Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

Abstract

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.

A Comparative Analysis of Multi-Probe Fluorescence <i>In Situ</i> Hybridisation (FISH) Karyotypes in 26 <i>Pinus</i> Species (Pinaceae)

“…The PCSR was amplified from the plasmid DNA of PDCD501 (accession no. AB051860, Hizume et al 2001) using a short repeat-specific primer (PD501RP1, GAA ACC CCA AAT TTT) and the M13 reverse primer (M13-1, AGC GGA TAA CAA TTT CAC ACA GGA), and then labelled with reactive isothiocyanate fluorescein (FITC) using FITC-High Prime (Roche). Our FISH procedure followed the methodology of Hizume et al (2002).…”

“…Not all homologous pairs in Pinus species were identified by probe signals in studies of karyotypes by FISH that used 35S rDNA and 5S rDNA probes (Doudrick et al 1995, Liu et al 2003, Cai et al 2006, Bogunić et al 2011, telomere sequences (Fuchs et al 1995, Shibata et al 2005, Islam-Faridi et al 2007, or simple sequence repeats (SSR) (Pavia et al 2014). Combined fluorescent banding and FISH investigations have identified interstitial and proximal CMA bands that are consistent with 35S rDNA and/or the short repetitive sequence proximal CMA band-specific repeat (PCSR) loci, and interstitial DAPI bands that are consistent with signals of repetitive sequences containing the Arabidopsis-type telomere sequence (Hizume et al 2001, Shibata et al 2005. A FISH probe combining 35S rDNA, 5S rDNA, the Arabidopsis-type telomere sequence, and the PCSR distinguished all homologous pairs in four Pinus species, thereby allowing karyotype comparisons among these species (Hizume et al 2002).…”

Fluorescent Chromosome Banding Patterns in Six Species of <i>Abies</i>, Pinaceae

“…In most probably all genera of Pinaceae, the secondary constriction showed CMA-band and FISH signal of 45S rDNA indicating the presence of rDNA repeats (Pinus: Hizume et al , 2001Hizume et al , 2002bPicea: Hizume and Kuzukawa 1995, Brown and Carlson 1997, Shibata et al 2004Larix: Hizume et al 1988, Liu et al 2007; Pseudotsuga: Hizume andAkiyama 1992, Amarasinghe andCedrus: Dagher-Kharrat et al 2001;Abies: Puizina et al 2008) as in other plant species (Schweizer 1976, Kuroki et al 2013. In the species of Larix, Pseudotsuga and Picea, one CMAband is consistent with the FISH signal of 5S rDNA (Hizume et al 1996, Amarasinghe and Carlson 1998, Liu et al 2007.…”

“…In order to draw whole taxonomical and phylogenetical pictures of Abies, more of the species should be analyzed for their chromosomes by fluorescent banding and FISH with not only rDNA but also other probes such as sequences cloned in Pinus densiflora (Hizume et al 2001), Larix leptolepis (Hizume et al 2002a) and Picea species (Brown et al1998, Vischi et al 2003, Shibata and Hizume 2008.…”

Multi-Color Fluorescence <i>in situ</i> Hybridization

“…The first use of a multi-colored FISH on chromosomes was reported by Gray et al (1991), after which it became an essential technique in chromosome analysis. The advantage of FISH was that many probes could be used at the same time, and these could be combined with fluorescent or other staining methods (Hizume et al 2001). DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in the DNA and is excited by UV.…”