Primer Design
Specimens of C. fleckeri , were collected from Horseshoe Bay
(19.1333° S, 146.8500° E), Magnetic Island, Queensland (1 November
2018), with other species having been collected either off the coast of
Magnetic Island, or Cairns, Queensland (Supplementary materials).
Genomic DNA (gDNA) was extracted using a Qiagen DNeasy Blood and Tissue
Kit (Venlo, The Netherlands). A 584 bp fragment of the mitochondrial 16S
rRNA gene was amplified via PCR using the “universal jellyfish
primers”, Forward-16SL, (Bayha et al . 2010)
Reverse-Aa_H16S_15141H (Ender & Schierwater 2003) and thermocycling
conditions reported for the forward and reverse primers respectively.
Purified PCR product was then sent to the Australian Genome Research
Facility (AGRF, Brisbane, Australia) to be Sanger sequenced. Returned
sequences were then edited and aligned in Geneious (Biomatters,
Auckland, New Zealand) and put into a n-Blast search downloaded from the
NCBI website to confirm their taxonomy and to identify likely
polymorphic sites among species suitable for the design of
species-specific eDNA primers (Appendix S1, Supporting information).
Species-specific eDNA primers were designed for C. fleckeri, C.
sivickisi , C. xaymacana and C. barnesi using Geneious
(Biomatters, Auckland, New Zealand) based on alignment of sequences and
identification of unique primer sequences for each species within the
mtDNA 16s rRNA gene. The specificity of species-specific primers was
then assessed against the genomic DNA of all other cubozoan taxa from
waters surrounding Magnetic Island, Queensland.
Using Real-Time PCR, technical replicates for each species were
performed. Amplicons were visualised on a 1.5% agarose electrophoresis
gel at 80 V for 40 min against other cubozoan species (Appendix S2,
Supporting information). The triplicate technical replicates of each
species were repeated for qPCR reactions. To deem specificity of
primers, exclusive amplification of the target species was completed.