Rabeya Begum
Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh
Falk Zakrzewski2,
Institute of Botany, Technische Universita¨t Dresden, D-01062 Dresden, Germany
Gerhard Menzel
Institute of Botany, Technische Universita¨t Dresden, D-01062 Dresden, Germany
Beatrice Weber
Institute of Botany, Technische Universita¨t Dresden, D-01062 Dresden, Germany
Sheikh Shamimul Alam
Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh
Thomas Schmidt*
Institute of Botany, Technische Universita¨t Dresden, D-01062 Dresden, Germany
Corchorus olitorius, Corchorus capsularis, Jute, karyotype, Satellite DNA, FISH, Physical mapping, DNA methylation, Immunolabelling.
Knowledge Management
Plant material and genomic DNA extraction Seeds of the cultivated jute species Corchorus capsularis ‘CVL-1’ and C. olitorius ‘O-4’ were kindly provided by the Bangladesh Jute Research Institute (Dhaka, Bangladesh). Seedlings were grown under greenhouse conditions. Genomic DNA was isolated from young leaves using the CTAB (cetyltrimethylammonium bromide) standard protocol (Saghai-Maroof et al., 1984). Generation of Corchorus-specific plasmid libraries and clone sequencing Genomic DNA of C. olitorius and C. capsularis was digested with the restriction endonuclease AluI and separated by agarose gel electrophoresis. DNA fragments corresponding to a size range from 100 to 500 bp were purified from the gel using the QIAquick gel extraction kit (Qiagen) and ligated in the SmaI site of the pUC18 vector. After transformation, clones of each species were collected and stored in 384-well plates. The plasmid libraries were transferred to Hybond-N+ nylon membrane (GE Healthcare) and hybridized with radioactively labelled genomic DNA from both Corchorus species. Plasmid clones containing repetitive sequences were identified by the signal strength and further analysed.
Sequence analyses Plasmid clones were sequenced on the CEQ 8000 capillary sequencer (Beckman) using M13 universal primers. Raw sequence data were analysed with Geneious software. Sequence alignments were generated by Geneious 6 (http://www. geneious.com/), using the MUSCLE algorithm (Edgar, 2004). The diversity of satellite repeats was analysed by the maximum parsimony algorithm using the MEGA software version 5 (Tamura et al., 2011).
PCR amplification and cloning Monomers and multimers of the satellite DNA family CoSat I were amplified from 100 ng of template DNA from C. olitorius and C. capsularis using the primer combination forward 5′ -TAGTTAGGCCATAAACAATGG-3′ and reverse 5′ -TCA TTTTGGTGAGTTAGTCC-3′. Polymerase chain reactions were performed using GoTaq DNA polymerase (Promega). Standard PCR conditions were 94 8C for 2 min, followed by 30 cycles of 94 8C for 20 s, annealing for 30 s at 54 8C, 72 8C for 20 s and a final incubation at 72 8C for 5 min. After gel electrophoresis, PCR fragments were purified with the QIAquick gel extraction kit (Qiagen) and ligated into the pGEM-T vector (Promega). Plasmid clones were sequenced on the CEQ 8000 capillary sequencer.
Chromosome preparation Primary roots were collected from seedlings after germination on wet filter paper, incubated in 2 mM 8-hydroxyquinoline for 2 h at room temperature, fixed in fixative solution (ethanol: glacial acetic acid 3:1) for 15 min at 4 8C, and stored in 70 % ethanol at 4 8C for use. Fixed roots were washed in enzyme buffer (10 mM citric acid–sodium citrate, pH 4.6) and macerated at 37 8C for 25 min in an enzyme mixture containing 1.25 % (w/ v) pectinase from Aspergillus niger(Sigma), 1.25 % (w/v) cellulase Onozuka R 10 (Serva) and 1.25 % (w/v) pectolyase from Aspergillus japonicus (Sigma). The suspension was dropped onto pre-cleaned glass slides as described by Schwarzacher and Heslop-Harrison (2000).
Labelling of FISH probes For physical mapping of rDNA sites, the probe pZR18S (accession no. HE578879) containing a 5066 bp fragment of the sugar beet 18S–5.8S–25S rRNA gene labelled with digoxigenin-11-dUTP by nick translation and the probe pXV1 (Schmidt et al., 1994) for the 5S rRNA gene labelled with biotin-11-dUTP by PCR were used. The telomeric probe pLT11 was labelled with digoxigenin-11-dUTP by nick translation. The satellite probe CoSat I was labelled with digoxigenin11-dUTP, and the retrotransposon sequences CoRetro I and CoRetro II were labelled with digoxigenin-11-dUTP and biotin-11-dUTP by PCR, respectively. For karyotyping, the following labelling was used: the probe pZR18S was labelled with DY-647-dUTP and the probe pXV1 was labelled with DY-415-dUTP (Dyomics, http://www.dyomics.com). The satellite probe CoSat I was labelled with biotin-11-dUTP and the retrotransposon sequence CoRetro I was labelled with digoxigenin-11-dUTP. Polymerase chain reaction labelling was performed with standard M13 primers using the following program: 5 min 95 8C, 35 cycles of (1 min 95 8C, 40 s 56 8C, 1 min 72 8C), 10 min 72 8C. Probes were purified from unincorporated nucleotides by precipitation and resuspended in water.
Annals of Botany 112: 123–134, 2013
Journal