Genetic Redundancy in Rye Shows in a Variety of Ways.

Fifty years ago Susumu Ohno formulated the famous C-value paradox, which states that there is no correlation between the physical sizes of the genome, i.e., the amount of DNA, and the complexity of the organism, and highlighted the problem of genome redundancy. DNA that does not have a positive effect on the fitness of organisms has been characterized as "junk or selfish DNA". The controversial concept of junk DNA remains viable. Rye is a convenient subject for yet another test of the correctness and scientific significance of this concept. The genome of cultivated rye, Secale cereale L., is considered one of the largest among species of the tribe Triticeae and thus it tops the average angiosperm genome and the genomes of its closest evolutionary neighbors, such as species of barley, Hordeum (by approximately 30-35%), and diploid wheat species, Triticum (approximately 25%). The review provides an analysis of the structural organization of various regions of rye chromosomes with a description of the molecular mechanisms contributing to their size increase during evolution and the classes of DNA sequences involved in these processes. The history of the development of the concept of eukaryotic genome redundancy is traced and the current state of this problem is discussed.

The origin and evolution of a two-component system of paralogous genes encoding the centromeric histone CENH3 in cereals.

BACKGROUND: The cereal family Poaceae is one of the largest and most diverse angiosperm families. The central component of centromere specification and function is the centromere-specific histone H3 (CENH3). Some cereal species (maize, rice) have one copy of the gene encoding this protein, while some (wheat, barley, rye) have two. We applied a homology-based approach to sequenced cereal genomes, in order to finally trace the mutual evolution of the structure of the CENH3 genes and the nearby regions in various tribes. RESULTS: We have established that the syntenic group or the CENH3 locus with the CENH3 gene and the boundaries defined by the CDPK2 and bZIP genes first appeared around 50 Mya in a common ancestor of the subfamilies Bambusoideae, Oryzoideae and Pooideae. This locus came to Pooideae with one copy of CENH3 in the most ancient tribes Nardeae and Meliceae. The ßCENH3 gene as a part of the locus appeared in the tribes Stipeae and Brachypodieae around 35-40 Mya. The duplication was accompanied by changes in the exon-intron structure. Purifying selection acts mostly on αCENH3s, while ßCENH3s form more heterogeneous structures, in which clade-specific amino acid motifs are present. In barley species, the ßCENH3 gene assumed an inverted orientation relative to αCENH3 and the CDPK2 gene was substituted with LHCB-l. As the evolution and domestication of plant species went on, the locus was growing in size due to an increasing distance between αCENH3 and ßCENH3 because of a massive insertion of the main LTR-containing retrotransposon superfamilies, gypsy and copia, without any evolutionary preference on either of them. A comparison of the molecular structure of the locus in the A, B and D subgenomes of the hexaploid wheat T. aestivum showed that invasion by mobile elements and concomitant rearrangements took place in an independent way even in evolutionarily close species. CONCLUSIONS: The CENH3 duplication in cereals was accompanied by changes in the exon-intron structure of the ßCENH3 paralog. The observed general tendency towards the expansion of the CENH3 locus reveals an amazing diversity of ways in which different species implement the scenario described in this paper.

Expression of Two Rye CENH3 Variants and Their Loading into Centromeres.

Gene duplication and the preservation of both copies during evolution is an intriguing evolutionary phenomenon. Their preservation is related to the function they perform. The central component of centromere specification and function is the centromere-specific histone H3 (CENH3). Some cereal species (maize, rice) have one copy of the gene encoding this protein, while some (wheat, barley, rye) have two. Therefore, they represent a good model for a comparative study of the functional activity of the duplicated CENH3 genes and their protein products. We determined the organization of the CENH3 locus in rye (Secale cereale L.) and identified the functional motifs in the vicinity of the CENH3 genes. We compared the expression of these genes at different stages of plant development and the loading of their products, the CENH3 proteins, into nucleosomes during mitosis and meiosis. Using extended chromatin fibers, we revealed patterns of loading CENH3 proteinsinto polynucleosomal domains in centromeric chromatin. Our results indicate no sign of neofunctionalization, subfunctionalization or specialization in the gene copies. The influence of negative selection on the coding part of the genes led them to preserve their conserved function. The advantage of having two functional genes appears as the gene-dosage effect.

