Recombinant genome of cereals: the pattern of formation and the role in evolution of polyploid species

The pivotal-differential model of evolution of polyploid species of cereals has been experimentally reproduced, and the pattern of the formation of a recombinant genome has been analyzed. It has been found that mutual substitution of chromosomes of the original genomes is subjected to selection pressure and, hence, is nonrandom. The selection occurs at the level of homeologs, whose selective advantages are determined by interactions between the genotype and the environment. If a homeolog has distinct selective advantages, the chromosomal composition of the corresponding homeologous group is completed rapidly, which leads to the formation of intergenomic recombination at the level of whole chromosomes. If homeologs have the same competitiveness, the composition of the group is stabilized more slowly. Domination of the genetic systems of the basic genome ensures a high rate of pairing of homeologous chromosomes of the recombinant genome during meiosis, which leads to recombinations at the level of chromosomal segments. It has been demonstrated that different combinations of chromosomes from original genomes are selected at different conditions of plant growth.     

Molecular analysis of the triticale lines with different Vrn gene systems using microsatellite markers and hybridization in situ

Hexaploid triticale (x Triticosecale Wittmack) lines were examined using molecular markers and the hybridization in situ technique. Triticale lines were generated based on wheat varieties differing by the Vrn gene systems and the earing times. Molecular analysis was performed using Xgwm and Xrms microsatellite markers with the known chromosomal localization in the common wheat Triticum aestivum, and rye Secale cereale genomes. Comparative molecular analysis of triticale lines and their parental forms showed that all lines contained A and B genomes of common wheat and also rye homeologous chromosomes. In the three lines the presence of D genome markers, mapped to the chromosomes 2D and 7D, was demonstrated. This was probably the consequence of the translocations of homeologous chromosomes from wheat genomes, which took part during the process of triticale formation. The data obtained by use of genomic in situ hybridization supported the data of molecular genetic analysis. In none of the lines wheat--rye translocations or recombinations were observed. These findings suggest that the change of the period between the seedling appearance and earing time in triticale lines compared to the initial wheat lines, resulted from the inhibitory effect of rye genome on wheat vernalization genes.     

Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica

The aim of this work was to find C genome specific repetitive DNA sequences able to differentiate the homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica, in order to assist in the physical identification of B. napus chromosomes. A repetitive sequence (pBo1.6) highly enriched in the C genome of Brassica was cloned from B. oleracea and its chromosomal organisation was investigated through fluorescent in situ hybridisation (FISH) in B. oleracea (2n = 18, CC), B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC) genomes. The sequence was 203 bp long with a GC content of 48.3%. It showed up to 89% sequence identity with telomere-like DNA from many plant species. This repeat was clearly underrepresented in the A genome and the in situ hybridisation showed its B. oleracea specificity at the chromosomal level. Sequence pBo1.6 was localised at interstitial and/or telomeric/subtelomeric regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus 18 to 24 chromosomes hybridised with pBo1.6. The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful for the determination of the chromosomal location of transgenic DNA in genetically modified oilseed rape.     

Meiotic segregation of a homeologous chromosome pair

During meiosis, the alignment of homologous chromosomes facilitates their subsequent migration away from one another to opposite spindle poles at anaphase I. Recombination is part of the mechanism by which chromosomes identify their homologous partners, and serves to link the homologs in a way that, in some organisms, has been shown to promote proper attachment to the meiotic spindle. We have built a diploid strain that contains a pair of homeologous chromosomes V': one is derived from Saccharomyces cerevisiae and one originates from S. carlsbergensis. Sequence analysis reveals that these chromosomes share 71% sequence identity. The homeologs experience high levels of meiotic double-stranded breaks. Despite their relatedness and their competence to initiate recombination, the meiotic segregation behavior of the homeologous chromosomes suggests that, in most meioses, they are partitioned by a meiotic segregation system that has been shown previously to partition non-exchange chromosomes and pairs with no homology. Though the homeologous chromosomes show a degree of meiotic segregation fidelity similar to that of other non-exchange pairs, our data provide evidence that their limited sequence homology may provide some bias in meiotic partner choice.     

Seventy million years of concerted evolution of a homoeologous chromosome pair, in parallel, in major Poaceae lineages

Whole genome duplication ~70 million years ago provided raw material for Poaceae (grass) diversification. Comparison of rice (Oryza sativa), sorghum (Sorghum bicolor), maize (Zea mays), and Brachypodium distachyon genomes revealed that one paleo-duplicated chromosome pair has experienced very different evolution than all the others. For tens of millions of years, the two chromosomes have experienced illegitimate recombination that has been temporally restricted in a stepwise manner, producing structural stratification in the chromosomes. These strata formed independently in different grass lineages, with their similarities (low sequence divergence between paleo-duplicated genes) preserved in parallel for millions of years since the divergence of these lineages. The pericentromeric region of this homeologous chromosome pair accounts for two-thirds of the gene content differences between the modern chromosomes. Both intriguing and perplexing is a distal chromosomal region with the greatest DNA similarity between surviving duplicated genes but also with the highest concentration of lineage-specific gene pairs found anywhere in these genomes and with a significantly elevated gene evolutionary rate. Intragenomic similarity near this chromosomal terminus may be important in hom(e)ologous chromosome pairing. Chromosome structural stratification, together with enrichment of autoimmune response-related (nucleotide binding site-leucine-rich repeat) genes and accelerated DNA rearrangement and gene loss, confer a striking resemblance of this grass chromosome pair to the sex chromosomes of other taxa.     

