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Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map

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Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map CopyTight 0 1996 by the Genetics Society of America Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map Stephen L. Johnson, Michael A. Gates, Michele Johnson, William S. Talbot, Sally Home, Kris Baik, Sunny Rude, Jamie R. Wong and John H. Postlethwait Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 Manuscript received October 12, 1995 Accepted for publication December 21, 1995 ABSTRACT The ease of isolating mutations inzebrafish will contribute to an understandingof a variety of processes common to all vertebrates. To facilitate genetic analysis of such mutations, we have identified DNA polymorphisms closely linked to each of the 25 centromeres of zebrafish, placed centromeres on the linkage map, increased the numberof mapped PCR-based markers to 652, and consolidated the number of linkage groups to the numberof chromosomes. Thiswork makes possible centromere-linkage analysis, a novel, rapid method to assign mutations to a specific linkage group using half-tetrads. ECENT studies have uncovered hundreds of muta- gaps in the linkage map so that the number of linkage R tions that disrupt specific events in zebrafish devel- groups would equal the number of chromosomes. Be- opment (KIMMEL 1989; MULLINS and NUSSLEIN-VOL- cause each chromosome has a single centromere, we HARD 1993; DRIEVERet al. 1994; HENIONet al. 1995; focused on localizing centromeres on the genetic map. JOHNSON and WESTON1995; JOHNSON et al. 199513) Be- Centromeres are attachment sites for spindle micro- cause the mechanisms of morphogenesis, organogene- tubules that mediate the segregation of chromosomes sis, and pattern formation are broadly shared among to daughter cells during mitosis and meiosis. Because vertebrates (KIMMEL 1989), most of these mutations will the centromeres of homologous chromosomes segre- identify the functions of genes whose homologues are gate from each other during the first meiotic division, involved inthe development of humansand other loci near their centromere will tend to segregate in mammals (CONCORDETand INGHAM1994; KAHN 1994). meiosis I (first division segregation),whereas crossovers To understand fully the processes disrupted by these between the centromere and more distal markers will mutations, precise phenotypic analysis afforded by opti- lead to segregation of markers in meiosis I1 (second cally clear, externally developing zebrafish embryos division segregation). The proportion of second divi- must be coupled to the molecular isolation of the gene sion segregation at alocus is a functionof the frequency eachmutation disrupts. Inthe candidate gene ap- of recombination between the locus and its centromere. proach for cloning a mutated locus, the genetic loca- Analysis of ordered tetrads-the four haploid products tion of a mutation may identify nearby candidates for of a single meiotic division-can reveal how often a the mutated gene (TALBOTet al. 1995). Alternatively, locus segregates at the first or second meiotic division, in the positional cloning approach, the identification and hence thedistance between the gene andits centro- of DNA polymorphisms tightly linked to the mutant mere (PERKINS1949, 1953). Because recombination locus can serve as entry points for a chromosome walk events are generally reciprocal, half-tetrads-one ofthe (WICKINGand WILLIAMSON1991; KINGSLEY et al. 1992). two products of the first meiotic division-give much These strategies are both facilitated by an extensive ge- the same information as anordered tetrad (STREI- netic linkage map. SINGER et al. 1986). Construction of the zebrafish linkage map has just In mice and humans,a rare error in the meiotic begun-before 1994, no two loci had been shown to arrest that follows meiosis I duringoogenesis can result be linked in the zebrafish genome. At that time, we in ovarian teratomas derived from a single meiotic half- reported a linkage map based on 425 genetic markers tetrad. Genetic analysis of these half-tetrads has led to distributed among 29 linkage groups (LGs) (POSTLE- the mapping of the centromeres of three mouse chro- THWNT et nl. 1994). Because the haploid chromosome mosomes (EPPIGand EICHER1983; ARTZT et nl. 1987; number is 25 (ENDO and INGALIS1968; DAGA et al. CHAKRAVARTIet al. 1989). [Other mouse centromeres 1996), the initial map had four more linkage groups have recently been localized by in situ hybridization than chromosomes, and hence contained at least four for a strainspecific centromere-associated satellite DNA gaps. A major goal of the current work was to fill these (CECIet al. 1994).] In several species of fish, half-tetrads can be grown into adult diploid individuals routinely Corresponding author: Stephen L. Johnson, Institute of Neurosci- ence, University of' Oregon, Eugene, OR 97403. in the laboratory (ALLENDOW et al. 1986; STREISINGER E-mail: [email protected] et al. 1986).Analysis of such animals has shown that the Gcnrtic-s 