Physical localisation of repetitive DNA sequences in Alstroemeria: karyotyping of two species with species-specific and ribosomal DNA

Abstract

Fluorescence in situ hybridization (FISH) was used to localise two species-specific repetitive DNA sequences, A001-I and D32-13, and two highly conserved 25S and 5S rDNA sequences on the metaphase chromosomes of two species of Alstroemeria. The Chilean species, Alstroemeria aurea (2n = 16), has abundant constitutive heterochromatin, whereas the Brazilian species, Alstroemeria inodora, has hardly any heterochromatin. The A. aurea specific A001-I probe hybridized specifically to the C-band regions on all chromosomes. The FISH patterns on A. inodora chromosomes using species-specific probe D32–13 resembled the C-banding pattern and the A001-I pattern on A. aurea chromosomes. There were notable differences in number and distribution of rDNA sites between the two species. The 25S rDNA probe revealed 16 sites in A. aurea that closely colocalised with A001-I sites and 12 in A. inodora that were predominantly detected in the centromeric regions. FISH karyotypes of the two Alstroemeria species were constructed accordingly, enabling full identification of all individual chromosomes. These FISH karyotypes will be useful for monitoring the chromosomes of both Alstroemeria species in hybrids and backcross derivatives.Key words: Alstroemeria, fluorescence in situ hybridization, FISH, repetitive DNA, ribosomal DNA, karyotype.

Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids

“…The repeats D32-13 (specific for A. inodora; EMBL accession number AJ228814) and A001-I (specific for A. aurea; EMBL accession number Y10977) were labelled by PCR with biotin-16-dUTP or digoxigenin-11-dUTP, respectively, and hybridized to the meiotic cells as described in Kamstra et al (1997Kamstra et al ( , 1999a. The biotinand digoxigenin-labelled probes were detected using Cy3-(Jackson ImmunoResearch Laboratories) and FITC/ Alexa 488-conjugated antibodies (Molecular Probes Inc.).…”

“…In addition, structurally altered chromosomes were detected in some of the BC1 plants (Kamstra et al, 1999b). These chromosomes contained hybridization sites for species-specific probes at unexpected locations, not found in parental species (Kamstra et al, 1997), indicating the presence of chromosomal rearrangements between the A. aurea and A. inodora genome (Kamstra et al, 1999b). This paper describes the meiotic analysis of four BC1 plants using FISH and GISH sequentially applied to spread pollen mother cells at metaphase I to telophase II.…”

Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea × A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes

“…The probes A001-I and D32-13, specific for A. aurea and A. inodora, respectively (De Yeu et al, 1997;Kamstra et al, 1997;G. J. Kuipers et al, unpubl.…”

“…The pretreatment and hybridization steps applied to meiotic and mitotic preparations were performed as described in Kamstra et al (1997). For meiotic preparations, the pretreatment with pepsin was increased from 10 min to 25 min at 37°C.…”

“…For meiotic preparations, the pretreatment with pepsin was increased from 10 min to 25 min at 37°C. Digoxigenin-11-dUTP-and biotin-1-dUTP-labelled probes were detected with fluorescein isothiocyanate (FITC)-and Cy3-conjugated antibodies respectively (for details, see Kamstra et al, 1997).…”