High-throughput DNA extraction for PCRbased genotyping of single Penaeus monodon embryos and nauplii

A simple technique is described for isolating DNA from individual embryos or nauplii of the Black Tiger shrimp, Penaeus monodon. The protocol used a commercial prepGEM(TM) insect DNA extraction kit in combination with water bath ultrasonic disruption. DNA quality and yields were sufficient for multiplex PCR discrimination of up to six DNA microsatellites in a single test. The utility of the method was demonstrated in defining the parentage of 1279 embryos (7 h old) spawned from 21 females and 684 newly-hatched N1 to N2 nauplii spawned from 12 females each inseminated artificially with the spermatophores of two different males. Overall, parentage assignments based on the six microsatellite markers were deduced to be 89.5 ± 6.7% and 93.0 ± 4.4% effective using DNA isolated from individual embryos and nauplii, respectively. The technique is simple, cost-effective and should provide a robust means of isolating DNA from individual embryos and nauplii of aquatic invertebrates suitable for PCR-based genotyping. [PUBLICATION ABSTRACT]