Biotin 14 datp

The 5S rDNA (200 bp) and 18S rDNA (1,400 bp) probes were obtained by PCR from the nuclear DNA of A. coeruleus individuals from Northeast Brazilian coast using primers A 5′-TAC GCC CGA TCT CGT CCG ATC-3′ and B 5′ -CAG GCT GGT ATG GCC GTA AGC-3' (Pendás et al., 1994 (link)) and NS1 5′-GTA GTC ATA TGC TTG TCT C-3′ and NS8 5′-TCC GCA GGT TCA CCT ACG GA-3′ (White et al., 1990 ), respectively. The 5S rDNA and 18S rDNA probes were labeled with biotin-14-dATP and digoxigenin-11-dUTP, respectively, using nick translation according to the manufacturer’s recommendations (Roche, Mannheim, Germany). Meanwhile, primers H2BAD 5′-CCC -CCC GAG ATG TGA TGG TAG A-3 ′ and H2BAR 5′-AGT ACA GCC TGG ATG TTT GGT AA-3′ were used to amplify the H2B-H2A sequences and primers H3D 5′-ATG GCT CGT ACC AAG CAG ACV GC-3′ and H3R 5′-ATA TCC TTR GGC ATR ATR GTG AC-3′ to amplify H3 sequences. Both primer sets were designed using the gene sequences of Mytilus edulis (Albig et al., 2003 (link)), and the genes were amplified according to Giribert and Distel (2003) . biotin-14-dATP and digoxigenin-11-dUTP were used to label H2B-H2A hisDNA and H3 hisDNA, respectively, using nick translation according to the manufacturer’s recommendations (Roche, Mannheim, Germany).

Fluorescence in situ hybridization

(FISH) followed the methods described by Pinkel et al. (1986) with an 18S rDNA probe obtained from Prochilodusargenteus Spix & Agassiz, 1829 (Hatanaka and Galetti Jr. 2004 (link)). The 18S rDNA probe was labeled with biotin-14-dATP (Roche Applied Science) by nick translation and the 5S rDNA probe from Leporinuselongatus Linnaeus, 1758 (Martins and Galetti Jr. 2001 (link)) was labeled with digoxigenin 11-dUTP (Roche Applied Science) by PCR. The hybridization signal was detected using avidin-FITC (fluorescein isothiocyanate) (Life Technologies) for the 18S rDNA probe and anti-digoxigenin-rhodamine (Roche Applied Science) for the 5S rDNA probe. The chromosomes were counterstained with propidium iodide or DAPI, respectively. All the images were acquired with a Leica DM 4500 B microscope equipped with a DFC 300FX camera and Leica IM50 4.0 software and optimized for best constrast and brightness with Adobe Photoshop CS6 software.

5S rDNA (~ 200 bp) and 18S rDNA (~ 1400 bp) probes were obtained by PCR from the nuclear DNA of Rachycentron canadum (Teleostei, Perciformes) using the primers A 5’-TAC GCC CGA TCT CGT CCG ATC-3’ and B 5’- CAG GCT GGT ATG GCC GTA AGC-3’ (Pendás et al., 1994 ), and NS1 5’-GTA GTC ATA TGC TTG TCT C-3’ and NS8 5’-TCC GGT GCA TCA CCT ACG GA-3’ (White et al., 1990 ), respectively. 5S rDNA and 18S rDNA probes were labeled by nick translation with biotin-14-dATP and digoxigenin-11-dUTP, respectively, according to the manufacturer’s specifications (Roche Mannheim, Germany). Tol2 (~ 200 bp) and Rex3 (~ 200 bp) probes were amplified using PCR from the nuclear DNA of E. itajara using the primers Tol2-5F 5′ -CTG TCA CTC TGA TGA AAC AG-3′ and Tol2-5R 5′ -CTT TGA CCT TAG GTT TGG GC-3′ (Kawakami and Shima, 1999 (link)) and Rex3-F5’ -YAA TGA CGG AGG GCC CGG CA-3′ and Rex3-5′-TGG GTG GTG GGG CAG GT ACN-3′ (Volff et al., 1999 (link); 2000 (link)) and labeled with digoxigenin-11-dUTP by nick translation (Roche Mannheim, Germany). In situ hybridizations with (CA)₁₅ and (GA)₁₅ microsatellites were performed as described by Kubat et al. (2008 (link)) using oligonucleotides labeled with Alexa Fluor 555 at the 5’ terminal position (InvitrogenTM, Thermo Fisher Scientific, California, USA).