The FISH experiments were carried out on specimens ZUEC 17569 and ZUEC 17579
(Table S2 ),
which represent the populations of Rio de Janeiro and Duque de Caxias,
respectively. The PcP190 satellite DNA sequence previously isolated from
C. gaudichaudii by Vittorazzi et al. (2014 (link)) was amplified to obtain chromosomal probes. For this,
one cloned fragment was amplified by PCR in the presence of digoxigenin-dUTP
(Roche) and primers T7 and SP6, which flank the connection site of the pGEM-T
Easy Vector (Promega). The probes were mixed with salmon DNA (1 ng/μL of probe)
and precipitated in ethanol. The DNA was dissolved in a hybridization buffer at
pH 7 composed of deionized formamide (50%), 2 x SSC, phosphate buffer (40 mM),
Denhardt’s solution, SDS (1%), and dextran sulfate (10%). The in
situ hybridization technique was based on Viegas-Péquignot (1992 ), with modifications for the detection
of digoxigenin-labeled probes with anti-DIG-Rhodamine (Roche).
The microsatellites (CA)15 and (GATA)8 oligonucleotides
were marked directly with Cy5-fluorochrome at the 5’ end during synthesis
(Sigma-Aldrich) and used as probes in FISH assays that followed the protocol of
Kubat et al. (2008 (link)), under high
stringency (77%) conditions. Images of the hybridized metaphase chromosomes were
captured with an Olympus BX-60 microscope and edited with the Image-Pro Plus
program (Media Cybernetics).
(
which represent the populations of Rio de Janeiro and Duque de Caxias,
respectively. The PcP190 satellite DNA sequence previously isolated from
C. gaudichaudii by Vittorazzi et al. (2014 (link)) was amplified to obtain chromosomal probes. For this,
one cloned fragment was amplified by PCR in the presence of digoxigenin-dUTP
(Roche) and primers T7 and SP6, which flank the connection site of the pGEM-T
Easy Vector (Promega). The probes were mixed with salmon DNA (1 ng/μL of probe)
and precipitated in ethanol. The DNA was dissolved in a hybridization buffer at
pH 7 composed of deionized formamide (50%), 2 x SSC, phosphate buffer (40 mM),
Denhardt’s solution, SDS (1%), and dextran sulfate (10%). The in
situ hybridization technique was based on Viegas-Péquignot (1992 ), with modifications for the detection
of digoxigenin-labeled probes with anti-DIG-Rhodamine (Roche).
The microsatellites (CA)15 and (GATA)8 oligonucleotides
were marked directly with Cy5-fluorochrome at the 5’ end during synthesis
(Sigma-Aldrich) and used as probes in FISH assays that followed the protocol of
Kubat et al. (2008 (link)), under high
stringency (77%) conditions. Images of the hybridized metaphase chromosomes were
captured with an Olympus BX-60 microscope and edited with the Image-Pro Plus
program (Media Cybernetics).