Recent accumulation of microbial genome data has demonstrated that lateral gene transfers constitute an important and universal evolutionary process in prokaryotes, while those in multicellular eukaryotes are still regarded as unusual, except for endosymbiotic gene transfers from mitochondria and plastids. Here we thoroughly investigated the bacterial genes derived from a Wolbachia endosymbiont on the nuclear genome of the beetle Callosobruchus chinensis. Exhaustive PCR detection and Southern blot analysis suggested that ∼30% of Wolbachia genes, in terms of the gene repertoire of wMel, are present on the insect nuclear genome. Fluorescent in situ hybridization located the transferred genes on the proximal region of the basal short arm of the X chromosome. Molecular evolutionary and other lines of evidence indicated that the transferred genes are probably derived from a single lateral transfer event. The transferred genes were, for the length examined, structurally disrupted, freed from functional constraints, and transcriptionally inactive. Hence, most, if not all, of the transferred genes have been pseudogenized. Notwithstanding this, the transferred genes were ubiquitously detected from Japanese and Taiwanese populations of C. chinensis, while the number of the transferred genes detected differed between the populations. The transferred genes were not detected from congenic beetle species, indicating that the transfer event occurred after speciation of C. chinensis, which was estimated to be one or several million years ago. These features of the laterally transferred endosymbiont genes are compared with the evolutionary patterns of mitochondrial and plastid genome fragments acquired by nuclear genomes through recent endosymbiotic gene transfers.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY(1)Y(2) in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4',6-diamidino-2-phenylindole bands of both Y chromosomes.

Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat ( PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.

SummaryThe genome sizes of 13 species of Taxodiaceae, 19 species of Cupressaceae s.s. and Sciadopitys verticillata were determined by flow cytometry of isolated nuclei stained with propidium iodide, using Hordeum vulgare nuclei as an internal standard. In Taxodiaceae, the genomes of Cunninghamia lanceolata (28.34 pg/2C) and Taiwania species (25.78, 26.80 pg/2C) were larger than those of other genera/species, which ranged from 19.85 to 22.87 pg/2C. In Cupressaceae s.s., genome size ranged from 20.03 to 27.93 pg/2C among 16 species. The Calocedrus species and Thujopsis had a larger genome than most other species. Sciadopitys verticillata had a large genome of 41.60 pg/2C. After comparing the diversity in genome size with previously reported cladograms constructed using nucleotide sequence data, the tendency of changes in genome size with phylogenetic differentiation is discussed.

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Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

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Summary Every species has a unique karyotype, but certain genera have common karyptypes among species. The markers (chromosome length and centromere position) used in traditional karyotyping do not distinguish all chromosome pairs in the genus Pinus. However, the application of multi-probe fluorescence in situ hybridisation (FISH) procedures allowed exact karyotyping of 26 Pinus congeners. We used these new data to examine species relationships. The 35S rDNA and 5S rDNA, Arabidopsis-type telomere repeat sequences, and the proximal CMA band-specific repeat (PCSR) of P. densiflora were used as FISH probes for our analysis of chromosomes in 26 Pinus species. Each species had a unique FISH karyotype and most homologous chromosome pairs were identified. The FISH karyotypes were used to compare corresponding or homologous chromosomes among the species. Common or similar FISH signal patterns appeared in closely related species. Species that had inherited common FISH signal patterns were classified into four karyotype groups. We used cluster analyses to compare quantitative differences in FISH signals within these groups. The results of these analyses were consistent with recent systematic interpretations and resolved differences among existing taxonomic systems based on diverse methodologies. Our results indicate that FISH signal patterns reflect the history of species differentiation and that comparative FISH karyotyping has potential as an important tool for studying the taxonomy or phylogeny of Pinus.