Retrotransposons and the Evolution of Genome Size in Pisum.

Here we investigate the plant population genetics of retrotransposon insertion sites in pea to find out whether genetic drift and the neutral theory of molecular evolution can account for their abundance in the pea genome. (1) We asked whether two contrasting types of pea LTR-containing retrotransposons have the frequency and age distributions consistent with the behavior of neutral alleles and whether these parameters can explain the rate of change of genome size in legumes. (2) We used the recently assembled v1a pea genome sequence to obtain data on LTR-LTR divergence from which their age can be estimated. We coupled these data to prior information on the distribution of insertion site alleles. (3) We found that the age and frequency distribution data are consistent with the neutral theory. (4) We concluded that demographic processes are the underlying cause of genome size variation in legumes.

Cytogenetic and molecular characteristics of rye genome in octoploid triticale (× Triticosecale Wittmack).

Alloploidization resulting from remote (interspecific or intergeneric) hybridization is one of the main factors in plant evolution, leading to the formation of new species. Triticale (× Triticosecale Wittmack, 1889) is the first artificial species created by crossing wheat (Triticum spp.) and rye (Secale cereale Linnaeus, 1753) and has a great potential as a grain and forage crop. Remote hybridization is a stress factor that causes a rapid reorganization of the parental genomes in hybrid progeny ("genomic shock") and is accompanied by abnormalities in the chromosome set of hybrids. The formation of the hybrid genome and its subsequent stabilization are directly related to the normalization of meiosis and the correct chromosome segregation. The aim of this work was to cytogenetically characterize triticale (× Triticosecale rimpaui Wittmack, 1899, AABBDDRR) obtained by crossing Triticum aestivum Linnaeus, 1753. Triple Dirk D × Secale cereale L. Korotkostebel'naya 69 in F3-F6 generations of hybrids, and to trace the process of genetic stabilization of hybrid genomes. Also, a comparative analysis of the nucleotide sequences of the centromeric histone CENH3 genes was performed in wheat-rye allopolyploids of various ploidy as well as their parental forms. In the hybrid genomes of octoploid triticale an increased expression of the rye CENH3 variants was detected. The octoploid triticale plants contain complete chromosome sets of the parental subgenomes maintaining the chromosome balance and meiotic stability. For three generations the percentage of aneuploids in the progeny of such plants has been gradually decreasing, and they maintain a complete set of the paternal rye chromosomes. However, the emergence of hexaploid and new aneuploid plants in F5 and F6 generations indicates that stabilization of the hybrid genome is not complete yet. This conclusion was confirmed by the analysis of morphological features in hybrid plants: the progeny of one plant having the whole chromosome sets of parental subgenomes showed significant morphological variations in awn length and spike density. Thus, we expect that the results of our karyotyping of octoploid triticales obtained by crossing hexaploid wheat to diploid rye supplemented by comparative analysis of CENH3 sequences will be applicable to targeted breeding of stable octo- and hexaploid hybrids.

Dynamics of the Centromeric Histone CENH3 Structure in Rye-Wheat Amphidiploids (Secalotriticum).

