A molecular cytogenetic analysis of chromosome behavior in Lilium ...

Small aberrant chromosomes and B chromosomes in Lilium hybridsand Appels 1986); 3) a probe of telomere repeat sequence generated by PCR according to Coxet al. (1993) with minor modifications. In brief, two oligomer primers 1fw (5’-TTTAGGG-3’) 5 and 1rev (5’-CCCTAAA-3’) 5 were synthesized by Isogen Life Science, Netherlands.Concatemers were produced during a PCR reaction in which the primers also serve astemplate. Each 100 μl reaction comprised 10μl of 10×Taq buffer (Promega) 1.5mM MgCl 2 , 2units of Taq polymerase (Promega), 2.5mM dNTPs and 10pmol of each primer. Temperaturecycling was performed according to Ijdo et al. (1991) with a final extension step of 10 min at72°C.Probes were labelled with either digoxigenin-11-dUTP or biotin-16-dUTP using standardnick translation according to the manufacturer’s instruction (Roche Diagnostics GmbH,Mannheim, Germany).In situ hybridizationGISH was carried out according to Barba-Gonzalez et al. (2005b) and Khan et al. (2009a), the40μl hybridization mixture contained 50% (v/v) deionized formamide, 10% (w/v) sodiumdextran sulphate, 2×SSC, 0.25% (w/v) sodium dodecyl sulphate, 0.6-1.0 ng/μL for each probeand 15-50 ng/μL block DNA. FISH was carried out according to Lim et al. (Lim et al. 2001b)with a 40μl hybridization mixture of 50% (v/v) deionized formamide, 10% (w/v) sodiumdextran sulphate, 2×SSC, 0.25% (w/v) sodium dodecyl sulphate, 2-2.5 ng/μL for each probeand 100-200 ng/μL sheared herring sperm DNA, the latter was used as block DNA. Thehybridization mixture for GISH or FISH was incubated at 73°C for 10 minutes and ice cooledfor at least 10 minutes, and then was added on each slide, the slides were covered with coverslips and denatured at 80°C for 5 minutes after which slides were transferred to a pre-warmedhybridization chamber for overnight incubation at 37°C. After hybridization, stringencywashing was performed using 0.1×SSC at 42 °C for 30 minutes. The probes labelled withdigoxigenin-11-dUTP or biotin-16-dUTP were detected with the anti-digoxigenin-FITC orCy3 respectively. After detection the slides were counterstained with 1 μg/mL 4’,6-diamidino-2-phenyl-indole (DAPI) and mounted with Vectashield (Vector Laboratories, Inc.,Builingame, USA). Preparations were photographed with a Canon camera attached to a ZeissAxiophot epifluorescence microscopy.Chromosome identification and karyotypingImages of mitotic metaphase chromosomes were measured using the computer programMicroMeasure (Reeves and Tear 2000). In all four genomes (L, A, O, T), the chromosomeswere put into sequence according to decreasing short arm length (Stewart 1947). In order toidentify the chromosome in each genome, the chromosome length, arm ratio, centromereindex (short arm length/ long arm length + short arm length), relative chromosome lengthindex (individual chromosome length/total length of a set of chromosomes) and 45S rDNAlocus were used as identification tools (Barthes and Ricroch 2001; Lim et al. 2001b).63