Genotyping live fish larvae: Non-lethal and noninvasive DNA isolation from 3-5 day old hatchlings.

Genotyping fish larvae is a valuable technique for numerous fields of study. While methods for collecting DNA from early stage larvae have been published, a non-lethal, non-invasive genotyping protocol for hatchlings that is amenable to high-throughput approaches is desirable. Here, we describe a method to individually genotype live, free-swimming, early fish larvae by characterizing their environmental DNA (eDNA). We demonstrate the utility of the method by assigning parentage to a sample (n = 50) of 3-5-day-old sheepshead minnow (Cyprinodon variegatus) larvae hatchlings, with very high rates of genotyping success (98%) and survival (92%) using mitochondrial and microsatellite DNA data. This method could be easily adapted to characterize early fish larvae from other model and non-model fish species, such as Danio rerio (zebrafish) and Medaka medaka.

Low mitochondrial DNA variation among American alligators and a novel non-coding region in crocodilians.

We analyzed 1317-1823 base pairs (bp) of mitochondrial DNA sequence beginning in the 5' end of cytochrome b (cyt b) and ending in the central domain of the control region for 25 American alligators (Alligator mississippiensis) and compared these to a homologous sequence from a Chinese alligator (A. sinensis). Both species share a non-coding spacer between cyt b and tRNA(Thr). Chinese alligator cyt b differs from that of the American alligator by 17.5% at the nucleotide level and 13.8% for inferred amino acids, which is consistent with their presumed ancient divergence. Only two cyt b haplotypes were detected among the 25 American alligators (693-1199 bp surveyed), with one haplotype shared among 24 individuals. One alligator from Mississippi differed from all other alligators by a single silent substitution. The control region contained only slightly more variation among the 25 American alligators, with two variable positions (624 bp surveyed), yielding three haplotypes with 22, two, and one individuals in each of these groups. Previous genetic studies examining allozymes and the proportion of variable microsatellite DNA loci also found low levels of genetic diversity in American alligators. However, in contrast with allozymes, microsatellites, and morphology, the mtDNA data shows no evidence of differentiation among populations from the extremes of the species range. These results suggest that American alligators underwent a severe population bottleneck in the late Pleistocene, resulting in nearly homogenous mtDNA among all American alligators today.