2.2 Primer design, DNA extraction, PCR, and sequencing
Reference nucleotide sequences (Ascothoracida and Cirripedia) of selected loci were downloaded from NCBI GenBank and aligned with in-house (see below) or NCBI Facetotecta sequences with MAFFT version 7.450 (–auto –maxiterate 1000; Katoh and Standley 2013). The alignments were imported, visualized, and used to design primers in Geneious Prime version 2022.0.2. To enhance specificity and amplification success, we strived to design longer (>20bp) primers with GC-contents between 30-60%, a GC-clamp of 2 (3’ terminating in at least one G/C to promote optimal binding), a melting temperature (Tm) between 55°C-68°C, and no runs of >4 bases (homopolymeric regions, e.g., TGGGGG). We tested a total of 28 primer pairs (23 new) and performed 2056 PCR reactions using these. Tables 1, 2, and S2 provide an overview of the primers, their sequences, and their amplification success with single specimens.
Our goal was to develop and test the efficacy of fast “filter-free” DNA-extraction methods that amplify single-specimen y-larvae while retaining their “exuviae” (here understood as any cuticular remains originating from an acutal molt of after tissue digestion during DNA extraction) as vouchers. We therefore tested and compared DNA-extraction using the GeneReleaser® kit (BioVentures, TN, USA), which has previously proven to be an efficient protocol for retrieving DNA from zooplankton (Schizas et al., 1997; Böttger-Schnack and Machida, 2011; Watanabe et al., 2016; Olesen et al., 2022) and a simplified DNeasy method using a subset of the reagents in the Blood and Tissue kit (QIAGEN, CA, USA). To this end, and as a part of a larger phylogenetic and barcoding campaign, we extracted DNA of a total 421 single y-larva specimens, of which nine were from the Azores, 326 from Sesoko Island (Japan), 4 from Malaysia, 105 from various locations in Taiwan, and 17 from the White Sea (Russia).
DNA extracts were kept in a freeze Eppendorf© vial rack during preparation of the PCR assay. We pipetted 12.2 µL filtered ddH2O, 5 µL “hot start” HOT FirePol® polymerase master mix (Solis BioDyne, Tartu, Estonia), 0.4 µL of each oligonucleotide primer (usually ordered as “desalted”), and 3 µL gDNA template to each vial. For multiplex PCR reactions, which use two or more primer pairs in combination, we subtracted the equivalent amount of added primer from the amount of ddH2O. To increase primer annealing sensitivity, specificity to complex templates (e.g., >60% G-C content), and yield we generally used a Touchdown PCR profile under the following conditions: initial denaturation for 15min at 95°C, then 10 cycles of denaturation at 95°C for 1min, annealing at Tm+10°C decreasing by 1°C/cycle for 30 seconds, extention at 72°C for 1kb/60secs, then 30 cycles of denaturation at 95°C for 1min, annealing at Tm-2°C for 30 seconds, extension at 72°C for 1kb/60secs, final extension at 72°C for 10min, and finally 20min at 4°C to halt the reaction. Primer details including sequences and annealing temperatures is listed in Supplementary Table 1.
All PCR reactions were conducted in a DNA Engine Thermal Cycler (Bio-Rad, Richmond, CA, United States) and PCR products were visualized in agarose gel with varying concentrations depending on fragment size. DNA sequencing was performed by Genomics BioSci & Tech Ltd. (New Taipei City, Taiwan) and Macrogen Europe BV (Amsterdam, The Netherlands).
We sequenced genomic DNA templates of three ribosomal loci (mitochondrial 16S and nuclear 18S, 28S) and two protein-coding genes (mitochondrial COX1 and nuclear Histone-3) of 74 specimens and added new sequences to NCBI GenBank (Tables 1, 2, S2). When we sequenced PCR amplicons, we defined “successful amplification” as those amplicons that yielded clear gel electrophoresis bands and clean chromatograms that aligned with our in-house database. Less than 10% of amplicons were not sequenced but nonetheless yielded clear bands, and these were computed as successful amplicons.