Rapid and repeated evolution of myosin copy number in threespine stickleback

Figures

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  Figure 1.. Myosin copy number increases are recurrent and rapid in freshwater stickleback populations (A) ChrXIX genomic region in the freshwater stickleback reference genome (stickleback v. 5)38 that includes four tandemly arrayed myosin heavy chain genes: MYH3C1 (orange), MYH3C2 (yellow), and two duplicated copies of MYH3C3 (green). NCBI gene annotations for nearby genes are shown in grey. The boundaries of the ~17.6 kb duplicated regions (light green shading with dashed boxes) include full duplications of MYH3C3 and partial duplications of MYH3C2 and SYT19. The duplicated region falls within a Global Sensitive EcoPeak (dark grey bar) and a Cheney-Scout-Loberg Sensitive TempoPeak (light grey bar) described by Roberts Kingman et al.26, indicating repeated SNP divergence between global marine and freshwater stickleback populations and rapid SNP allele frequency changes in three populations of marine fish introduced into freshwater lakes, respectively. (B) MYH3C3 read depth normalized by mean autosomal read depth determined for female stickleback collected from different populations in the North American Pacific region (n = 10 marine, n = 66 freshwater) and Northern Europe (n = 5 marine, n =…

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  Figure 2.. Myosin haplotypes and sequence variants from marine and freshwater stickleback (A) Structure of MYH3C copy number haplotypes with one C1 copy (orange), one C2 copy (yellow), and one to four C3 copies (green). All expansion haplotypes contain the same duplicated ~17.6 kb region (dashed boxes) with breakpoints in C2 and SYT19 arranged in tandem. The 3-copy haplotypes come from marine (red) fish while 4-, 5-, and 6-copy haplotypes come from freshwater (blue) populations and show characteristic marine versus freshwater SNP divergence. The C3 duplications occur exclusively on ChrXIX while a singular marine-like 3-copy haplotype with a pseudogenized C1 is present on ChrY in all surveyed populations. HARBI1 is also present between NLRC5 and C1 in freshwater 4- to 6-copy alleles but the first exon is deleted in BLAU and it is completely absent from marine 3-copy haplotypes. See Table S2 for sample abbreviations, source locations, and assembly information. (B) Phylogenetic tree based on coding region nucleotide sequences of threespine stickleback MYH3C copies and putative orthologs from Danio rerio, Orzias melastigma, and Pungitius pungitius. Bootstrap values of ≥70 are labeled below each…

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  Figure 3.. Expression of different MYH3C copies and alleles in developing stickleback (A) F1 hybrids were generated by crossing two 3-copy marine RABS (red) females with a 5-copy freshwater BEPA (blue) male and raising offspring for either 1 or 5 months. RNA was extracted from whole juvenile fish (no caudal fin) from 1-month females, or from flank muscle, abductor pectoral muscle, and adductor pectoral muscle dissected from 5-month young adult females. K-mers were used to detect total expression or allele-specific expression from RNA-seq reads (n = 6 per condition; Table S4). Alignment of Kinnex reads (n = 2 per condition) to a marine 3-copy and freshwater 5-copy composite genome was used to identify relative expression of each C3 copy. For simplicity, the small fragments of the C2 and SYT19 genes are not diagrammed in the RABS and BEPA haplotypes. (B) Box plots showing the relative expression levels of MYH3C copies in each tissue based on seven sets of MYH3C copy expression (MCE) 27-mers. Reads per million (RPM) was calculated by dividing the number of paired reads detected by each k-mer by the total number of paired reads from each sample. C1 (orange) has no to low expression in all…

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  Figure 4.. The duplicated C2-C3 intergenic region contains a muscle enhancer (A) Schematic of the negative control empty vector, SALR, and BEPA constructs used to generate transgenic fish. The SALR (red) and BEPA (blue) putative regulatory regions, which include the intergenic region between C2 and C3 (dark grey highlight), were inserted upstream of a minimal promoter (Hsp70) driving green fluorescent protein (GFP). The candidate regulatory intergenic region is present in one copy in the marine SALR 3-copy haplotype and three copies in the freshwater BEPA 5-copy haplotype—upstream of C3A, C3B, and C3L. For the expression constructs, a single copy of either the SALR or BEPA sequences was cloned and tested. (B) Representative images of GFP expression in transgenic stickleback. The SALR and BEPA constructs drive expression in the lens of the eye (left image) and in body skeletal muscle fibers of 3-week old larval fish (right image). Muscle expression begins at about 13 dpf and continues past 3 months of age (not shown). The control empty vector construct drives expression predominantly in the lens, a known site of activity of the Hsp70 promoter84. Scale bar, 1mm. (C) Box plots of normalized…

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  Figure 5.. Mechanisms of MYH3C copy number expansion (A) Hypothesized mechanism for 3- to 4-copy expansion via microhomology break-induced replication (MMBIR). During replication, a double strand break occurs near the SYT19 breakpoint (1, grey) of a 3-copy haplotype, followed by 5’ to 3’ resection. Annealing to the sister chromatid or homologue using shared microhomology (fuchsia) within C2 (2, yellow) restarts replication and produces a duplication with an intermediate breakpoint (3) that fuses the SYT19 and C2 sequences. Perfect microhomology of 4 bp and imperfect microhomology of 8 bp occurs at the breakpoints in all haplotypes (BOULxBDGB sequence shown here). (B) Hypothesized mechanism for 4- to 5-copy expansion and 5- to 6-copy expansion via non-allelic homologous recombination (NAHR). Inter-chromosomal or inter-chromatid recombination between two misaligned 4-copy haplotypes (C3A with the C3L duplicated region) could produce reciprocal duplication (5-copy) and deletion (3-copy) products (top). The new intermediate duplication gene would be more similar to the L region at the 5’ end and the A region at the 3’ end. If inter-chromosomal or inter-chromatid recombination occurred between…