Loss and recovery of Arabidopsis-type telomere repeat sequences 5'-(TTTAGGG)(n)-3' in the evolution of a major radiation of flowering plants.

Fluorescent in situ hybridization and Southern blotting were used for showing the predominant absence of the Arabidopsis-type telomere repeat sequence (TRS) 5'-(TTTAGGG)(n)-3' (the 'typical' telomere) in a monocot clade which comprises up to 6300 species within Asparagales. Initially, two apparently disparate genera that lacked the typical telomere were identified. Here, we used the new angiosperm phylogenetic classification for predicting in which other related families such telomeres might have been lost. Our data revealed that 16 species in 12 families of Asparagales lacked typical telomeres. Phylogenetically, these were clustered in a derived clade, thereby enabling us to predict that the typical telomere was lost, probably as a single evolutionary event, following the divergence of Doryanthaceae ca. 80--90 million years ago. This result illustrates the predictive value of the new phylogeny, as the pattern of species lacking the typical telomere would be considered randomly placed against many previous angiosperm taxonomies. Possible mechanisms by which chromosome end maintenance could have evolved in this group of plants are discussed. Surprisingly, one genus, Ornithogalum (Hyacinthaceae), which is central to the group of plants that have lost the typical telomere, appears to have regained the sequences. The mechanism(s) by which such recovery may have occurred is unknown, but possibilities include horizontal gene transfer and sequence reamplification.

Genome reorganization from polyteny to polyploidy in the nurse cells found in onion fly (Delia antiqua) and cabbage root fly (Delia radicum) ovaries (Diptera, Anthomyiidae).

The material required to ensure successful embryogenesis in the onion fly (Delia antiqua) and the cabbage root fly (Delia radicum) (Diptera, Anthomyiidae) is supplied by 15 nurse cells, while the oocyte chromosomes enter a quiescent stage during prophase I of meiosis. This level of transcription is achieved by the polyploidization of the nurse cell DNA. Elongate polytene chromosomes form in both species, but lack the banding and conspicuous puffing commonly seen in other dipteran tissues. The polytene chromosomes contract until they finally appear as small, densely staining spheres. These fragment into large numbers of endochromosomes that are much smaller than their mitotic counterparts, which then despiralize, resulting in the flocculate appearance of the nurse cell nucleus. Photodensitometry revealed a gradient of DNA values between nurse cells near the oocytes and those further away. Final DNA values 1000 times the haploid level were recorded in the nurse cell nearest to the oocyte compared with 336 times the C-value in the most distal cell. At lower temperatures (< 10 degrees C), the polytene chromosomes become banded and longer. None of the onion flies kept in these conditions produced viable eggs, though there was some reproductive success among the cabbage root flies.

Female meiosis in the onion fly and cabbage root fly (Diptera: Anthomyiidae).

The sequence of female meiosis was investigated in two populations of the cabbage root fly (Delia radicum) and three populations of the onion fly (D. antiqua). In contrast with the completely achiasmate males, both species showed high levels of recombination in females. However, significant differences in chiasma frequency occurred between individuals within populations and between the populations. It was not uncommon to find aneuploidy of the X chromosomes. The autosomes occasionally showed asynapsis or desynapsis, but normal disjunction of univalents was facilitated by distance pairing.

Combined PI-DAPI staining (CPD) reveals NOR asymmetry and facilitates karyotyping of plant chromosomes.

This paper presents a preparative and staining procedure for plant mitotic chromosomes that uses a combination of PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindol) and which reveals a pattern of high-affinity regions for these fluorochromes. Nucleolar organiser regions (NORs), telomeres and centromeric regions exhibit high PI affinity (red), whereas other chromosomal regions exhibit high affinity for either PI (red) or DAPI (blue). NOR-bearing and other chromosomes are readily distinguished, facilitating karyotyping. The dual staining pattern was observed in all the plants tested. Aspects of NOR size, number and occurrence are discussed. A karyotype of rice metaphase chromosomes is presented, based on their fluorescent banding patterns.

Intergeneric somatic hybrids of rice [Oryza sativa L. (+) Porteresia coarctata (Roxb.) Tateoka].

Somatic hybrid plants were obtained following the electrofusion of rice (Oryza sativa L. cv 'Taipei 309', 2n = 2x = 24) cell suspension-derived protoplasts with non-dividing leaf protoplasts of Porteresia coarctata (2n = 4x = 48), a saline-tolerant wild species. Fusion-treated protoplasts were plated on the surface of cellulose nitrate filter membranes, overlaying Lolium multiflorum nurse cells. The nurse cells were embedded in KPR medium containing 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid and semi-solidified with SeaPlaque agarose. Putative somatic hybrid cell colonies were selected on the basis of their growth, whereby faster growing colonies were transferred preferentially to MS-based medium with 2.0 mg l(-1) kinetin, 0.5 mg l(-1)α-naphthaleneacetic acid, 30 g l(-1) sucrose and 4.0 g l(-1) SeaKem agarose to induce shoot regeneration. One hundred and nineteen regenerated plants were micropropagated clonally on MS-based medium containing 2.0 mg l(-1) 6-benzylaminopurine, 50 g l(-1) sucrose and 4.0 g l(-1) SeaKem agarose, prior to DNA extraction of plant samples. Putative somatic hybrids were initially identified by RAPD analysis, and 8 plant lines were selected for further investigation by flow cytometric ploidy determination and cytology. Plants of one line had an allohexaploid chromosome complement (2n = 6x = 72) and, following examination of its vegetative clones by GISH, were confirmed as somatic hybrids containing full chromosome complements of both O. sativa and P. coarctata.

A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.

A preparation technique has been developed for plants with small chromosomes, which produces large numbers of good-quality mitotic preparations. The technique employs a hydrochloric acid treatment to hydrolyse the cytoplasm, facilitating the subsequent removal of cytoplasmic debris. The evaporative force of a methanol-based fixative is exploited to disperse the cytoplasm and to deposit the chromosomes in a single optical plane. This technique permits detailed observations of chromosome morphology and karyotyping. The mitotic preparations are also suitable for the complex analysis associated with in-situ hybridization, as in studies of genome interaction in plant hybrids.