The centromeres perform integral control of the cell division process and proper distribution of chromosomes into daughter cells. The correct course of this process is often disrupted in case of remote hybridization, which is a stress factor. The combination of parental genomes of different species in a hybrid cell leads to a "genomic shock" followed by loss of genes, changes in gene expression, deletions, inversions, and translocations of chromosome regions. The created rye-wheat allopolyploid hybrids, which were collectively called secalotriticum, represent a new interesting model for studying the effect of remote hybridization on the centromere and its components. The main feature of an active centromere is the presence of a specific histone H3 modification in the centromeric nucleosomes, which is referred to as CENH3 in plants. In this paper the results of cytogenetic analysis of the secalotriticum hybrid karyotypes and the comparison of the CENH3 N-terminal domain structure of parent and hybrid forms are presented. It is shown that the karyotypes of the created secalotriticum forms are stable balanced hexaploids not containing minichromosomes with deleted arms, in full or in part. A high level of homology between rye and wheat enables to express both parental forms of CENH3 gene in the hybrid genomes of secalotriticum cultivars. The CENH3 structure in hybrids in each crossing combination has some specific features. The percentage of polymorphisms at several amino acid positions is much higher in one of the secalotriticum hybrids, STr VD, than in parental forms, whereas the other hybrid, STr VM, inherits a high level of amino acid substitutions at the position 25 from the maternal parent.

Chromosomal assignment of centromere-specific histone CENH3 genes in rye (Secale cereale L.) and their phylogeny.

Centromeres are essential for correct chromosome segregation during cell division and are determined by the presence of centromere-specific histone 3 (CENH3). Most of the diploid plant species, in which the structure and copy number of CENH3 genes have been determined, have this gene as a singleton; however, some cereal species in the tribe Triticeae have been found to have CENH3 in two variants. In this work, using the set of the wheat-rye addition lines we wanted to establish the chromosomal assignment of the CENH3 genes in the cultivated rye, Secale cereale (Linnaeus, 1753), in order to expand our knowledge about synteny conservation in the most important cereal species and about their chromosome evolution. To this end, we have also analyzed data in available genome sequencing databases. As a result, the αCENH3 and ßCENH3 forms have been assigned to rye chromosomes 1R and 6R: specifically, the commonest variants αCENH3v1 and ßCENH3v1 to chromosome 1R, and the rare variants, αCENH3v2 and probably ßCENH3v2, to chromosome 6R. No other CENH3 variants have been found by analysis of the rye genome sequencing databases. Our chromosomal assignment of CENH3 in rye has been found to be the same as that in barley, suggesting that both main forms of CENH3 appeared in a Triticeae species before the barley and wheatrye lineages split.

The roles of the monomer length and nucleotide context of plant tandem repeats in nucleosome positioning.

Similar to regularly spaced nucleosomes in chromatin, long tandem DNA arrays are composed of regularly alternating monomers that have almost identical primary DNA structures. Such a similarity in the structural organization makes these arrays especially interesting for studying the role of intrinsic DNA preferences in nucleosome positioning. We have studied the nucleosome formation potential of DNA tandem repeat families with different monomer lengths (ML). In total, 165 plant tandem repeat families from the PlantSat database (http://w3lamc.umbr.cas.cz/PlantSat/) were divided into two classes based on the number of nucleosome repeats in one DNA monomer. For predicting nucleosome formation potential, we developed the Phase method, which combines the advantages of multiple bioinformatics models. The Phase method was able to distinguish interfamily differences and intrafamily monomer variation and identify the influence of nucleotide context on nucleosome formation potential. Three main types of nucleosome arrangement in DNA tandem repeat arrays--regular, partially regular (partial), and flexible--were distinguished among a great variety of Phase profiles. The regular type, in which all nucleosomes of the monomer array are positioned in a context-dependent manner, is the most representative type of the class 1 families, with ML equal to or a multiple of the nucleosome repeat length (NRL). In the partially regular type, nucleotide context influences the positioning of only a subset of nucleosomes. The influence of the nucleotide context on nucleosome positioning has the least effect in the flexible type, which contains the greatest number of families (65). The majority of these families belong to class 2 and have nonmultiple ML to NRL ratios.

The genetic diversity and evolution of field pea (Pisum) studied by high throughput retrotransposon based insertion polymorphism (RBIP) marker analysis.

