Carolyn R. Leach

A repetitive DNA sequence common to the different B chromosomes of the genus Brachycome

Abstract. Dot-like micro B chromosomes of Brachycome dichromosomatica were analysed for their sequence composition. Southern hybridization patterns of a total micro B probe to genomic DNA from plants with and without micro Bs demonstrated that the micro Bs shared sequences with the A chromosomes. In addition to telomere, rDNA and common A and B chromosome sequences, a new B-specific, highly methylated tandem repeat (Bdm29) was detected. After in situ hybridization with Bdm29 the entire micro B chromosome was labelled and clustering of the condensed micro Bs could be observed at interphase. A high number of Bdm29-like sequences were also found in the larger B chromosomes of B. dichromosomatica and in other Bs within the genus Brachycome.

Organisation and origin of a B chromosome centromeric sequence from Brachycome dichromosomatica

Brachycome dichromosomatica is an Australian native daisy that has two pairs of A chromosomes and up to three B chromosomes in some populations. A putative B-specific tandem repeat DNA sequence (Bd49) was isolated previously. Here we describe further characterisation of this sequence and investigate its possible origin. Southern analysis showed that all individual B chromosomes examined have highly methylated tandem repeats of Bd49 but differences in banding pattern for distinct B isolates suggested that the sequence is in a state of flux. Using in situ hybridisation, the sequence was shown to be located at the centromeric region of the B chromosome. Southern analysis of genomic DNA with Bd49 demonstrated that multiple copies of the sequence exist in the genomes of B. eriogona, B. ciliaris, B. segmentosa and B. multifida (none of which have B chromosomes) whereas other species tested (including 0B plants of B. dichromosomatica and 0B B. curvicarpa and B. dentata) have few or no copies. Genomic clones and Bd49-like sequences derived by the polymerase chain reaction (PCR) were obtained from five species but determination of phylogenetic relationships within the genus and inference as to the possible origin of the B chromosome were problematic because of extensive intragenomic heterogeneity of the sequences.

The genomic complexity of micro B chromosomes of Brachycome dichromosomatica

Abstract. A major sequence component of the micro B chromosome of Brachycomedichromosomatica (2n=4) is the tandem repeat Bdm29, which was found by in situ hybridisation to be distributed along the entire length of the chromosome. A high copy number of this sequence does not occur as a regular feature of the A chromosomes in this species but it was found in infrequent individuals in two wild populations that were analysed. In these instances Bdm29 is localised within heterochromatic, polymorphic segments on the long arm of chromosome 1. The origin of the micro B chromosomes was investigated by determining whether they are related to this A chromosome polymorphism by simple excision and/or integration. Results obtained by using Bdm29, together with a newly isolated repeat sequence, Bdm54, and a number of other sequences known to occur on the micro B chromosome, as probes in in situ hybridisation and Southern analysis demonstrated that the formation of micro B chromosomes is a complex multistep process. The observation that the genomic organisation of the micro B chromosome is unlike anything found on the A chromosomes precludes their origin by simple excision and also indicates that micro Bs do not integrate directly into the A complement to form polymorphic heterochomatic segments.

Differences of histone H4 acetylation and replication timing between A and B chromosomes of Brachycome dichromosomatica

Differences are demonstrated between A (transcriptionally active) and B (transcriptionally inactive) chromosomes that are characterized by a different level of histone H4 acetylation and a different timing of DNA replication. These differences between the chromatin of A and B chromosomes were found after immunolabelling of chromsomes of Brachycome dichromosomatica with antibodies specific for different acetylated forms (lysine 5, 8, 12 and 16) of histone H4. In contrast to the A chromosomes, which are labelled brightly in their entirety, the transcriptionally inactive B chromosomes are faintly labelled with antibodies against H4Ac5 and H4Ac8. No such difference between the chromosomes is found after immunostaining with the other antibodies H4Ac12 and H4Ac16. Analysis of DNA replication timing in root-tip meristems suggests that B chromosomes are labelled late in S-phase compared with A chromosomes. After C-banding the B chromosome appeared to have a similar amount of heterochromatin to the A chromosomes.

