FISH with the BoR300 probe on mitotic and polytene nuclei of <i>Bactrocera oleae.</i>

<p>Chromosomes were counterstained with DAPI (blue). Female (a) and male (b) metaphase showing strong hybridization signals (red) on the centromeres of chromosomes 4 and 5. Polytene complement (c) showing strong hybridization signals (red) on the centromeric heterochromatic blocks (C) of chromosomes III and IV (arrows). Bar  = 3 µm (a, b), 20 µm (c).</p

CENP-B like motif.

<p>Comparison of the CENP-B like motif (237–252 bp) found in BoR300 with the degenerate motif considered to bind the CENP-B protein.</p

qPCR analysis data of the relative estimation of BoR300 repeats using a reference standard curve.

a<p>Initial template concentration of the <i>B</i>. <i>oleae</i> genomic DNA (pg) used at the qPCR reactions.</p>b<p>Mean copy number of BoR300 repeats which was estimated for the initial template concentration of the genomic DNA (a) based on the standard curve.</p>c<p><i>B. oleae</i> haploid genome size: 0.352 pg.</p>d<p>Standard Error (SE) for the triplicate measurements (n = 3).</p

Species distribution of BoR300 repeats.

<p>HaeIII-digested genomic DNA of representative species of the Diptera family and the genus Bactrocera was analyzed by Southern hybridization using as probe the biotinylated monomer (298 bp) of the repeat. (<b>A</b>) Southern blot analysis of digested genomic DNA from the following dipteran species: <i>B. oleae</i> (lane 1), <i>C. capitata</i> (lane 2), <i>D. melanogaster</i> (lane 3) and <i>An. gambiae</i> (lane 4). <b>B</b>) Southern blot analysis of digested genomic DNA from the following Bactrocera species: <i>B. oleae</i> (lane 1), <i>B. correcta</i> (lane 3), <i>B. cucurbitae</i> (lane 4), and <i>B. dorsalis</i> (lane 5). In Lane 2 is the cloned monomer of the repeat. M represents the molecular marker (SM0331, Fermentas).</p

Curvature analysis of BoR300.

<p>Curvature-propensity plot of the consensus sequence of BoR300 showing that the maximum peak is located at the beginning of the monomer (red line).</p

Standard curves for the reference single copy gene (<i>ace</i>) and the repeats of BoR300.

<p>The construction of the curves was based on serial 10-fold dilutions of the genomic DNA template used (10 pg, 100 pg, 1 ng). For each amplicon, qPCR determined Ct values were plotted against the logarithm of their initial concentration (1, 2 and 3 values respectively).</p

Nucleotide sequence of the monomer BoR300.

<p>The arrows indicate the outfacing primer pair: the reverse (BoR300-R) and the forward (BoR300-F) primer respectively. The restriction sites of the restriction endonucleases HaeIII and TaqI are also highlighted.</p

Comparative analysis data of the qPCR amplification curves of BoR300 repeats and the single copy gene (<i>ace</i>), for the absolute estimation of BoR300 copies.

a,b<p>Intercept and slope of the linear regression lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079393#pone-0079393-g006" target="_blank">Figure 6</a>).</p>c<p>The efficiency of the reaction was estimated by the equation: E = 10^−1/slope-1.</p>d<p>ΔCt value is obtained by comparing the y-axis intercepts derived from the two lines-of-best-fit of the target sequences BoR300 and <i>ace</i>.</p>e<p>According to the mean efficiency of the reactions (94.05%) the amplification factor F was 1.881.</p

Primer sequences and parameters of the RT-PCR and qPCR assay.

<p>Primer sequences and parameters of the RT-PCR and qPCR assay.</p

Analysis of BoR300 transcription.

<p>Total RNA from male (lane 1) and female (lane 2) adult <i>B. oleae</i> flies were extracted and reverse-transcribed using random oligonucleotides. Satellite transcripts were amplified by PCR using BoR300-F and BoR300-R primers. M represents the molecular marker (1000 bp/1 kb BLUE DNA Ladder, GeneON). The epic175F and epic175R primers were also used, to check the presence of any DNA contamination on both male and female cDNAs (lanes 4 & 5 respectively). The amplification results were compared to those obtained using the genomic DNA as template (lane 7) at which the product size was 550 bp.</p