The cytology of Brachycome

Over 1,000 plants of B. dichromosomatica have been counted. 10% of these carried one, two or three B chromosomes. The B chromosome is large, and though it is not heteropycnotic it condenses precociously at mitosis. It behaves regularly at mitosis, and when two are present they pair regularly at meiosis. Non-disjunction and preferential distribution of the B to the generative nucleus occurs at the first pollen grain mitosis, with very high frequency. This is corroborated by data from crosses, which also indicate that the transmission of the B through the female gamete is normal. — The frequency of B chromosomes in marginal populations of var. dichromosomatica is significantly higher than in central populations. In one population of var. alba the frequency of Bs increased significantly after two very dry seasons. It is suggested that both these cases of increased frequency were in response to a selective advantage of plants with Bs under arid conditions. — Plants with one B chromosome appear to be less fit than plants with 2 Bs. The combination of the calculated effects of the nondisjunction mechanism and the inferred relative fitness of the 0B, 1B and 2B plants, provides a reasonable explanation of the observed frequencies of the 0B, 1B and 2B plants in the populations studied.     

The cytology of Brachycome lineariloba

Meiotic behaviour in PMC's and in ESMC's, the development of pollen and embryo sacs, and the chromosome constitution of endosperm in this species (species E) define its unique genetic system. It is not a normal diploid, and is perhaps best defined as a quasidiploid. — The somatic complement consists of a diploid set of four chromosomes plus a haploid set of two nonhomologous chromosomes. The latter are inherited solely via the pollen. — In meiosis in both PMC'c and ESMC's the two univalents divide at the first division and lag at second anaphase. Pollen grains which do not receive them are non-functional. The embryo sacs are of the normal Asteroid type, and each is derived from a chalazal megaspore. They do not transmit the univalent chromosomes. Following fusion of the polar nuclei and double fertilisation, the endosperm has a constitution of 14 chromosomes. — The system parallels the maternal inheritance of univalents in Rosa canina and in Leucopogon juniperinus, except that the univalents do not constitute a full haploid set, and except that their transmission is paternal.     

The cytology of Brachycome lineariloba

A study of approximately 300 plants in the taxonomic species B. lineariloba is reported. Five biological species differing in chromosome number exist in this species complex. The species with the lowest number (species A, n = 2) often carries B chromosomes, which may be large, or minute. It also exists in three racial forms which differ in karyotype. Observations on naturally occurring hybrids in zones of overlap show that two of the races differ by an unequal interchange. — The species with n = 8 (species C) is probably of amphidiploid origin from the cross A X B. Species B, E and D, with n = 6, 5 and 4 respectively, may represent a series of reductions in chromosome number. They show close karyotypic relationships. The relationship of species A with D, E and B is obscure.     

The cytology of Brachycome lineariloba

The frequencies and behaviour of two types of accessory chromosome in Brachycome lineariloba species A (n=2) are reported. These include a larger (ca. 4.0 macro-B chromosome, and a minute (<1.0 ) micro-B. Twelve per-cent of the 452 plants studied were carriers of one, two or more rarely three macro-B chromosomes. These were usually mitotically stable, but showed some meiotic irregularity when present in odd number. An equilibrium situation is inferred, and loss must be balanced against mechanisms tending to increase B frequency. A surplus of plants with two B chromosomes also suggests that mechanisms of doubling may operate. A possible source of the macro-B's is considered to be hybridisation between the several species of the B. lineariloba complex. — The micro-B chromosomes were found in 3.8% of two only of the three known cytological races of the species. They showed extreme irregularity in both mitotic and meiotic behaviour. Their relationship to the normal (A) chromosomes and to the macro-B's is obscure.     

The cytology of Brachycome lineariloba

Brachycome lineariloba race A (n=2) contains at least four cytodemes designated A₁, A₂, A₃ and A₄ which differ in karyotype and geographical distribution. Data from chromosome morphology, patterns of early and late condensing chromatin and heterochromatin and meiotic pairing are presented.—These show that A₂, A₃ and A₄ differ by loss or suppression of nucleolar organisers. A₄ has a nucleolar organiser on both chromosomes and is likely to be primitive. A₁ differs by interchange. There has been some conversion of early to late condensing chromatin. All the cytodemes show karyotypic polymorphism.     

