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Methods for Karyotyping and for Localization of Developmentally Relevant Genes on the Chromosomes of the Purple Sea Urchin, Strongylocentrotus purpuratus

Celeste C. Eno, Stefanie A. Böttger^* and Charles W. Walker

Molecular, Cellular and Biomedical Sciences, University of New Hampshire, 46 College Road, Durham, New Hampshire 03824

To whom correspondence should be addressed. E-mail: cwwalker{at}unh.edu

The purple sea urchin, Strongylocentrotus purpuratus, is the only non-chordate deuterostome model with a fully sequenced genome. Chromosomal localization of individual genes and resulting gene maps are unavailable for this or for any sea urchin. As a result, the purple sea urchin genome has not been mapped onto specific chromosomes and remains inaccessible to genome-wide approaches addressing questions that require positional information for particular genes. Here we describe the first successful methods for karyotyping and localizing specific gene loci on chromosomes of Strongylocentrotus purpuratus and those of the phylogenetically related Strongylocentrotus droebachiensis. Both species have 42 chromosomes in their diploid genomes (n = 21). There are 2 large, 8 medium, and 10 small pairs, plus one putative sex pair. In both species, bindin genes were localized to 2 pair of homologous chromosomes by fluorescent in situ hybridization. Fluorescently labeled bacterial artificial chromosome clones generated from S. purpuratus for the functionally related genes brachyury, foxa, and foxb were localized to different chromosomes. Our protocols provide previously unavailable tools for developing a gene map for the purple sea urchin genome.

Abbreviations: BAC, bacterial artificial chromosomes • BAC-FISH, fluorescently labeled bacterial artificial chromosome in situ hybridization • DAPI, 4'-6-diamidino-2-phenylindole • FISH, fluorescent in situ hybridization • FSW, filtered seawater • PABA, p-aminobenzoic acid • SSC buffer, 3.5% NaCl, 0.89% sodium citrate • PI, propidium iodide