(A) Polymorphic markers (z21170, kdr_e28, 5’scfd2, z14614) were used to map meiotic recombination events, narrowing the region containing the ref mutation to <0.1 cM on linkage group (LG) 20. (See also Table 3). Fractions indicate frequencies of proximal (magenta) and distal (green) recombination between markers and ref. Six annotated genes are present in this region (GRCv10); sequence analysis of kdr, kita, gsx2, lnx1, and fip1l1a in ref mutants revealed only missense mutations that led to conserved amino acid changes. (B) RT-PCR spanning exons 13–19 of pdgfra generates a single, properly spliced product from homozygous wt embryos and multiple, smaller products from ref mutant embryos. Sequencing revealed that exon 14 was either omitted or truncated in these smaller products; in all cases, the observed missplicing would result in a frameshift followed by a premature stop codon. Although we did not detect any normally spliced pdgfra products in ref mutants, we cannot rule out the presence of low levels of wild-type mRNA. RT-PCR of gapdh demonstrates use of comparable amounts of template. (C,D) Sequencing the e14i15 exon-intron boundary of pdgfra revealed that ref mutant genomic DNA contains a G-to-A mutation in a conserved intronic nucleotide required for proper splicing. Chromatograms (C) and sequence alignment (D) show position of the mutation relative to reference sequences. Schematics (D) depict the proteins predicted to result from the wt, ref, and b1059 alleles of pdgfra; immunoglobulin (magenta), transmembrane (green), and tyrosine kinase (blue) domains are shown. (E,F) Lateral views depict expression of pdgfra at 11 s. Expression levels are lower in ref mutants (F; n = 5/5) than in wt (E). (G–J) Dorsal views, anterior to the top, of myl7 expression at 22 s. In contrast to wt (G), cardiac fusion defects are evident in embryos injected with a pdgfra morpholino (MO) (H), b1059 homozygous mutant embryos (I), and ref/b1059 transheterozygous mutant embryos (J). See Figure 2—figure supplement 2 for additional information on the prevalence of each of these phenotypes. Scale bars: 60 μm.