['zebrafish', 'because:', '(1)', 'the', 'zebrafish', 'linkage', 'map', 'is', 'twice', 'the', 'length', 'of', 'the', 'mouse', 'map;', '(2)', 'zebrafish', 'have', '25', 'chromosomes', 'compared', 'with', '20', 'in', 'mouse;', 'and', '(3)', 'the', 'ends', 'of', 'zebrafish', 'chromosomes', 'have', 'not', 'yet', 'been', 'placed', 'on', 'the', 'linkage', 'map.', 'Coupling', 'PCR', 'to', 'the', 'genetic', 'tricks', 'Streisinger', 'et', 'al.!', 'developed', 'for', 'zebrafish', '(Box', '1)', 'has', 'permitted', 'the', 'design', 'of', 'two', 'efficient', 'methods', 'for', 'mapping', 'zebrafish', 'genes', 'with', 'far', 'fewer', 'PCRs,', 'bulked', 'segregant', 'analysis??45', 'and', 'centromere-linkage', 'analysis', 'using', 'half-tetrad', 'animals3244,', 'Bulked', 'segregant', 'analysis', '(BSA)', 'identifies', 'dominant', 'or', 'co-dominant', 'markers', 'tinked', 'to', 'mutations', 'by', 'the', 'dif-', 'ferential', 'amplification', 'of', 'fragments', 'from', 'two', 'genomic', 'DNA', 'pools,', 'one', 'from', 'wild-type', 'animals', 'and', 'the', 'other', 'from', 'their', 'mutant', 'siblings', '(Fig.', '1),', 'Both', 'alleles', 'of', 'mark-', 'ers', 'unlinked', 'or', 'distantly', 'linked', 'to', 'the', 'mutation', 'are', 'equally', 'represented', 'in', 'both', 'phenotypic', 'pools', 'because', 'of', 'independent', 'assortment', 'and', 'crossing-over.', 'Hence,', 'unlinked', 'marker', 'fragments', 'are', 'amplified', 'from', 'both', 'pools.', 'In', 'contrast,', 'fragments', 'from', 'markers', 'linked', 'to', 'the', 'mutation', 'amplify', 'in', 'only', 'one', 'pool', 'because', 'these', 'alleles', 'co-segregate', 'with', 'the', 'mutation', 'on', 'which', 'the', 'pools', 'are', 'based.', 'Although', 'the', 'pools', 'can', 'consist', 'of', 'either', 'haploid', 'or', 'diploid', 'individuals,', 'the', 'use', 'of', 'haploids', 'simplifies', 'the', 'analysis', 'of', 'dominant', 'markers', 'such', 'as', 'RAPDs', 'because,', 'in', 'haploids,', 'dominant', 'alleles', 'do', 'not', 'obscure', 'their', 'recess-', 'ive', 'counterparts', 'as', 'they', 'do', 'in', 'diploid', 'heterozygotes.', 'Centromere-linkage', 'analysis', '(CLA)', 'relies', 'on', 'diploid', 'halftetrad', 'animals', 'to', 'assign', 'mutations', 'to', 'linkage', 'groups', 'rapidly', '(Fig.', '2)32,', 'Half-tetrad', 'analysis', 'reveals', 'the', 'distance', 'between', 'genes', 'and', 'their', 'centromeres*.4?', 'because', 'homo-', 'zygous', 'mutant', 'half-tetrads', 'result', 'almost', 'exclusively', 'from', 'meioses', 'with', 'no', 'cross-overs', 'between', 'the', 'mutant', 'locus', 'and', 'its', 'centromere', '(Fig.', '2,', 'Step', '1).', 'Exceptions', 'due', 'to', 'multiple', 'cross-overs', 'are', 'rare', 'because', 'of', 'high', 'recombi-', 'national', 'interference', 'in', 'zebrafish*.', 'The', 'identification', 'of', 'PCR-based', 'markers', 'near', 'each', 'of', 'the', '25', 'centromeres', 'permits', 'a', 'mutation', 'to', 'be', 'tested', 'for', 'linkage', 'to', 'each', 'cen-', 'tromere*,42,', 'This,', 'in', 'turn,', 'allows', 'the', 'mutation', 'to', 'be', 'assigned', 'to', 'one', 'of', 'the', '25', 'linkage', 'groups', 'with', 'a', 'mini-', 'mum', 'number', 'of', 'PCR', 'assays', '(Fig.', '2,', 'Step', '2).', 'CLA', 'allows', 'a', 'mutation', 'to', 'be', 'assigned', 