Analysis of positive selection within <i>SYNGR2</i> across mammalian species using site-specific models of evolution.

For each model, the dN/dS ratio was estimated for the entire gene (dN/dS) and across each site within SYNGR2 using the corresponding gene tree. A significant likelihood ratio test (LRT) statistic indicates the model allowing positive selection (M8, ω≤1 or ω>1) is a better fit than the null model (M7, ω≤1). The number of positive selection sites (PSS) is reported for each cluster.</p

<i>SYNGR2</i> haplotypes identified across <i>Suidae</i> groups.

(*187 = SYNGR2 p.Arg63Cys; DM = Domestic, WB = Wild Boar, SR = Sus Relatives).</p

Integer-joining network of <i>SYNGR2</i> haplotypes within <i>Suidae</i>.

Haplotype frequencies within each S. scrofa subgroup were applied to hypothetical sample sets of equal size. Node size represents haplotype frequency across S. scrofa, and different colored segments represent the proportion within each subgroup: European Domestic (EUD), European Wild Boar (EUW), Asian Domestic (ASD), Asian Wild Boar (ASW), Sus relatives (SUR). Black nodes represent inferred intermediate haplotypes not present in any samples within the dataset. Single nucleotide changes are represented as hatch marks.</p

PCV2 genome copy number in wildtype and edited PK15 clones following <i>in vitro</i> infection.

Log10 transformed viral genome copy number per well in the cell (top) and per uL in the supernatant (bottom) fractions across timepoints post infection with PCV2b or PCV2d (MOI = 0.00025). Error bars represent one standard error from the mean for three independent infection replicates. *P<0.05, **P<0.01, ***P<0.001.</p

Proposed scenario of <i>SYNGR2 p</i>.<i>63Cys/Hap1</i> origin in European domestic swine.

Solid lines reflect well-documented and experimentally supported directions of gene flow between populations throughout swine domestication. Dashed lines represent less documented directions of gene flow necessary for the proposed scenario of origin in EUD (red lines). Pie charts represent the frequency of Hap1 (orange) and Hap2 (blue) in each S. scrofa subgroup. (ASW = Asian Wild Boar, ASD = Asian Domestic, EUW = European Wild Boar, EUD = European Domestic).</p

Parameters of genetic diversity across domestic groups with alternate homozygous <i>SYNGR2 p</i>.<i>Arg63Cys</i> genotypes.

Average number of alleles (Num), effective number of alleles (Eff_num), observed heterozygosity (Ho), expected heterozygosity (Hs), and inbreeding coefficient (Gis) were estimated for each group.</p

Supporting figures and tables.

Fig A. CRISPR-Cas9 guide RNA and template design for generation of clones homozygous for alternate SYNGR2 p.63Cys allele. The selected guide RNA (sg_AF) targeted a PAM site within exon 2 (gray box) that included the SYNGR2 p.63Arg allele (red). An 80 bp ssDNA sequence (thick black line) homologous to the non-targeting strand and encoding the alternate SYNGR2 p.63Cys allele (yellow) was used as a template for homology-directed repair. Fig B. Alignment of the SYNGR2 coding sequence for wildtype and edited PK15 cell lines. Sequences were obtained by targeted amplification of the SYNGR2 transcript and Sanger sequencing. Position of the substitution of p.63Arg (C) to p.63Cys (T) in the edited PK15 clones, emSYNGR2+p.63Cys and 2emSYNGR2+p.63Cys, is highlighted in red. The 106 bp deletion in the predicted SYNGR2 knock-out PK15 clone, emSYNGR2-del, is represented by dashes. Fig C. Expression of SYNGR2 in wildtype and edited PK15 following PCV2b infection mock-infected control cells. Expression represented as Log10 transformed mean normalized expression (MNE) across three independent replicates with error bars representing one standard error from the mean. Samples collected from control and infected cells across timepoints post PCV2b infection (MOI = 0.00075). Letters denote significant differences in gene expression between cell lines within treatment group (C = control, I = infected) or between treatment groups within cell lines (wt = wildtype, em = edited). *PFig D. Frequency of SYNGR2 haplotypes across geographic and domestic/wild S. scrofa subgroups. The two haplotypes that differ by only the SYNGR2 p.Arg63Cys allele, Hap1 (Cys) and Hap2 (Arg), are represented as independent pie segments. Hap3-Hap10 and rare haplotypes were combined into a single category denoted as “Other”. (ASW = Asian Wild Boar, ASD = Asian Domestic, EUW = European Wild Boar, EUD = European Domestic). Fig E. Frequency of SYNGR2 haplotypes across domestic breeds. The two haplotypes that differ by only the SYNGR2 p.Arg63Cys SNP, Hap1 (Cys) and Hap2 (Arg), are represented as independent pie segments. Hap3-Hap10 and rare haplotypes were combined into a single category denoted as “Other”. (BR = Berkshire, DR = Duroc, IB = Iberian, PI = Pietrain, LR = Landrace, LW = Large White, YR = Yorkshire, EUDO = European Domestic Other, EH = Erhualian, MS = Meishan, ASDO = Asian Domestic Other). Table A. Mammalian SYNGR2 transcript sequences and taxonomic groups. Table B. SYNGR2 SNP identified across Suidae sequences. Each SYNGR2 SNP is denoted by nucleotide position within the SYNGR2 coding sequence. (*187 = SYNGR2 p.Arg63Cys; SSC12 = chromosome 12, S.scrofa11.1). Table C. Allelic frequencies for SYNGR2 SNP across Sus scrofa subgroups. (*187 = SYNGR2 p.Arg63Cys; EUD = European Domestic, EUW = European Wild, ASD = Asian Domestic, ASW = Asian Wild). Table D. Allelic frequencies for SYNGR2 SNP across domestic breeds. (*187 = SYNGR2 p.Arg63Cys; BR = Berkshire, DR = Duroc, IB = Iberian, PI = Pietrain, LR = Landrace, LW = LargeWhite, YR = Yorkshire, EUDO = European other, EH = Erhualian, MS = Meishan, ASDO = Asian other). Table E. Frequency of SYNGR2 haplotypes across S. scrofa subgroups. (EUD = European Domestic, EUW = European Wild, ASD = Asian Domestic, ASW = Asian Wild). (DOCX)</p

Position of putative positive selection sites identified across mammals within the <i>SYNGR2</i> protein.

Schematic depicting the four transmembrane domains (gray boxes), two intraluminal loops, one cytoplasmic loop domain and cytoplasmic N- and C-termini. Amino acid residues identified as positive selection sites within one (green) or more (purple) taxonomic groups are represented relative to the SYNGR2 p.Arg63Cys polymorphism (red).</p

SYNGR2 expression and viral titer data following in vitro PCV2 infection of PK15 cells.

SYNGR2 expression and viral titer data following in vitro PCV2 infection of PK15 cells.</p

Allelic frequencies for <i>SYNGR2</i> SNPs between domestic groups with alternate homozygous <i>SYNGR2 p</i>.<i>Arg63Cys</i> genotypes.

Bottom portion of table denotes the average (Avg), highest (Max), and lowest (Min) minor allele frequency (MAF) in each group.</p