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

Chapter 4FISH experiments were performed using two different probes, 1) clone pTa71 which containsthe EcoRI fragment of 45S ribosomal DNA from wheat (9kb) (Gerlach and Bedbrook 1979);2) a probe of telomere repeat sequence generated by PCR according to Cox et al. (1993) withminor modifications. In brief, two oligomer primers 1fw (5’-TTTAGGG-3’) 5 and 1rev (5’-CCCTAAA-3’) 5 were synthetized by Isogen Life Science, the Netherlands. PCR reactionswere set-up in the absence of template DNA. Each 100 µL PCR reaction comprised of 10 µLof 10 × Taq buffer (Promega), 1.5 mM MgCl 2 , 2 units of Taq polymerase (Promega), 2.5 mMdNTPs and 10 pmol of each primer 1fw and 1rev. Temperature cycling was performedaccording to Ijdo et al. (1991) with a final extension step of 10 min at 72°C. Probes ofdifferent genomic DNA were labelled with either digoxigenin-11-dUTP or biotin-16-dUTP bynick translation according to the manufacturer’s instruction (Roche Diannostics GmbH,Mannheim, Germany).In situ hybridizationThe procedure of in situ hybridization was carried out according to Khan et al. (2009a) andXie et al. (2010) with minor modification. For GISH, the hybridization mixture contained50% formamide, 10% dextransulphate, 2 × saline sodium citrate (SSC), 0.25% sodiumdodecyl sulphate (SDS), 1.0-1.5 ng/μL for the probe and 25-50 ng/μL block DNA. While forFISH, the hybridization mixture contained 50% formamide, 10% dextransulphate, 2 × salinesodium citrate (SSC), 0.25% sodium dodecyl sulphate (SDS), 2.0-2.5 ng/μL for the probe and50-100 ng/μL block DNA. The mixture was incubated at 73°C for 10 minutes and ice cooledfor 10 minutes, then 60μl hybridization mixture was added on each slide. After denaturationfor 5 minutes at 80°C , slides were left in a pre-warmed box for overnight hybridization at37°C. After hybridization, the slides were washed in 2 × SSC for 15 minutes then stringencywashing was followed with 0.1 × SSC at 42°C for 30 minutes. The probe labelled withdigoxigenin-11-dUTP was detected with the anti-digoxigenin detection system and probelabelled with biotin-16-dUTP was detected by cy3-streptavidin system. Then the slides werecounterstained with DAPI and mounted with Vectashield. At last, photographs were takenwith a Canon digital camera attached to a Zeiss Axiophot epifluorescence microscopy.50