2.4. DNA amplification via PCR using RAPD, ISSR and ITS primers
PCR-RAPD reactions were performed using 12 primers (Table 2) from Operon Technologies Inc., Alameda, CA, USA (OP06, OPG07, OPG10, OPC11, OPS03, OPG19, OPA02, OPG03, OPG13, OPG15, OPG06 and OPM12) suggested by Mondragon-Jacob et al. [11] for detection of genetic polymorphism in cactus pear. RAPD reactions were prepared with a final volume of 25 µl containing 50.0 ng DNA, 1X PCR buffer [100 mM Tris-HCl (pH 8.5)], 3.0 mM MgCl2; 200 µM dNTP's; 1 µM of primer, 1 unit of Taq DNA polymerase) and ultra-pure water to complete the volume. The protocol for DNA amplification, after preliminary tests, consisted of an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 minute; annealing the primers at 40°C for 1 min; extension at 72°C for 1 min and final extension at 72°C for 5 min using a thermal cycler model TC-Plus Techne Bibby Scientific Ltd.
Table 2
List of RAPD, ISSR and ITS primers used
| Marker | Primer | Sequence 5’------- 3’ |
| RAPD | OPG 07 | GAA CCT GCG G |
| RAPD | OPG 10 | AGG GCC GTC T |
| RAPD | OPC 11 | AAA GCT GCG G |
| RAPD | OPS 03 | CAG AGG TCC C |
| RAPD | OPG 19 | GTC AGG GGC A |
| RAPD | OPA 02 | TGC CGA GCT G |
| RAPD | OPG 03 | GAG CCC TCC A |
| RAPD | OPG 13 | CTC TCC GCC A |
| RAPD | OPG 15 | ACT GGG ACT C |
| RAPD | OPG 06 | GAA CGG ACT C |
| RAPD | OPM 12 | GGG ACG TTG G |
| ISSR | UBC 01 | ACA CAC ACA CAC ACA CT |
| ISSR | UBC 02 | GAG AGA GAG AGA GAG AT |
| ISSR | UBC 857 | ACA CAC ACA CAC ACA CYG |
| ISSR | UBC 808 | AGA GAG AGA GAG AGA GC |
| ISSR | UBC 810 | GAG AGA GAG AGA GAG AT |
| ISSR | UBC 827 | AC AC AC AC AC AC AC AC G |
| ISSR | UBC 830 | TG TG TG TG TG TG TG TG G |
| ISSR | UBC 842 | GA GA GA GA GA GA GA GA YG |
| ISSR | UBC 849 | GT GT GT GT GT GT GT GT YA |
| ISSR | UBC 855 | AC AC AC AC AC AC AC AC YT |
| ISSR | UBC 862 | AGC AGC AGC AGC AGC AGC |
| ISSR | UBC 866 | CTC CTC CTC CTC CTC CTC |
| ITS | ITS 1 | TCCGTAGGTGAACCTGCGG |
| ITS | ITS 4 | TCCTCCGCTTATTGATATGC |
PCR-ISSR reactions were performed using 12 primers (Table 2) from the Nucleic Acid-Protein Service Unit, University of British Columbia, USA (UBC01, UBC02, UBC857, UBC808, UBC810, UBC827, UBC830, UBC842, UBC849, UBC855, UBC862 and UBC866) suggested by Mergulhão et al. [12] and Alves et al. [13] for detection of genetic polymorphism in cactus pear. PCR reactions were prepared with a final volume of 25 µl containing 50.0 ng of DNA, 1X PCR buffer [100 mM Tris-HCl (pH 8.5)], 3.0 mM MgCl2, 200 µM dNTP's, 1 µM primer, 1.0 unit of Taq DNA polymerase and ultra-pure water to complete the volume.
The protocol for DNA amplification consisted of an initial denaturation of 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 seconds; annealing the primers at 52°C for 45 seconds; extension at 72°C for 2 min and final extension at 72°C for 5 min [12] using a thermal cycler model TC-Plus Techne Bibby Scientific Ltd.
The ITS region of nuclear ribosomal DNA was amplified using the ITS1 and ITS4 primers (Table 2) suggested by Lyra et al. [14] for molecular studies in cactus pear. PCR reactions were prepared with a final volume of 25 µl containing 20.0 ng of DNA; 1.0 µl 1X PCR buffer [100 mM Tris-HCl (pH 8.5), 0.25 mM dNTP's, 3.0 mM MgCl2; 1.0 µM of primers, 1.25 units of Taq DNA polymerase and ultra-pure water to complete the volume [15]. The protocol for DNA amplification consisted of an initial denaturation of 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 seconds; annealing at 62°C for 1 min; extension at 72°C for 2 min and final extension at 72°C for 5 min using a thermal cycler model TC-Plus Techne Bibby Scientific Ltd.
2.5. Amplicons separation by gel electrophoresis
Amplicons separation was performed by horizontal electrophoresis in agarose gel (1.5%) with ethidium bromide dye (0.5 ug mL-1). Amplicon migration was performed in 0.5X TBE running buffer (Tris, boric acid, EDTA) at a voltage of 100 V for 120 min. A 100 bp molecular weight standard (Invitrogen™ 1 Kb Plus DNA Ladder) was used. The gel was visualized under ultraviolet light and photodocumented with the aid of the Gel Logic 112Pro imaging system (Carestream, Rochester, NY).
2.6. Data analysis
The bands generated by each primer were classified as monomorphic and polymorphic and their presence/absence computed for all sampled. Subsequently, the total number of amplified bands, number of monomorphic bands, number of polymorphic bands and percentage of polymorphism for each primer were calculated.
The presence/absence data were organized into a binary matrix (1 = presence and 0 = absence) which was used to determine the binary Sokal distance using the Genes software [16]. Using the Sokal binary distance values, a dissimilarity dendrogram was constructed based on Ward [17] clustering method. The definition of the number of groups was performed based on the criterion proposed by Mojena [18] using bootstrap analysis to verify and provide statistical support to the internal nodes of the dendrograms. The validation of the clusters was determined by calculating the cophenetic correlation coefficient [19]. Additionally, a principal component analysis was performed using the Past program [20].