'to', 'a', 'linkage', 'group', 'by', 'scoring', '12', 'homozygous', 'mutant', 'half-tetrads', 'for', 'markers', 'linked', 'to', 'each', 'of', 'the', '25', 'centromeres', '(300', 'PCRs),', 'assuming', 'that', 'stocks', 'are', 'inbred', 'and', 'polymorphic', 'with', 'respect', 'to', 'cen-', 'tromeric', 'markers.', 'If', 'one', 'uses', 'polymorphic', 'co-dominant', 'markers', 'and', 'prepares', 'pools', 'of', 'DNA', 'from', 'half-tetrad', 'diploids', '(ie.', 'one', 'pool', 'from', '12', 'wild-type', 'animals', 'and', 'the', 'other', 'from', '12', 'mutant', 'animals)', 'then', '50', 'PCRs', 'are', 'sufficient', 'to', 'localize', 'the', 'mutation', 'to', 'a', 'linkage', 'group', '(25', 'centromere', 'markers', 'X', 'two', 'phenotypic', 'pools).', 'After', 'the', 'linkage', 'group', 'is', 'determined,', 'additional', 'markers', 'are', 'used', 'to', 'localize', 'it', 'on', 'a', 'chromosome', 'arm', 'within', 'a', 'few', 'centimorgans', '(Fig.', '2,', 'Step', '3).', 'The', 'genetic', 'map', 'and', 'the', 'methodologies', 'of', 'BSA', 'and', 'CLA', 'can', 'expedite', 'the', 'genomic', 'localization', 'of', 'mutations,', 'but', 'how', 'can', 'mutations', 'best', 'be', 'cloned?', 'Three', 'approaches', 'have', 'proven', 'fruitful:', 'insertional', 'mutagenesis,', 'candidate', 'genes', 'and', 'positional', 'cloning.', ' ', 'Insertional', 'mutagenesis', 'Recent', 'studies', 'show', 'that', 'insertional', 'mutagenesis', 'with', 'retroviral', 'vectors', 'is', 'feasible', 'in', 'zebrafish!*4,', 'Screens', 'of', 'F,', 'progeny', 'from', 'F,', 'animals', 'bearing', 'transgenes', 'identified', 'four', 'lethal', 'mutations', 'linked', 'to,', 'and', 'presumably', 'in-', 'duced', 'by.', 'retroviral', 'insertions.', 'In', 'three', 'of', 'these', 'cases,', 'sequence', 'analysis', 'of', 'flanking', 'DNA', 'rapidly', 'identified', 'the', 'gene', 'disrupted', 'by', 'the', 'insertion,', 'demonstrating', 'an', 'impor-', 'tant', 'advantage', 'of', 'insertional', 'mutagenesis.', 'One', 'appli-', 'cation', 'of', 'retroviral', 'vectors', 'is', 'whole-genome', 'mutation', 'screens,', 'but', 'the', 'inefficiency', 'of', 'insertional', 'mutagenesis', 'will', 'make', 'it', 'difficult', 'to', 'isolate', 'mutations', 'in', 'as', 'many', 'genes', 'as', 'identified', 'in', 'the', 'chemical', 'screens.', 'Approximately', '35', 'F,', 'families,', 'with', 'two', 'insertions', 'each,', 'must', 'be', 'screened', 'to', 'identify', 'each', 'embryonic', 'lethal', 'mutation', 'because', 'about', '1', 'in', '70', 'retroviral', 'insertions', 'disrupts', 'an', 'essential', 'gene!2.', 'By', 'contrast,', 'mutagenesis', 'screens', 'employing', 'the', 'chemical', 'mutagen', 'ethylnitrosourea', '(ENU)', 'identified', 'a', 'lethal', 'mutation', 'in', 'almost', 'all', 'F,', 'families’.', 'Another', 'application', 'of', 'retroviral', 'mutagens', 'is', 'the', 'iso-', 'lation', 'of', 'tagged', 'alleles', 'of', 'previously', 'identified', 'loci', 'by', 'non-complementation', 'of', 'existing', 'mutations.', 'It', 'is', 'likely', 'that', 'more', 'than', '70000', 'insertions', 'must', 'be', 'tested', 'to', 'find', 'an', 'insertional', 'allele', 'of', 'a', 'known', 'locus', 'because', 'most', 'loc!', 'identified', 'in', 'ENU', 'mutagenesis', 'screens', 'were', 'recovered', 'at', 'a', 