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.     

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.     

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.     

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     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.     

Variability of the 5S and 45S rDNA sites in Passiflora L. species with distinct base chromosome numbers

Cytologically, the species of Passiflora with known chromosome number can be divided into four groups: (1) 2n = 12, 24, 36; (2) 2n = 24; (3) 2n = 18, 72; and (4) 2n = 20. The base chromosome number proposed for the genus is x = 6, with x = 9, x = 10 and x = 12 being considered secondary base numbers. In the present study, variability of 5S and 45S rDNA sites was investigated in 20 species of these four groups to check the reliability of this hypothesis. In the group with x = 6, five diploid species (2n = 12) exhibit two 5S rDNA sites and two (P. capsularis, P. morifolia and P. rubra) or four (P. misera 2x and P. tricuspis) 45S rDNA sites. The hexaploid cytotype of P. misera had 12 45S rDNA sites and six 5S rDNA. A tetraploid species, P. suberosa, had ten 45S rDNA sites and four 5S rDNA sites, both in the same chromosomes as the 45S rDNA sites. In the group with x = 9, P. actinia, P. amethystina, P. edmundoi, P. elegans, P. galbana, P. glandulosa and P. mucronata displayed six 45S rDNA sites, whereas P. alata, P. cincinnata, P. edulis f. flavicarpa, P. edulis var. roxo and P. laurifolia had four sites. In this group, all species were diploid (2n = 18) and had only two 5S rDNA sites. Passiflora foetida, the only species with 2n = 20, had six 45S rDNA sites and four 5S rDNA sites. The species with x = 12 (2n = 24), P. haematostigma and P. pentagona, showed four 45S rDNA sites and two 5S rDNA. In general, the number and location of 5S and 45S rDNA sites were consistent with the hypothesis of x = 6 as the probable ancestral genome for the genus, while the groups of species with x = 9, x = 10 and x = 12 were considered to be of tetraploid origin with descending dysploidy and gene silencing of some redundant gene sites, mainly those of 5S rDNA.     

Physical mapping of the 5S and 45S rDNA in teosintes

The physical locations of the 5S and 45S rDNA sequences were examined in three types of teosinte, Zea mays ssp. mexicana (2n = 20), Zea diploperennis (2n = 20) and Zea perennis (2n = 40) by biotinylated fluorescence in situ hybridization (FISH). The tested materials only showed one hybridization site of 5S rDNA on their genomes, but they were different in the position of the signals. The hybridization site of Zea mays ssp. mexicana was located on the long arm of chromosome 2, indicating that it is the same as the cultivated maize in the position of 5S rDNA, while the sites of Zea diploperennis and Zea perennis were on the short arms of other chromosomes. For 45S rDNA, one hybridization site was detected at secondary constriction region of the satellite chromosomes in Zea mays ssp. mexicana and Zea diploperennis, while in Zea perennis, besides the site located at the secondary constriction region, a second site on the short arm of another chromosome pair was observed. Our results provide additional evidence for Zea mays ssp. mexicana being a subspecies of Zea mays.     

18S rDNA sequences and the holometabolous insects

The Holometabola (insects with complete metamorphosis: beetles, wasps, flies, fleas, butterflies, lacewings, and others) is a monophyletic group that includes the majority of the world's animal species. Holometabolous orders are well defined by morphological characters, but relationships among orders are unclear. In a search for a region of DNA that will clarify the interordinal relationships we sequenced approximately 1080 nucleotides of the 5' end of the 18S ribosomal RNA gene from representatives of 14 families of insects in the orders Hymenoptera (sawflies and wasps), Neuroptera (lacewing and antlion), Siphonaptera (flea), and Mecoptera (scorpionfly). We aligned the sequences with the published sequences of insects from the orders Coleoptera (beetle) and Diptera (mosquito and Drosophila), and the outgroups aphid, shrimp, and spider. Unlike the other insects examined in this study, the neuropterans have A-T rich insertions or expansion regions: one in the antlion was approximately 260 bp long. The dipteran 18S rDNA evolved rapidly, with over 3 times as many substitutions among the aligned sequences, and 2-3 times more unalignable nucleotides than other Holometabola, in violation of an insect-wide molecular clock. When we excluded the long-branched taxa (Diptera, shrimp, and spider) from the analysis, the most parsimonious (minimum-length) trees placed the beetle basal to other holometabolous orders, and supported a morphologically monophyletic clade including the fleas+scorpionflies (96% bootstrap support). However, most interordinal relationships were not significantly supported when tested by maximum likelihood or bootstrapping and were sensitive to the taxa included in the analysis. The most parsimonious and maximum-likelihood trees both separated the Coleoptera and Neuroptera, but this separation was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta)

BACKGROUND AND AIMS: Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers. METHODS: The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites. KEY RESULTS: The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20). CONCLUSIONS: The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently.     