142: 1277- I288 (April, 1996) 1278 S. L. Johnson et nl. pigment pattern mutation rose is tightly linked to the Markers and nomenclature: Formal locus names for RAPD et al. 1990)markers in zebrafish consist of the centromere of LG I (JOHNSON et nl. 1995a), the CA- (WILLIAMS name of the 10 nucleotide long primer, followed by the ap- repeat variant ssrl2 to thecentromere ofLG XWI proximate size of the amplification product. Thus, the locus (KAUFFMAN et nl. 1995), and theembryonic lethal muta- lOC.9550, on LG 20, is amplified by primer GI0 (Operon Tech- tion no tail to the centromere of LG XIX (HAL.PEKNst nologies; Alameda, CA), andresults in a 950-bp amplification nl. 1993). product. The formal locus name is followed by a letter in Because half-tetrad zebrafish express mutant pheno- parentheses to indicate the parental origin of markers (A, AB; C, C32; D, DAR; S, SJD). Following this scheme, IOG.950 types appropriate to their genotypes, we have proposed was originally designated as lOG.95O(A), indicating that the an efficient method for the initial mapping of muta- AB-derived allele produced theamplification product. A slash tions using centromere-linkage analysis (JOHNSON st al. separating two letters indicates that the markeris codominant 1995a). In a family of half-tetrad zebrafish, some will in the two genetic backgrounds shown (Z.P., the locus pro- be homozygous for the mutant locus; in those homozy- duces slightly different sixd PCR products from the different alleles, and haploid individuals in the mapping panel have gotes, most centromeres will segregate randomly, indi- one or the other sized product, but never both and never cating that the mutation is unlinked to these centro- neither). To help present the datain Table 1, we have distin- meres. In contrast, for the centromere linked to the guished between the two alleles of codominant loci by indicat- mutation, most or all of the selected homozygous mu- ing the size of their amplification products in the marker tant half-tetrads will have inherited the centromere al- name. When a marker has been genotyped in both AB X DAR and C32 X SJD mapping panels, only the parental origin lele in coupling to the mutant locus. Although we have for the latter mapping panel, based on inbred strains C32 used variations on this strategy to map several muta- and SJD, is shown on the map. In some cases, the RAF’D tions, including the pigment pattern mutations rose (to marker has been converted to a sequence tagged site (STS, LC; I) and jaguar (to LC; 15; JOHNSON PL al. 1995a), see below, and Table 2). Inthese cases, the formal RAPD efficient application of this technique will require the locus name remains the same, butis appended by a *, indicat- identification of markers tightly linked to the centro- ing that the marker may usually be detected in most genetic backgrounds, and the parentalorigin of the markeris omitted meres for each of the linkage groups of the zebrafish (for instance, 10G. 950”). genetic map. Because consolidation of linkage groups has led to chang- In work presented here, we report the identification ing of some linkage group designations, and because the num- of DNA polymorphisms closely linked to each of the 25 ber of linkage groups, each with a mapped centromere,equals centromeres. This analysis plus the addition of 235 new the number of chromosomes, we have renamed the linkage groups with Arabic, rather than Roman,numerals. Thus, for- markers to the map resulted in the consolidation of the merly named LGs I-XVII, XX, XXII, XXIV and XXV (POST- number of linkage groups to the number of chromo- LEI‘HWAIT rt nl. 1994) have been renamed LG 1-17, 20,22, somes. The application of centromere-linkage analysis 24, and 25. In this study, former LGs XVIII and XXVIII were to the large collection of unmapped zebrafish muta- combined and renamed LG 18, LGs XIX and XXVI were tions promises to facilitate their genetic localization, combined and renamed LG 19, LGsXXI and XXIX were combined and renamed I,G 21, and LGs XXIII and XXVII and hence, themolecular genetic analysis of vertebrate were combined and renamed LC; 23. development. Mapping: Methods for PCR amplification of RAPDs, simple sequence repeat (SSR) polymorphisms, and sequence tagged sites (STSs), as well the genotyping and the construction of MATERIALS AND METHODS the map have been described previously (POSTI.I.’THM’AITrt al. Stocks: Mappingstrains have been described previously 1994). WDprimers were chosen for genotyping this map- (STREISINGERet al. HE TW JOHN SON et al. 1994, 1995a). Briefly, ping panel based on their amplification of markers well dis- C32 (S’I‘KEISINGEK et al. 1981) is a clonal, presumably homozy- tributed on the previous mapping panel (POSI‘l.l~.TlWAlTet gous, derivative of STKEISINGEK’Soutbred population AB. The nl. 1994), and included primers that amplified markers on DAR (Darjee1ing;JoHNsON et al. 1994) strain was isolated from LG XXTrI to XXIX or markers linked to spa, ros or /eo, to fish captured in the wild in India in 1987, and subsequently integrate the two maps.
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