Positional cloning of the zdfl8a12 allele. (A) Genetic map of the grechetto region on LG 19. Top panel shows low- and intermediate-resolution mapping carried out with SSLPs. Bottom panel shows fine mapping obtained by sequencing of intronic and 3′UTR SNPs. Solid black line indicates chromosome 19 (not in scale); red lines, bacterial artificial chromosomes (BAC); arrows, genes. The distance in centimorgans (cM) between markers was calculated assuming 1.5 recombination events per meiosis. (B-C) Chromatograms from cpsf1 cDNA sequencing of genotypic WT and mutant grechetto siblings, respectively, showing a T → A mutation in the latter resulting in a premature stop codon. (D) Identity at the protein level between the human (hs), mouse (mm), and zebrafish (dr) CPSF1 protein. The nuclear localization signal (NLS) is schematically represented by a green box and the putative RNA-binding domain by a red box. (E-G) Complementation experiment performed by crossing a cpsf1^hi2675 heterozygous fish (carrying a viral insertion in exon 2 of the cpsf1 gene) with a cpsf1^zdfl8a12 heterozygous fish (carrying a premature stop codon in exon 9 of the cpsf1 gene) depicted schematically in panel E. (F-G) WISH for mpx. All lateral views, anterior to the left, and dorsal upward, showed that the compound cpsf1^{zdfl8a12/hi2675} heterozygote offspring lacked myelopoiesis in the CHT at 5 dpf (compare the inset in panel G to a WT sibling in panel F), a typical feature of the grechetto phenotype.