R. Neil Jones

Molecular cytogenetic characterisation of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale)

The terminal heterochromatic segments of the long arms of 20 rye B-chromosomes were isolated by means of laser microdissection technology. Also the remaining portions of the long arms, along with the short arms of the same chromosomes were isolated. Each sample was used for degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplification reactions. The resulting products were used as probes for chromosome in situ hybridisation experiments, and in Southern hybridisation to digests of 0B and +B DNA. Competition hybridisation of these probes with 0B DNA allowed the detection of B-specific sequences. The terminal heterochromatin of the rye B-chromosome contains both B-specific sequences and sequences also present on the A-chromosomes of rye. The B-specific D1100 family is the major repeat species located in the terminal heterochromatin. Primers designed to the cloned sequence (E1100) were used to search for related low copy sequences in 0B DNA. The sequences of the PCR products revealed no similarities to that of the clone E1100 except for the primer sequences. The possible origin of this sequence is discussed in the context of models for the evolution of the rye B-chromosome.

Transcriptionally active heterochromatin in rye B chromosomes.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type–dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.

Isolation of a sequence common to A- and B-chromosomes of rye (Secale cereale) by microcloning

The techniques of microdissection and microcloning have been applied to the isolation of B-chromosome DNA from rye. We have identified a DNA sequence on the rye B-chromosome which is homologous to an A-chromosome sequence, and which is dispersed and moderately repeated on the A- and B-chromosomes. This demonstrates that the rye B-chromosome is heterogeneous in the nature of its DNA sequence composition, containing sequences which are present on the A-chromosomes in addition to those not present on the A-chromosomes.

The influence of B-chromosomes upon the nuclear phenotype in rye

Quantitative studies on the ffects of B-chromosomes in rye upon variation of the nuclear phenotype showed that Bs: (i) influence the phenotype of the A-chromosomes at metaphase of mitosis, (ii) increase the amount of nuclear DNA in proportion to their number present, (iii) have a structural organisation different to that of the A-chromosomes, (iv) influence the structure of the interphase nucleus in respect of non-permanent chromosome material.

Analysis of rye B-chromosome structure using fluorescencein situ hybridization (FISH)

Fluorescencein situ hybridization (FISH) has been used to analyse the structure of the rye B chromosome. Genomicin situ hybridization (GISH) demonstrates the high level of overall similarity between A and B chromosomes of rye, as well as the presence of a number of specific sequences. The B-specific repeat families D1100 and E3900 have been analysed in terms of their physical location and possible contiguity. Rye Bs contain members of the rye-specific dispersed repetitive family R173, as well as centromeric regions similar to those of the As. The B chromosomes analysed in our study lack detectable rDNA sequences. Anomalous results have been obtained with a number of subtelomeric repetitive probes from rye. Bs usually lack these sequences, but evidence is presented that in some cases A–B translocation events may relocate such sequences from the As to the Bs. These data are discussed in the context of current models for the origin of the B chromosome.

PHYLETIC HOT SPOTS FOR B CHROMOSOMES IN ANGIOSPERMS

Abstract We determined whether supernumerary B chromosomes were nonrandomly distributed among major angiosperm lineages and among lineages within families, as well as the identity of lineages with unusually high B‐chromosome frequencies (hot spots). The incidence of B chromosomes for each taxon was gathered from databases showing species with and without these chromosomes (among species with known chromosome numbers). Heterogeneity was found at all ranks above the species level. About 8% of monocots had B chromosomes versus 3% for eudicots; they were rare in nonmonocot basal angiosperms. Significant heterogeneity in B‐chromosome frequency occurred among related orders, families within orders, and major taxa within families. There were many B‐chromosome hot spots, including Liliales and Commelinales at the order level. At the family level, there was a trend suggesting that B‐chromosome frequencies are positively correlated with genome size.

B chromosomes are more frequent in mammals with acrocentric karyotypes: support for the theory of centromeric drive.

The chromosomes of mammals tend to be either mostly acrocentric (having one long arm) or mostly bi–armed, with few species having intermediate karyotypes. The theory of centromeric drive suggests that this observation reflects a bias during female meiosis, favouring either more centromeres or fewer, and that the direction of this bias changes frequently over evolutionary time. B chromosomes are selfish genetic elements found in some individuals within some species. B chromosomes are often harmful, but persist because they drive (i.e. they are transmitted more frequently than expected). We predicted that species with mainly acrocentric chromosomes would be more likely to harbour B chromosomes than those with mainly bi–armed chromosomes, because female meiosis would favour more centromeres over fewer in species with one–armed chromosomes. Our results show that B chromosomes are indeed more common in species with acrocentric chromosomes, across all mammals, among rodents, among non–rodents and in a test of independent taxonomic contrasts. These results provide independent evidence supporting the theory of centromeric drive and also help to explain the distribution of selfish DNA across species. In addition, we demonstrate an association between the shape of the B chromosomes and the shape of the typical (‘A’) chromosomes.

Interphase arrangement of rye B chromosomes in rye and wheat

Probes for B chromosome-specific sequences in the distal region of the long arm of the rye B have been used to investigate the interphase arrangement of the Bs in rye and in hexaploid wheat. The Lindstro¨m strain of wheat carries the rye Bs as additions. The number of in situ signals in nuclei with two, three and four Bs is often less than the maximum B number, and it seems that the Bs may be grouped together in various ways rather than being randomly dispersed throughout the nucleus. The degree of physical association is greater in rye than in the alien wheat background. The results are discussed in relation to the pairing and recombination preferences of the Bs in rye and in Lindstro¨m wheat.

The Plant Nucleus at War and Peace: Genome Organization in the Interphase Nucleus

The interphase nucleus is highly dynamic, anything but the ‘resting stage’ of the cell cycle. In terms of genome organisation the diploid nucleus is the most ‘peaceful’, but contrasting structural arrangements may be found in apparently comparable plants at every physical level, from the conservation or not of Rabl organisation as genome size increases through to the presence or absence of expressed genes within a species. This plasticity may mean that virtually every individual of a species represents a unique combination of gene copy numbers, heterochromatic and mobile element content and interchromosomal associations. Tensions in the nucleus are most apparent in newly created interspecific hybrids and allopolyploids, where two genomes share a common cytoplasm and experience numerous and rapid interactions, including: loss or gain of sequences, transposon activation, epigenetic changes, interaction of regulatory elements, genome drift and modifications to cell cycling. The story of order and chaos in the plant nucleus is thus incomplete and open-ended, since our current knowledge is based on only a small number of model species. We also have to bear in mind recent findings that many diploids, if not all, have a history of having passed through earlier cycles of ploidy events, and still bear the duplications as evidence. New discoveries on genome readjustment in hybrids and allopolyploids have implications for our understanding of genome change in evolution, as well as presenting opportunities for the release of new forms of genetic and epigenetic variation in crop plants.