A monophyletic origin of the B chromosomes of Brachycome dichromosomatica (Asteraceae)

The A and B chromosomes of different karyotype variants (cytodemes A1, A2, A3 and A4) ofBrachycome dichromosomatica were analysed by computer-aided chromosome image analysis and fluorescencein situ hybridisation (FISH). Ribosomal DNA and the B chromosome-specific sequence Bd49 were detected on all B chromosomes. In addition to minor size variation of the Bs, polymorphism of the rDNA and Bd49 position and copy number revealed two major types of B chromosomes. The B chromosomes of all the cytodemes were indistinguishable from each other in length, but that of A3 showed evidence of rearrangements consistent with its long-term geographic isolation. The results presented suggest a monophyletic origin of the B chromosomes ofB. dichromosomatica.

Ribosomal RNA genes and the B chromosome of Brachycome dichromosomatica

Fluorescence in situ hybridization (FISH) with biotinylated rDNA revealed the presence of an rRNA gene cluster on both the A and B chromosomes of Brachycome dichromosomatica, an Australian native ephemeral plant of the arid regions of south-eastern Australia. This species contains only two pairs of A chromosomes and up to three B chromosomes. The regular attachment of the B chromosome to a nucleolus suggests that these ribosomal RNA genes are transcribed. Southern hybridization of DNA from 0B and +B plants digested with a variety of restriction enzymes indicates that the rRNA genes on the A and B chromosomes are the same in sequence and methylation status.

The B chromosomes in Brachycome

This review presents a historical account of studies of B chromosomes in the genus Brachycome Cass. (synonym: Brachyscome) from the earliest cytological investigations carried out in the late 1960s though to the most recent molecular analyses. Molecular analyses provide insights into the origin and evolution of the B chromosomes (Bs) of Brachycome dichromosomatica, a species which has Bs of two different sizes. The larger Bs are somatically stable whereas the smaller, or micro, Bs are somatically unstable. Both B types contain clusters of ribosomal RNA genes that have been shown unequivocally to be inactive in the case of the larger Bs. The large Bs carry a family of tandem repeat sequences (Bd49) that are located mainly at the centromere. Multiple copies of sequences related to this repeat are present on the A chromosomes (As) of related species, whereas only a few copies exist in the A chromosomes of B. dichromosomatica. The micro Bs share DNA sequences with the As and the larger Bs, and they also have B-specific repeats (Bdm29 and Bdm54). In some cases repeat sequences on the micro Bs have been shown to occur as clusters on the A chromosomes in a proportion of individuals within a population. It is clear that none of these B types originated by simple excision of segments from the A chromosomes.

The molecular organisation of a B chromosome tandem repeat sequence from Brachycome dichromosomatica

A high copy, tandemly repeated, sequence (Bd49) specific to the B chromosome and located near the centromere inBrachycome dichromosomatica was used to identify lambda genomic clones from DNA of a 3B plant. Only one clone of those analysed was composed entirely of a tandem array of the B-specific repeat unit. In other clones, the Bd49 repeats were linked to, or interspersed with, sequences that are repetitious and distributed elsewhere on the A and B chromosomes. One such repetitious flanking sequence has similarity to retrotransposon sequences and a second is similar to chloroplast DNA sequences. Of the four separate junctions analysed of Bd49-like sequence with flanking sequence, three were associated with the same A/T-rich region in Bd49 and the fourth was close to a 25 bp imperfect dyadic sequence. No novel B-specific sequences were detected within the genomic clones.

The incompatibility loci as indicators of conserved linkage groups in the Poaceae

Linkage between an incompatibility locus and the locus of the gene encoding the enzyme glucose phosphoisomerase has been demonstrated for the grasses Alopecurus myosuroides, Phalaris coerulescens, Festuca pratensis, Holcus lanatus and Secale cereale suggesting the conservation of a common chromosomal element in the evolution of the Poaceae.

Internal transcribed spacer sequence analyses indicate cytoevolutionary patterns within Brachycome Cass. (Asteraceae)

The sequences of the Internal Transcribed Spacer regions (ITS1 and ITS2) within the genes coding for cytoplasmic ribosomal (r) RNAs on the A chromosome complement of 34 members of the higher plant genus Brachycome (synonym Brachyscome) have been compared. The ITS1 sequence of species within the B. lineariloba complex contains a 56 bp tract that is absent from at least 12 Brachycome species but is present in other species within Brachycome as well as other Asteraceae. Phylogenetic data support the suggestion that the number of chromosomes reduced in several independent Brachycome lineages during speciation. Comparisons with the B chromosome ITS2 of B. dichromosomatica cytodeme A1 suggests an origin of the B chromosome at a time prior to the divergence of the four cytodemes of B. dichromosomatica.