PCR-RFLP
The amplification program used for the three loci was 5 minutes at 94oC, followed by
35 cycles of 20 seconds at 94oC, 45 seconds at 54oC, 80 seconds at 72oC, and a final extension
step of 9 minutes at 72oC. The PCR product was digested with the enzymes DraI and RsaI
(CM12), HaeIII (CM14) and BstoI (CM28) following manufacturer instructions. Since the
combination of these three loci can only discriminate with confidence E. imbricata and C.
caretta, we genotyped 53 individuals previously detected as hybrids with mtDNA analysis by
Lara-Ruiz et al.(Lara-Ruiz et al. 2006), 133 individuals classified by mtDNA and morphology as
E. imbricata and 40 individuals classified by mtDNA and morphology as C. caretta (Reis et al.
2010) to certify the occurrence of private alleles in each species. After this initial screening,
some individuals were chosen as controls and were amplified in all PCR reactions. The results
were visualized in 0.8% agarose gel stained with ethidium bromide (loci CM-12 and CM-28) or
in 8% acrylamide gel (CM-14) stained with silver nitrate (Neto et al. 1993).
Autosomal haplotypes detected by sequencing
PCR reaction mixes of 15 μL included 50 ng of genomic DNA, 1 U of Taq polymerase
(Phoneutria), 200 μM of dNTPs, 1X Tris-KCl buffer with 1.5 mM MgCl2 (Phoneutria) and 1 μM of
each primer. PCR enhancers used for each amplified locus are shown in supplementary table 1.
The amplification program consisted of 3 min at 94o C, followed by 35 cycles of 40 s at 94o C,
45 s at 45-50o C, 50 s at the annealing temperature of each primer and a final extension step of
10 min at 72o C. After amplification, PCR products were checked by running in a 0.8% agarose
gels and stained with ethidium bromide. These products were cleaned by precipitation using
20% polyethyleneglicol and 2.5M NaCl before loading to the sequencing reactions, which were
performed using either of the amplification primers. The sequencing reaction was performed
with ET DYE Terminator Kit (GE Healthcare) according to the manufacturer’s instructions. Then,
sequencing products were precipitated and run in the automatic sequencer MegaBACE 1000
(GE Healthcare).
High quality consensus sequences were obtained using the programs Phred (Ewing et
al. 1998), Phrap (Ewing & Green 1998) and Consed 16.0 (Gordon et al. 1998). The consensus
sequences of the autosomal loci were aligned by the Clustal X algorithm implemented in MEGA
5 (Kumar et al. 2008) together with the two E. imbricata and C. caretta reference sequences
published by Naro-Maciel et al. (2008). Polymorphic sites were either identified by visual
inspection in Consed or using Polyphred 6.11 (Nickerson et al. 1997; Stephens et al. 2006).
Microsatellites
Polymerase chain reaction (PCR) mixes of 9 µL included 1 µL of genomic DNA (~40 ng),
1 U of Taq Platinum polymerase (Invitrogen), 200 µM of deoxynucleoside triphosphates, 1 X
Tris–KCl buffer (Invitrogen), 1.5 mM MgCl2 (Invitrogen), 1 mM of the forward primer labeled
with a m13 tail, 10 mM of the reverse primer and 10 mM of the m13 primer with fluorescence
FAM or HEX (Schuelke 2000). The amplification program consisted of 3 min at 94o C, followed
by 30 cycles of 30 s at 94o C, 30s at annealing temperatures depending on the locus (55o C for
OR1, OR2 and OR3 and 60o C for CC1G02 and Cc1G03), 30 s at 72o C, and a final extension step
of 30 min at 72o C. The amplicons were diluted fivefold with Milli-Q water. Genotyping reaction
mixes of 10 µL included 2 µL of diluted amplicon, 0.25 µL of ET 550-R (GE Healthcare) and 7.75
µL of Tween20 0.1%. The running conditions followed the manufacturer’s recommendations
(GE Healthcare) for genotyping in an automated MegaBACE 1000 DNA analysis system. The
peaks were analyzed in the Fragment Profiler Program (GE Healthcare) for allele scoring.
The allele distribution was analyzed for the presence of null alleles with the program
MicroChecker version 2.2.3 (Van Oosterhout et al. 2004) and the observed and expected
heterozygosities were calculated with Arlequin version 3.5 (Excoffier & Lischer 2010).
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