BACKGROUND: The genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus. RESULTS: 3020 Pisum germplasm samples from the John Innes Pisum germplasm collection were genotyped for 45 retrotransposon based insertion polymorphism (RBIP) markers by the Tagged Array Marker (TAM) method. The data set was stored in a purpose-built Germinate relational database and analysed by both principal coordinate analysis and a nested application of the Structure program which yielded substantially similar but complementary views of the diversity of the genus Pisum. Structure revealed three Groups (1-3) corresponding approximately to landrace, cultivar and wild Pisum respectively, which were resolved by nested Structure analysis into 14 Sub-Groups, many of which correlate with taxonomic sub-divisions of Pisum, domestication related phenotypic traits and/or restricted geographical locations. Genetic distances calculated between these Sub-Groups are broadly supported by principal coordinate analysis and these, together with the trait and geographical data, were used to infer a detailed model for the domestication of Pisum. CONCLUSIONS: These data provide a clear picture of the major distinct gene pools into which the genus Pisum is partitioned and their geographical distribution. The data strongly support the model of independent domestications for P. sativum ssp abyssinicum and P. sativum. The relationships between these two cultivated germplasms and the various sub-divisions of wild Pisum have been clarified and the most likely ancestral wild gene pools for domesticated P. sativum identified. Lastly, this study provides a framework for defining global Pisum germplasm which will be useful for designing core collections.

Identification of yessotoxin in mussels from the Caucasian Black Sea Coast of the Russian Federation.

The toxin load of shellfish hepatopancreas harvested from the Caucasian Black Sea Coast of the Russian Federation was investigated. The majority of the toxin load was shown to be yessotoxin (YTX), 45-hydroxy-yessotoxin (45-OH-YTX), and homoyessotoxin (homoYTX). Concurrent with the mussel intoxication, the dinoflagellates Lingulodinium polyedrum and Gonyaulax spinifera were found in high concentrations.

Insertional polymorphism and antiquity of PDR1 retrotransposon insertions in pisum species.

Sequences flanking 73 insertions of the retrotransposon PDR1 have been characterized, together with an additional 270 flanking regions from one side alone, from a diverse collection of Pisum germ plasm. Most of the identified flanking sequences are repetitious DNAs but more than expected (7%) lie within nuclear gene protein-coding regions. The approximate age of 52 of the PDR1 insertions has been determined by measuring sequence divergence among LTR pairs. These data show that PDR1 transpositions occurred within the last 5 MY, with a peak at 1-2.5 MYA. The insertional polymorphism of 68 insertions has been assessed across 47 selected Pisum accessions, representing the diversity of the genus. None of the insertions are fixed, showing that PDR1 insertions can persist in a polymorphic state for millions of years in Pisum. The insertional polymorphism data have been compared with the age estimations to ask what rules control the proliferation of PDR1 insertions in Pisum. Relatively recent insertions (< approximately 1.5 MYA) tend to be found in small subsets of the Pisum accessions set, "middle-aged" insertions (between approximately 1.5 and 2.5 MYA) vary greatly in their occurrence, and older insertions (> approximately 2.5 MYA) are mostly found in small subsets of Pisum. Finally, the average age estimate for PDR1 insertions, together with an existing data set for PDR1 retrotransposon SSAP markers, has been used to derive an estimate of the effective population size for Pisum of approximately 7.5 x 10(5).

Chromosome ends: different sequences may provide conserved functions.

The structures of specific chromosome regions, centromeres and telomeres, present a number of puzzles. As functions performed by these regions are ubiquitous and essential, their DNA, proteins and chromatin structure are expected to be conserved. Recent studies of centromeric DNA from human, Drosophila and plant species have demonstrated that a hidden universal centromere-specific sequence is highly unlikely. The DNA of telomeres is more conserved consisting of a tandemly repeated 6-8 bp Arabidopsis-like sequence in a majority of organisms as diverse as protozoan, fungi, mammals and plants. However, there are alternatives to short DNA repeats at the ends of chromosomes and for telomere elongation by telomerase. Here we focus on the similarities and diversity that exist among the structural elements, DNA sequences and proteins, that make up terminal domains (telomeres and subtelomeres), and how organisms use these in different ways to fulfil the functions of end-replication and end-protection.

