mtCOI, EF1α PCR and cloning
Primers and annealing temperatures are listed in Tab. 2.3 . PCR amplification for mitochondrial cytochrome oxidase c I (mtCOI)and elongation factor 1α (EF1α) loci was done using Q5 Hot Start High-Fidelity DNA Polymerase (NEB) with a 30 s extension time and annealing performed at 50°C. PCR reaction mix consisted of buffer (5x, Qiagen, 5 µl), dNTPs (0.5 µl), forward and reverse primers (1.25 µl of 10 µM for both), 0.25 µl polymerase and ddH2O (to 25 µl). ‘Pan-molluscan’ barcoding primers (Folmer et al. 1994) did not amplifymtCOI allele in L. balthica. Therefore, degenerate primers designed to match the same region of L. balthica mtCOIwere used (Metzger et al. 2018), detailed in Tab. 2.3 . We identified two transcripts with an annotation of EF1α in a published L. balthica transcriptome (Yurchenko et al. 2018). These transcripts aligned to one another with a high degree of polymorphism, indicating these may represent multiple copies ofEF1 -like genes in the L. balthica genome rather than different isoforms or haplotypes. We assumed intron placement would likely be conserved in the EF1α gene among bivalves, so we aligned these transcripts to an oyster genome (Crassostrea_gigas : oyster_v9 genome version) EF1αregion using Geneious alignment with default settings. We then chose primer sites to target coding sequence and amplify across the 5th intron. We chose primers that would only target one of the EF1αtranscripts (transcript ID: ”evgsoapLocus_712298”) to control for the possibility of multiple EF1α copies. In all cases, 25-50 ng of genomic DNA was amplified for 35 cycles with initial denaturation performed at 98°C for 30 s.
PCR products were gel extracted using QIAquick Gel Extraction Kit (Qiagen) and either directly sequenced, or, when multiple alleles at a locus could not be resolved by direct sequencing, were cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). Plasmids were transformed into TOP10 or DH5α competent Escherichia coli (Invitrogen) and at least 6 clones were picked for further sequencing using M13F and M13R primers (Genewiz) using the difficult template protocol, due to the hairpin found in some of the EF1α alleles. In cases where a single clone was sequenced that was 1 SNP different from another clone found in multiple clones from the same animal, or a single clone was found to be consistent with recombination between two other clones found in multiple clones in the same animal, the single clones were assumed to be PCR artefacts. In cases where differences were found between the sequence results of haemolymph and tissue from the same individual, the alleles found more often in the haemolymph sample were called as the haemolymph alleles and those found more often in tissue were considered the tissue alleles. The primer binding regions were excluded from sequence analysis and all unique alleles were identified.