DOI: 10.5061/dryad.wdbrv15q7
title: Zona pellucida (ZP3) sequence data from 230 Pacific cod (phased)

description: Genetic differentiation has been observed in marine species even when no
 obvious barriers to gene flow exist, and understanding such
 differentiation is essential for effective fisheries management. Highly
 differentiated outlier loci can provide information on how genetic
 variation might contribute to local adaptation but may also be affected by
 historical demographic events. A locus which aligned to a predicted zona
 pellucida sperm-binding protein 3 gene (ZP3) in Atlantic cod (Gadus
 morhua) was previously identified as the highest outlier based on FST in a
 RADseq study of Pacific cod (Gadus macrocephalus) across the West Coast of
 North America. However, because of the limited length of the RAD sequence
 and restricted geographic area of sampling, no conclusion on the
 functional significance of the observed variation was possible. In other
 marine species, ZP3 is involved in reproductive isolation, local
 adaptation, and has neofunctionalized as an antifreeze gene, and so it may
 provide important insights in functional population structure of Pacific
 cod. Here, we sequenced a 544 bp region of ZP3 in 230 Pacific cod
 collected from throughout their geographic range. We observed striking
 patterns of spatial structuring of ZP3 haplotypes, with a sharp break near
 Kodiak, Alaska, USA where populations within ~200 km of each other are
 nearly fixed for different haplotypes, contrasting a pattern of isolation
 by distance at other genetic markers in this region (FST = 0.003).
 Phylogenetic analysis of ZP3 haplotypes revealed that the more southern
 haplotypes appear to be ancestral, with the northern haplotype evolving
 more recently, potentially in response to a novel selective pressure as
 Pacific cod recolonized northern latitudes after glaciation. The sharp
 break in haplotype frequencies suggests strong selective pressures are
 operating on small spatial scales and illustrates that selection can
 create high divergence even in marine species with ample opportunities for
 gene flow.
description: PCRs for the variable region of the putative zona pellucida
 subunit 3 gene (primer ZP_GM, F: 3' gcaatctgagggtaggacca 5', R:
 3' aacgcagtgatccacaaaga 5') were carried out in a 25 µl volume,
 with Phusion 5X buffer (New England Biolabs, Ipswich, MA), 10mM dNTPs,
 10µM forward and reverse primers, 0.2µL Phusion <i>Taq
 </i>polymerase and approximately 200ng DNA. Thermal cycling
 conditions were 98°C for 30 sec, followed by 5 cycles of 98°C for 10 sec,
 63-59°C touchdown for 30 seconds (-1°C each cycle for 5 cycles) and 72°C
 for 30 seconds, and then by 30 cycles of 98°C for 10 seconds, 58°C for 30
 seconds and 72°C for 30 seconds, and concluded at 72°C for 5
 minutes. A total of 230 Pacific cod were sequenced to
 screen 544 bp of the variable region of ZP3 using primers ZP_GM (Table 1).
 Sanger sequencing was performed bidirectionally using forward and reverse
 primers at MCLAB (320 Harbor Way, South San Francisco, CA). Contigs were
 aligned using Sequencher v. 5.0 (Gene Codes Corporation, Ann Arbor, MI)
 and scores below 80% quality were discarded. Sequence calls were double
 checked and confirmed by two readers when ambiguities were present.
 Consensus sequences were aligned in BioEdit v. 7.2 (Hall, 1999). Sanger
 DNA sequence data was transformed from unphased sequences to fasta files
 with two haplotypes for all individuals. Haplotypes for ambiguous
 (heterozygous) nucleotides at ZP3 segregating sites were inferred using
 Bayesian methodology with a priori expectations based on coalescent theory
 in PHASE v2.1.1 and SeqPHASE (Stephens, Smith, & Donnelly, 2001;
 Flot, 2010). The most likely pair of haplotypes was selected for each
 individual based on the highest posterior probability.
 Flot, F.J. (2010). SeqPHASE: a web tool for interconverting PHASE
 input/output files and FASTA sequence alignments, <i>Molecular
 Ecology Resources</i>, 10 (1), 162–166. Stephens, M., Smith, N.J.,
 & Donnelly, P. (2001). A new statistical method for haplotype
 reconstruction from population data. <i>The American Journal of
 Human Genetics</i>, 68(4), 978–989.