Genetic characterization of CHO production host DG44 and derivative recombinant cell lines

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.     

Effect of chromosomal instability on the mutation-selection balance in unicellular populations

This paper develops a mathematical model describing the evolutionary dynamics of a unicellular, asexually replicating population exhibiting chromosomal instability. Chromosomal instability is a form of genetic instability characterized by the gain or loss of entire chromosomes during cell division. We assume that the cellular genome is divided into several homologous groups of chromosomes, and that a single functional chromosome per homologous group is required for the cell to have the wild-type fitness. If the fitness is unaffected by the total number of chromosomes in the cell, our model is analytically solvable, and yields a mean fitness at mutation-selection balance that is identical to the mean fitness when there is no chromosomal instability. If this assumption is relaxed and the total number of chromosomes in the cell is not allowed to increase without bound, then chromosomal instability leads to a reduction in mean fitness. The results of this paper provide a useful baseline that can inform both future theoretial and experimental studies of chromosomal instability.     

Cytogenetics of Triticum x Dasypyrum hybrids and derived lines

Genomic in situ hybridization was used to study Triticum x Dasypyrum wide hybrids and derived lines. A cytogenetic investigation was carried out in progenies of (i) amphiploids derived from T. turgidum var. durum (T. durum; 2n = 14; genomes AABB) x D. villosum (2n = 14; genome VV), (ii) three-parental hybrids (T. durum x D. villosum) x T. aestivum (2n = 42, genomes A'A'B'B'D'D'), and (iii) T. aestivum aneuploid lines carrying D. villosum chromosomes or chromatin. The amphiploids derived from T. durum x D. villosum showed a stable chromosomal constitution, made up of 14 V chromosomes, 14 chromosomes carrying the wheat A genome and 14 chromosomes carrying the B genome. High karyological instability was observed in the progenies of three-parental hybrids ([T. durum x D. villosum] x T. aestivum). Plants having the expected 14 A chromosomes, 14 B chromosomes, 7 D chromosomes, and 7 V chromosomes were rather rare (4.5%). Many progeny plants (45.5%) had the hexaploid wheat genome with 42 chromosomes and lacked any detectable D. villosum chromatin. Other plants (50%) had 14 A chromosomes and 14 B chromosomes, plus variable numbers of D and V chromosomes, the former being better retained than the latter in most cases. Some T. aestivum lines carrying D. villosum chromosomes or chromatin, as the result of addition, substitution, or recombination events or even a combination of these karyological events, were found to be stable. Other lines were unstable, and these lines carried 1V, 3V, or 5V chromosomes or their portions. Substitution or recombination events where 1V chromosomes were involved could concern the homeologous counterparts in both the A and B and D genomes of wheat. No line could be recovered where the shorter arm of 3V chromosomes was present. Changes in the morphology and banding pattern of V chromosomes were observed in hybrids that did not carry the entire D. villosum complement. By comparing the results of our cytogenetic analyses with certain phenotypic characteristics of the lines studied, genes for discrete traits could be assigned to specific V chromosomes or V chromosome arms. From the frequency of V chromosomes that were involved in chromatin exchanges with or substituted for one of their homeologous counterparts in the A, B, and D wheat genomes, it was inferred that D. villosum belongs to the same phyletic lineage as T. urartu (donor of the A genome of wheat) and Aegilops speltoides (B genome), and that Ae. squarrosa (D genome) diverged earlier from D. villosum.     

A genetic map of tomato based on BC(1) Lycopersicon esculentum x Solanum lycopersicoides reveals overall synteny but suppressed recombination between these homeologous genomes

