight on the origin of Y genome and its relationship to other genomes in Elymus.     

Hop,an active Mutator-like element in the genome of the fungus Fusarium oxysporum

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.