Sample processing
To evaluate the biodiversity and molecular identity of the endolithic
algae associated with the collected Porites , we used the CTAB DNA
extraction protocol (Coffroth et al. 1992), adopting modifications to
increase the quality of the DNA associated with Ostreobium spp.,
for example, the coral tissue was removed using a WaterPik, leaving the
bare skeleton to avoid contamination of the host’s DNA. A piece of coral
skeleton was crushed to became a powder and we continued with extraction
process. The resulting product was quantified and pass to a centration
of 30 ng/ul so that it reacted adequately in the amplification process.
Next, the rbcL gene was amplified with primers rbcLF250
[5’GATATTGARCCTGTTGTTG GTGAAGA 3 ’], and rbcLR670 [5’
CCAGTTTCAGCTTGWGCTTTATAAA 3 ’], based on the sequence of amino acids
(Massé et al. 2020). The mixture to carry out the PCR had a total of 15
ul distributed as follows, 6.35 ul of water, 2 ul of
MgCl2, 3 ul of buffer, 0.75 ul BSA (Bovine Serum
Albumin, 20ng /ul), 0.6/ul of each primer, 1 ul Taq DNA polymerase, and
1 ul of genomic DNA. Subsequently, the following conditions were
established for amplification, starting at a temperature of 94 °C for 2
minutes, followed by 40 cycles of 94 °C for 90 s, 54 °C for 90 s, and 72
°C for 60 seconds. When the cycles ending, proceed with a temperature of
72 °C for 5 minutes. The amplified fragments were visualized in a 1%
agarose gel using Gelgreen (Invitrogen) to finally be sequenced by the
Sanger method in the GenCore laboratory of the Universidad de los Andes.