The cytology of Brachycome lineariloba

A comparison of the karyotypes of races D (2n=8), E (2n=10), B (2n=12) and C (2n=16) of B. lineariloba suggests that these races have in common a basic set of four chromosome pairs, and that the higher chromosome number races are related to race D by successive chromosome addition. — A study of meiosis in B × C and A₁ × B hybrids supports this contention and elucidates the homologies of the additional chromosomes. — Meiotic pairing in hybrids between A and C is very complex. At present it can only be stated that there are extensive interchromosomal homologies between the two races. — Two phyletic schemes of the relationships of the races are considered. The second, which is favoured, involves successive chromosome addition, with the quasidiploid race E (2n = 10) giving rise to race B by diploidisation of the univalent chromosomes. This scheme is supported by features of univalent behaviour in the various races and their hybrids. — The ecogeographic distribution pattern of the races shows replacement of D by E by B by C as the species extends into more arid and more harsh environments. This replacement is also associated with increasing vigour. — It seems most likely that the addition chromosomes are derived from a race A (2n=4) source since they are added always by twos, and each addition increases both vigour and drought tolerance. Race A is the most vigorous and one of the most drought tolerant of the five races.It is suggested that the evolution of the races can be related to the increasing aridity of the Late Pleistocene and Recent geological epochs.     

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 genomic complexity of micro B chromosomes of Brachycome dichromosomatica

A major sequence component of the micro B chromosome of Brachycome dichromosomatica (2 n=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.     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

A sequence specific to B chromosomes of Brachycome dichromosomatica

Supernumerary B chromosomes represent one of many causes of numerical chromosome variation that exist in higher plants and animals. Sequences of DNA unique to B chromosomes of Brachycome dichromosomatica were enriched prior to cloning and resultant clones hybridizing only to plants containing B chromosomes were further investigated. Sequences of DNA that were characterised include members of a family of 176-bp tandem repeats that are specific to the B chromosomes of B. dichromosomatica, an annual Australian native plant species with only two pairs of A chromosomes and up to three dispensable B chromosomes. Sequence analysis of these six related clones indicated that some regions of the sequence are more highly conserved than others or, alternatively, that some adenine residues at the NdeII site are methylated. The repeat is homologous to DNA from Brachycome ciliaris var. languinosa but not to DNA from other related taxa growing in the vicinity of the B. dichromosomatica populations.     

Brachycome lineariloba: A species for experimental cytogenetics

A chromosome number ofn=2 is reported forBrachycome lineariloba. The chromosomes are 6–8 μ long in mitotic metaphase, and first meiotic prophase stages are amenable to study. A B-chromosome is sometimes present. The species is an ephemeral annual and should be easily cultivated. It may prove to be a suitable organism for experimental cytogenetics.     

Cloning and characterisation of polymorphic heterochromatic segments of Brachycome dichromosomatica

After selective enrichment and differential hybridisation of C_ot-1 DNA fractions of plants with and without polymorphic heterochromatic segments, a repetitive sequence (called Bds1) specific to the polymorphic chromosome segments of Brachycome dichromosomatica (Brachyscome dichromosomatica) was isolated. A single repeat unit of Bds1 is 92 bp long and is organised in tandem arrays at three different polymorphic segment sites on the chromosomes of cytodeme A2. Although all three sites showed extensive polymorphism between plants, the karyotypes of all analysed mitotic root cells were stable within a single plant. Electron microscopy revealed heavily condensed chromatin structures at the most obvious polymorphic site. The mechanisms that generate and maintain the observed chromosome structure polymorphisms are discussed. Received: 16 March 1999; in revised form: 28 September 1999 / Accepted: 11 November 1999     

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.