Topo 2 vector

A total of 14 isolates (five from apple, five from quince and four from pear), which showed distinct RFLP profiles, were chosen for further analysis. The PCR amplicons were purified using a matrix gel extraction system (Marligen Bioscience, Ijamsville, MD, USA) and cloned into the TOPO-II vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Positive clones were Sanger sequenced in both orientations using the universal M13 forward and reverse primers.

Embryonic eyes were collected as described above and punctured in the posterior region of the globe to permit perfusion fixative. Samples were fixed overnight at 4 ˚C in modified Carnoy’s fixative (60% ethanol, 30% formaldehyde and 10% glacial acetic acid). Tissues were dehydrated in an ethanol series, embedded in paraffin, and sectioned at 10–12 µm thickness. Probes were PCR amplified from cDNA templates containing transcripts of the gene of interest and cloned into TOPO-II vector (Invitrogen). Primers used for amplifying the cDNA gene segment to clone into TOPO-II are listed in Supplementary Table 1. Anti-sense digoxygenin labeled riboprobes were generated from the TOPO-II clones by in vitro RNA transcription (DIG Labeling Kit, Roche). Section in situ hybridization was performed as previously described^{156 (link)}.

The head extracts were prepared in a buffer containing: 20 mM Tris-HCl, (pH 8.0), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.1% Triton X-100, 1.5 mM DTT, 0.2 mg/ml heparin, 0.2 mg/ml tRNA, 0.25% BSA, complete mini protein inhibitor (Roche), 40 units/ml RNase plus inhibitor (Promega). The RNA and protein (~2 mg of total protein) were incubated in the buffer with rotation for 40 min at RT. Pre-equilibrated Streptavidin Paramagnetic Beads (Roche) were added to each binding reaction, and the mixture was incubated for a further 40 min. The beads were then captured with a magnet, washed five times for 10 min with extraction buffer, and boiled in Laemmli SDS-PAGE sample buffer. The supernatant and pellet fractions were prepared as described above. To obtain biotin-labelled RNA, the 3’ UTR of the Oscar, Tequila, Murashka, and Actin88F genes were cloned into the TopoII vector (Invitrogen), and biotin-labelled RNA was prepared using Megascript RNA synthesis kit (Ambion) in the presence of Bio-17-ATP and Bio-11-CTP. The RNA was purified, and ~ 2 μg of biotin-labelled RNA was incubated with the supernatant and pellet protein extracts. The protein/RNA complexes were recovered with streptavidin magnetic beads (Dynal). The amount of supernatant and lysate used in the control lanes is 5% the amount used for RNA binding.