Genotyping individual amphimedon embryos, larvae, and adults

INTRODUCTIONThe distribution of Amphimedon queenslandica is patchy on coral reefs in the Great Barrier Reef, with small, localized populations detected in shallow, still water reef-flat environments. A. queenslandica is a spermcast spawner, in which fertilization occurs internally. Sperm presumably originate from neighboring reproductive individuals within the population. The ability to genotype individual embryos within a single brood chamber has the potential to shed light on the fertilization biology and generation/maintenance of genetic diversity in this sessile invertebrate. Here, we describe a protocol for rapidly genotyping individuals using polymorphic microsatellite loci. The loci are amplified by PCR using a pair of primers specifically designed for the region of interest with a fluorescent dye attached to the 5'-end to enable easy detection of the amplified product. An advantage of this procedure is that fluorescently labeled PCR products can be combined (i.e., multiplexed) to reduce time and cost when using the genotyping machine. The dye label and size of the product must be taken into consideration when multiplexing. For example, three differently labeled PCR products can be multiplexed, or PCR products with the same label can be multiplexed as long as the allelic size ranges do not overlap. The amount of each cleaned, labeled PCR product added to the multiplex must be optimized depending on the dye and the PCR efficiency.