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Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes
Published on: January 6, 2015
Characterization, evolution and chromosomal distribution of two satellite DNA sequence families in Lathyrus species
M Ceccarelli1, V Sarri, E Polizzi
1Dipartimento di Biologia Cellulare e Ambientale, Sezione di Biologia Cellulare e Molecolare, Università degli Studi di Perugia, Perugia, Italy.
Abstract:
DNA clones containing highly repetitive DNA sequences were selected from partial libraries of Lathyrus sativus and L. sylvestris. Two satellite DNA sequence families were isolated from the genome of the former species. A first family was made up of repeats that varied in length from 54-56 bp, and shared 51.7-94.8% nucleotide sequence similarity. The repeats of the second sequence family were 52-62 bp in length, and shared a 58.5-78.5% nucleotide sequence similarity. All the repeat units contained in a clone from L. sylvestris were 41 bp in length and showed an almost perfect structural conservation (95.1-100% nucleotide sequence similarity). The evolution of the first sequence family from L. sativus and of that isolated from L. sylvestris was studied by dot-blot hybridization to the genomic DNA of these species and 3 other Lathyrus species, L. clymenum, L. latifolius and L. odoratus. The former repeats were found to be species-specific and their redundancy was calculated to be 2.9 x 10(7). The satellite DNA sequence isolated from L. sylvestris was present also in L.latifolius, with a redundancy of 1.4 x 10(7) and 1.1 x 10(7), respectively. Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal distribution of the two sequence families and of 45S and 5S ribosomal genes. The species-specific sequences of L. sativus were located around the centromere of chromosome pair IV, where they occupied a very broad region, and, in a much smaller amount, close to the centromere in the short arm of pair II. Sequences related to the repeat units isolated from L. sylvestris were found, both in this species and L. latifolius, in all of the chromosome pairs at terminal and interstitial regions, where they co-localize with the vast majority of DAPI bands. The pattern of distribution of the satellite DNA sequences investigated, together with that of DAPI bands and ribosomal DNA, allowed each chromosome pair of the 3 complements studied to be identified unambiguously.
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