Qubit broad range assay

Lissoclinum verrilli adult zooids were digested in a solution of CTAB and proteinase K in a 65°C water bath, and DNA was extracted and purified with a magnetic‐bead protocol (SprintPrep DNA purification kit, Agencourt Bioscience Corporation Beverly). The DNA of larval offspring was extracted and purified using the protocol of the QIAamp DNA Micro Kit (Qiagen). DNA concentrations were quantified with a NanoDrop ND‐1000 spectrophotometer (Thermo Fisher Scientific) and with a Qubit broad‐range assay (Thermo Fisher Scientific).
A genomic DNA library enriched for microsatellite loci was developed for L. verrilli by the Evolutionary Genetics Core Facility at Cornell University. Ninety potential contigs were screened from this library and 10 loci were selected based on repeat motif, product size, and degree of polymorphism (Table S2). Loci were amplified with the Qiagen Type‐it Microsatellite PCR Kit under the following cycling parameters: 95°C for 4 min, 30 cycles of 95°C for 30 s, 57°C for 40 s, 72°C for 45 s, 9 cycles of 95°C for 30 s, 53°C for 40 s, 72°C for 40 s, and a final extension of 72°C for 10 min. Fragment analysis was performed by the DNA Sequencing Facility at Florida State University with GeneScan 500 ROX standard (Thermo Fischer Scientific) and alleles were called in the program Geneious 9.1.8 (Biomatters).

After the first step of PCR, the quantity and quality of PCR products were checked in 2% Agarose gel. PCR products were then digested with 0.5 µl DpnI (to remove methylated DNA of plasmid template to prevent its interference in the second PCR step) and 2 µl ExoSAP (to hydrolyze excess primers and nucleotides) at 37°C for 1 h. The enzymes were then inactivated at 80°C for 20 min. Next, the fragments were purified by QIAGEN MinElute spin column as per manufacturer’s protocol and were eluted in 15 µl Molecular Grade water. The purified products were checked in 2% agarose gel. The concentrations of purified PCR products were then measured by Qubit Broad Range assay (Invitrogen). Molar mass of each PCR fragment was determined using Sequence Manipulation Suite which calculated the number of moles present per microlitre of solution.