Evolution and Meiotic Maps by Massively Parallel DNA Sequencing

Teleost fish provide useful models for investigating vertebrate development, physiology, toxicology, evolution, and disease but possess anciently duplicated genomes that complicates genome connectivity. Genomic information for a lineage of ray-fin fish that diverged from teleosts before the teleost genome duplication (TGD) would provide an outgroup for inferring mechanisms of evolution after whole-genome duplication. To determine whether spotted gar (Lepisosteus oculatus) provides an outgroup for the TGD, we used massively parallel DNA sequencing (RAD-tags) to develop a meiotic map. We genotyped F1 offspring of two individual gar fish collected directly from nature (Allyse Ferrara, Nichols State University) and utilized polymorphisms existing in these two wild individuals, thus shortcutting the usual F2 map cross design. To analyze millions of RAD-tag sequences, we developed novel software (Stacks) that automatically recognizes polymorphisms and calls genotypes. Using Stacks, we constructed a meiotic map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Comparative genomics showed that the gar lineage diverged from the teleost lineage before the TGD and that gar genome organization is more similar to that of humans than teleosts. Results show that spotted gar provides a link between teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of this rapid, thrifty F1 dense mapping strategy to species with no prior genome information can facilitate comparative genomics and help order the numerous contigs arising from next generation genome sequencing.