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.

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.