Diverse patterns of the tandem repeats organization in rye chromosomes.

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.

Transposable elements reveal the impact of introgression, rather than transposition, in Pisum diversity, evolution, and domestication.

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.

Retroelements, transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation.

We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed, and plants regenerated from tissue culture that show mantling, an abnormality leading to flower abortion. Using PCR assays, reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of En/Spm transposons were cloned. Sequence analysis revealed the presence of a major family of LINEs in oil palm, with other diverged copies. Gypsy-like retrotransposons form a single homologous group, whereas En/Spm transposons are present in several diverged families. Southern analysis revealed their presence in low (LINEs) to medium (gypsy and En/Spm) copy numbers in oil palm, and in situ hybridization showed a limited number of distinct loci for each class of transposable element. No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected, but different levels of sequence methylation were observed. During tissue culture, McrBC digestion revealed the genome-wide reduction in DNA methylation, which was restored to near-normal levels in regenerated trees. HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent. The genomic organization of the transposable DNA elements in different oil palm species, accessions and individual regenerated trees was investigated revealing only minor differences. The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components.

Various organizations of the complex repeats in vole sex chromosome heterochromatin.

Different patterns of the DNA sequences organization were revealed in the vole (Rodentia) sex chromosome heterochromatin using dual-label fluorescence in-situ hybridization on extended DNA fibers with different repetitive DNA sequences as probes. In Microtus rossiaemeridionalis, the basic type represents the homogeneous relatively short tracks consisting of tandemly reiterated monomers of the MS3 family alternating with similar tracks of MS4 monomers and with non-fluorescent spacers. These tracks varied in the length of both repeats, with an average size of 12-22 kb or 3-5 copies. Apart from this, some continuous tracks of both families spanning 100-200 kb were interrupted by short spacers or single signals from the sequences with homology to LINEs. These results, together with that obtained by the analysis of phage clones of the genomic library, unequivocally demonstrate a variable large-scale DNA structural organization in heterochromatin of the M. rossiaemeridionalis sex chromosome. The dominant type of large-scale DNA organization in M. transcaspicus heterochromatin represents the unicolor relatively long tracks consisting of monotonous but not alternating monomers of MS3 or MS4 with sizes ranging from 15 to 40 kb and separated by extended spacers with an average length of 20 kb. Thus, the formation of the vole sex chromosome heterochromatic regions occurred relatively recently during speciation.

LINEs and gypsy-like retrotransposons in Hordeum species.

LINE and gypsy-like retroelements were studied in the genome of Hordeum vulgare, and compared with the representatives of the major sections of the genus Hordeum. We isolated reverse transcriptase (RT) genes from four gypsy-like and three LINE families using PCR primers specific for the corresponding conserved domains. A full-length barley LINE of 6295 bp, named BLIN, was isolated from a BAC genomic library. BLIN looks alien in the barley genome because its G+C content is 62% compared to an average of 45%. The BLIN nucleotide sequence showed it was structurally intact with the features typical of non-LTR retrotransposons, including 16 bp target site duplications, two short cysteine motifs, and two degenerate open reading frames (ORFs). The high degeneracy was also found in RT domain of both gypsy-like and, particularly, LINE families. The copy numbers of the gypsy-like families were relatively low compared to well-characterized copia-like element BARE-1. Each gypsy-like family gave unique RFLP patterns when hybridized to genomic DNA from each of the four basic Hordeum genomes. H. vulgare (I genome) had accumulated more copies than the wild Hordeum species (H, X, Y genomes), with the other I genome species, H. bulbosum, being intermediate. Analysis of the BAC library and in situ hybridization with LINE RT domains showed the low copy number of the LINE families, but there was little correlation between hybridization patterns and the division of the genus into four basic genomes. The distribution and content of gypsy retrotransposons in the BAC library indicated that a few copies are nested, although most are present as single, distinct, copies. Our results suggest that the major groups of retroelements make individual contributions to the shape of the plant genome; the factors involved in their amplification and distribution are independent, also varying among species.