F(1) hybrids between the cultivated tomato (Lycopersicon esculentum) and the wild nightshade Solanum lycopersicoides are male sterile and unilaterally incompatible, breeding barriers that impede further crosses to tomato. Meiosis is disrupted in 2x hybrids, with reduced chiasma formation and frequent univalents, but is normal in allotetraploid hybrids, indicating the genomes are homeologous. In this study, a partially male-fertile F(1) was backcrossed to tomato, producing the first BC(1) population suitable for genetic mapping from this cross. BC(1) plants were genotyped at marker loci to study the transmission of wild alleles and to measure rates of homeologous recombination. The pattern of segregation distortion, in favor of homozygotes on chromosomes 2 and 5 and heterozygotes on chromosomes 6 and 9, suggested linkage to a small number of loci under selection on each chromosome. Genome ratios nonetheless fit Mendelian expectations. Resulting genetic maps were essentially colinear with existing tomato maps but showed an overall reduction in recombination of approximately 27%. Recombination suppression was observed for all chromosomes except 9 and 12, affected both proximal and distal regions, and was most severe on chromosome 10 (70% reduction). Recombination between markers on the long arm of this chromosome was completely eliminated, suggesting a lack of colinearity between S. lycopersicoides and L. esculentum homeologues in this region. Results are discussed with respect to phylogenetic relationships between the species and their potential use for studies of homeologous pairing and recombination in a diploid plant genome.     

Identification of Chromosomes from Multiple Rice Genomes Using a Universal Molecular Cytogenetic Marker System

To develop reliable techniques for chromosome identification is critical for cytogenetic research, especially for genomes with a large number and smaller-sized chromosomes. An efficient approach using bacterial artificial chromosome （BAC） clones as molecular cytological markers has been developed for many organisms. Herein, we present a set of chromosomal arm-specific molecular cytological markers derived from the gene-enriched regions of the sequenced rice genome. All these markers are able to generate very strong signals on the pachytene chromosomes of Oryza sativa L. （AA genome） when used as fluorescence in situ hybridization （FISH） probes. We further probed those markers to the pachytene chromosomes of O. punctata （BB genome） and O. officinalis （CC genome） and also got very strong signals on the relevant pachytene chromosomes. The signal position of each marker on the related chromosomes from the three different rice genomes was pretty much stable, which enabled us to identify different chromosomes among various rice genomes. We also constructed the karyotype for both O. punctata and O. officinalis with the BB and CC genomes, respectively, by analysis of 10 pachytene cells anchored by these chromosomal arm-specific markers.     

In vitro androgenesis response of durum wheat disomic substitution

The variety 'Langdon' and its substitution series were used to evaluate the effect of each substituted chromosome of the A and B genomes on the in vitro androgenetic potential. This study showed the implication of chromosomes 1B and 5B in repressing embryogenesis. Genes located on these chromosomes seem to have an inhibitor effect. The substitution of these chromosomes by their homeologous ones from the D genome increased the number of embryos while with the presence of the original genes the number of embryos was less than in the control. Chromosome 5B is also especially involved in the regeneration of green plants. The genetic control is inhibitory; this explains the difficulty of obtaining good levels of in vitro androgenesis in durum wheat. In this study no effect of the D genome on the androgenetic response of the substitution lines was observed.     

Chromosome evolution

The idea of evolution as a principle for the origin of biodiversity fits all phenomena of life, including the carriers of nuclear inheritance, the chromosomes. Insights into the evolutionary mechanisms that contribute to the shape, size, composition, number and redundancy of chromosomes elucidate the high plasticity of nuclear genomes at the chromosomal level, and the potential for genome modification in the course of breeding processes. Aspects of chromosome fusion, as exemplified by karyotype evolution of relatives of Arabidopsis, have recently received special attention.     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Relationship between Chromosome 9 of Maize and Wheat Homeologous Group 7 Chromosomes

Comparison of the genetic map of maize chromosome 9 with maps of wheat chromosomes has revealed a high degree of colinearity between maize chromosome 9 and the group 4 and 7 chromosomes of wheat. The order of DNA markers on the short arm and a proximal region of the long arm of the genetic map of maize chromosome 9 is highly conserved with the marker order on the short arm and proximal region of the long arm of the genetic maps of the wheat homeologous group 7 chromosomes. A major part of the long arm of the genetic map of maize chromosome 9 is homeologous with a short segment in the proximal region of the long arm of the genetic map of the wheat group 4 chromosomes. Evidence is also presented that maize chromosome 9 has diverged from the wheat group 7 chromosomes by both a pericentric and a paracentric inversion. The paracentric inversion is probably unique to maize among the major cereal genomes.     

Inter- and intra-genomic homology of the Brassica genomes: implications for their origin and evolution

In order to determine the homologous regions shared by the cultivated Brassica genomes, linkage maps of the diploid cultivated B. rapa (A genome, n = 10), B. nigra (B genome, n = 8) and B. oleracea (C genome, n = 9), were compared. We found intergenomic conserved regions but with extensitve reordering among the genomes. Eighteen linkage groups from all three species could be associated on the basis of homologous segments based on at least three common markers. Intragenomic homologous conservation was also observed for some of the chromosomes of the A, B and C genomes. A possible chromosome phylogenetic pathway based on an ancestral genome of at least five, and no more than seven chromosomes, was drawn from the chromosomal inter-relationships observed. These results demonstrate that extensive duplication and rearrangement have been involved in the formation of the Brassica genomes from a smaller ancestral genome.