SHORT COM M UNICATION Centromere-Linked Microsatellite Markers for Linkage Groups 3, 4, 6, 7, 13, and 20 of Zebrafish (Danio rerio) Manzoor-Ali P. K. Mohideen,* Jessica L. Moore,* and Keith C. Cheng * , ² ,1 * The Jake Gittlen Cancer Research Institute, Department of Pathology, and ² Department of Biochemistry and Molecular Biology, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033 Received February 14, 2000; accepted April 18, 2000 A large number of interesting mutations affecting development and organogenesis have been identified through genetic screens in zebrafish. Mapping of these mutations to a chromosomal region can be rapidly accomplished using half-tetrad analysis. However, knowledge of centromere-linked markers on every chromosome is essential to this mapping method. Cen- tromeres on all 25 linkage groups have been mapped on the RAPD zebrafish genetic map. However, species specificity and the lack of codominance make RAPD markers less practical for mapping than microsatel- lite-based markers. On the microsatellite-based ge- netic map, centromere-linked markers have been identified for 19 linkage groups. No direct evidence has been published linking microsatellite markers to the centromeres of linkage groups 3, 4, 6, 7, 13, and 20. Therefore, we compared the microsatellite-based ge- netic map with the RAPD map to identify markers most likely linked to the centromeres of these 6 link- age groups. These candidate markers were tested for potential centromere linkage using four panels of half- tetrad embryos derived by early-pressure treatment of eggs from four different female zebrafish. We have identified microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 to within 1.7 cM of their centromeres. These markers will greatly facilitate the rapid map- ping of mutations in zebrafish by half-tetrad analysis. © 2000 Academic Press Genetic screens have identified over 600 genes in zebrafish that are critical in early development and organogenesis (2, 6, 7). To facilitate the mapping and positional cloning of these genes, zebrafish genetic re- sources such as genetic maps (3, 10, 13, 17), radiation hybrid maps (4, 8), and YAC (20), BAC (Genome Sys- tems, Inc.), and PAC libraries (1) are available. Map- ping of the mutant genes is typically accomplished by bulked segregant analysis as the first step in positional cloning. In bulked segregant analysis, DNA pools of mutant and wildtype embryos are genotyped with markers representing each of the 25 linkage groups. In one approach, genome scanning, the pooled DNA is genotyped with 150 to 230 markers that span the ge- nome, with each marker separated by 10 to 15 cM. In the second approach, half-tetrad mapping, the DNA pools obtained from gynogenetic half-tetrad mutant and wildtype embryos are genotyped with markers linked to each of the 25 centromeres of zebrafish (9, 10, 15). The distance of the mutant locus from its centro- mere is estimated from the frequency of mutant half- tetrads (9, 10, 12, 18). In both mapping methods, po- tential linkages are confirmed by genotyping individual mutant embryos using progressively closer markers. However, for polymorphic markers, initial mapping of one mutant locus by genome scanning re- quires 300 – 460 PCRs while the half-tetrad procedure requires only 50 PCRs. Thus, when a mutant pheno- type can be reliably scored in half-tetrad embryos, half- tetrad mapping is an efficient way to place a zebrafish mutation on the genetic map. Our laboratory has identified a number of mutations that affect genomic instability and cell differentiation in two screens of gynogenetic half-tetrads in zebrafish, (J. Moore, G. Tsao-Wu, K. Cheng, et al., to be published elsewhere). Knowledge of markers linked to each of the 25 centromeres is essential to mapping these muta- tions using half-tetrads (12). On the RAPD map, which is a female-biased, haploid meiotic map, centromere- linked markers for all 25 linkage groups (LG) are known (10). However, on the microsatellite map, which is a sex-averaged, diploid meiotic map, markers tightly linked to centromeres have not been identified for 6 linkage groups: LG 3, 4, 6, 7, 13, and 20 (13, 17). In most instances, including ours, the use of RAPD mark- ers is not possible because mutations were scored in 1 To whom correspondence should be addressed at The Jake Git- tlen Cancer Research Institute, H059, The Pennsylvania State Uni- versity College of Medicine, 500 University Drive, Hershey, PA 17033. Telephone: (717) 531-5635. Fax: (717) 531-5634. E-mail: kcheng@psu.edu. All articles available online at http://www.idealibrary.com on Genomics 67, 102–106 (2000) doi:10.1006/geno.2000.6233 102 0888-7543/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.