Characterization of diploid,tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthuswere performed using Feulgen staining, Giemsa C and CMA₃ (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x=17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.     

Direct and sensitive fluorescence in situ hybridization of 45S rDNA on tomato chromosomes.

We describe a direct and sensitive fluorescence in situ hybridization protocol for plant chromosomes. We labelled 45S rDNA with fluorescein-12-dUTP and hybridized to somatic chromosomes of four tomato genotypes. This technique does not require posthybridization immunocytochemical amplifications. The improved signal sensitivity with this technique allowed identification of new rDNA loci on three pairs of chromosomes, in addition to the previously known locus on chromosome 2. We discuss favorable features of direct fluorescence in situ hybridization for chromosomes fixed on a slide and chromosomes or cells in suspension.     

45S rDNA基因的不稳定性及其与转录的相关性

45S rDNA基因由串联重复序列构成,是遗传不稳定性的热点区域,易于发生DNA断裂和重组。以Hela和CHO细胞系为研究对象,运用荧光原位杂交技术检测有丝分裂不同时期的45S rDNA基因的不稳定性表型。结果表明,位点特异性的染色体浓缩失败是其在中期染色体上不稳定性的主要表型。具有这种表型的染色体在后期可能会出现落后或粘连现象,甚至有可能引发断裂,形成卫星核。同时,免疫荧光双染色技术检测表明DNA双链断裂的标记蛋白(γH2AX)和RNA聚合酶I的上游结合因子(UBF)在有丝分裂的不同时期都存在共定位现象。该结果为探讨45S rDNA基因的不稳定性与转录的关系提供了直观的细胞学证据。     

Physical mapping of 5S and 45S rDNA loci in pufferfishes (Tetraodontiformes)

Chromosomal features, location and variation of the major and minor rDNA genes cluster were studied in three pufferfish species: Sphoeroides greeleyi and Sphoeroides testudineus (Tetraodontidae) and Cyclichthys spinosus (Diodontidae). The location of the major rDNA was revealed with an 18S probe in two loci for all species. The minor rDNA loci (5S rDNA) was found in one chromosome pair in tetraodontid fishes and four sites located on two distinct chromosomal pairs in C. spinosus. A syntenical organization was not observed among the ribosomal genes. Signal homogeneity for GC/AT-DNA specific fluorochromes was observed in diodontid fish except in the NORs regions, which were CMA₃-positive. Giemsa karyotypes of tetraodontid species presents 2n = 46, having the same diploid value of other Sphoeroides species that have been investigated. On the other hand, the karyotype of C. spinosus, described for the first time, shows 2n = 50 chromosomes (4m + 18sm + 12st + 16a). The foreknowledge of the karyotypic structure of this group and also the physical mapping of certain genes could be very helpful for further DNA sequence analysis.     

薏苡45S和5S rDNA的染色体定位研究(简报)

通过荧光原位杂交的方法确定了45S和5S正NA序列在薏苡前中期染色体上的位置.尽管具有20条染色体的薏苡是四倍体植物,但它的基因组中只有一个45S和5S rDNA位点.根据薏苡前中期染色体的核型,确定45S rDNA序列位于薏苡第2号染色体短臂上的次级缢痕区和随体上,5S rDNA序列位于第7号染色体长臂靠近着丝粒处,5S rDNA位点到着丝粒的百分距离是29.13±1.76.     

Physical mapping of 5S and 18S-25S rDNA and repetitive DNA sequences in Aegilops umbellulata.

An accurate physical map of the location of the 5S and the 18S-5.8S-25S rRNA genes and a repetitive DNA sequence has been produced on Aegilops umbellulata Zhuk., (2n = 2x = 14) chromosomes by in situ hybridization. Chromosome morphology together with the hybridization pattern of pSc119.2, a DNA sequence from rye, allowed identification and discrimination of different chromosomes; pSc119.2 hybridizes with all Ae. umbellulata chromosomes at the telomeres, except for the short arm of chromosome 6U, and shows intercalary sites on the long arms of chromosomes 6U and 7U. The 5S and 18S-25S rDNA have been mapped physically only on the short arms of chromosomes 1U and 5U. On chromosome 1U the order of the genes is 5S rDNA subterminal and 18S-25S rDNA more proximal, while on chromosome 5U the position of the genes is reversed. The relative order of the genes, together with the hybridization pattern of the pSc119.2, is useful in identifying whole chromosomes or chromosome segments from Ae. umbellulata in recombinant or addition lines with wheat. The data help link the physical organization of chromosomes to the genetic map. Other members of the Triticeae vary in the presence and order of the 5S and 18S-25S rDNA sequences on groups 1 and 5, indicating multiple and complex evolutionary rearrangements of the chromosome arms.