'rate', 'of', 'less', 'than', 'one', 'allele', 'in', '1000', 'lethal', 'mutations’?', 'and', 'because', 'about', '1', 'in', '70', 'retroviral', 'insertions', 'hits', 'an', 'essential', 'gene.', 'The', 'germline', 'of', 'an', 'insertionally', 'muta-', 'genized', 'GO', 'individual', 'transmits,', 'on', 'average,', 'about', '11', 'insertions!2.13,', 'therefore,', 'on', 'average,', 'about', '7000', 'pair', 'matings', 'between', 'GO', 'individuals', 'and', 'heterozygous', 'testers', 'need', 'to', 'be', 'screened', 'to', 'find', 'an', 'insertional', 'allele', 'at', 'a', 'previously', 'identified', 'locus.', 'In', 'addition,', 'insertion', 'alleles', 'of', 'some', 'loci', 'might', 'be', 'difficult', 'or', 'impossible', 'to', 'find', 'because', 'the', 'vector', 'might', 'have', 'a', 'target-site', 'bias', 'as', 'with', 'P', 'elements', 'in', 'Drosophila.', 'In', 'spite', 'of', 'these', 'con-', 'cers,', 'improvements', 'in', 'the', 'breeding', 'and', 'screening', 'protocols,', 'coupled', 'with', 'more', 'efficient', 'vectors,', 'per-', 'haps', 'inchiding', 'mobilizable', 'transposons,', 'will', 'make', 'insertional', 'mutagenesis', 'an', 'attractive', 'strategy', 'for', 'the', 'cloning', 'of', 'mutations.', ' ', ' ', 'The', 'candidate', 'gene', 'approach:', 'exploiting', 'mouse', 'and', 'human', 'maps', 'The', 'systematic', 'testing', 'of', 'candidate', 'genes', 'has', 'resulted', 'in', 'the', 'mclecular', 'isolation', 'of', 'four', 'zebrafish', 'mu-', 'tations:', 'no', 'tail', '(Ref.', '22),', 'floating', 'bead', '(fh)', '(Ref.', '23),', 'no', 'isthmus', '(Ref.', '24)', 'and', 'nici', '(Ref.', '48).', 'In', 'this', 'approach,', 'the', 'map', 'location', 'of', 'a', 'mutation', 'is', 'compared', 'with', 'the', 'map', 'location', 'of', 'cloned', 'genes', 'that', 'are', 'expressed', 'in', 'or', 'near', 'tissues', 'phenotypically', 'altered', 'by', 'the', 'mutation,', 'This', 'cloning', 'approach', 'benefits', 'from', 'rapid', 'methodologies', 'to', 'map', 'mutations', 'and', 'from', 'a', 'genetic', 'map', 'with', 'many', 'cloned', 'genes', 'to', 'serve', 'as', 'candidates,', 'The', 'future', 'success', 'of', 'the', 'candidate', 'gene', 'approach', 'will', 'be', 'improved', 'by', 'increasing', 'the', 'density', 'of', 'cloned', 'genes', 'on', 'the', 'zebrafish', 'map.', 'The', 'mapping', 'of', 'cloned', 'genes', 'in', 'zebrafish', 'began', 'in', '1994', '(Ref.', '29)', 'with', 'the', 'lo-', 'calization', 'of', 'mbcdab,', 'snaill', 'and', 'msxb.', 'To', 'map', 'cloned', 'genes,', 'sequences', 'expected', 'to', 'be', 'polymorphic,', 'such', 'as', 'introns', 'or', '3’', 'untranslated', 'regions,', 'are', 'amplified', 'by', 'PCR.', 'Polymorphisms', 'are', 'revealed', 'either', 'by', 'a', 'restriction', 'enzyme,', 'by', 'size', 'differences', 'or', 'by', 'single-strand', 'confor-', 'mation', 'polymorphism', '(SSCP)', 'analysis*®.', 'The', 'map', 'cur-', 'rently', 'contains', 'more', 'than', '120', 'cloned', 'genes,', 'most', 'of', 'which', 'are', 'of', 'interest', 'to', 'developmental', 'biologists', 'and,', 'hence,', 'provide', 'a', 'source', 'of', 'candidate', 'genes', 'for', 'embry-', 'onic', 'mutations', '(J.H.', 'Postlethwait', 'et', 'a/.,', 'unpublished).', 'TIG', 'May', '1997', 'VoL.', '13', 'No.', '5', '185', ' ', '50734591']