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CHARACTERIZATION OF THE PITX1 GENE OF GOODEID FISHES**

Abstract

Homoplastic, regulatory defects in the nuclear PitX1 gene have been implicated in pelvic-appendage loss in manatees (Mammalia) and stickleback fishes (Teleostei), and affect this complex, multi-organ structure in yet other vertebrates. Here we continue investigating the mechanism of pelvic-fin loss in an independent vertebrate group by generating PitX1 DNA sequences for representative goodeid fishes of the sister-clades Empetrichthyinae, which lack pelvic fins, and Goodeinae, which have these fins. To this end, bioinformatics approaches are being used to generate primers for PCR in and around this four-exon gene using the publicly available genomic scaffold of Fundulus heteroclitus (Fundulidae). PitX1 comprises more than 12 kb in this taxon, and the pel regulatory region, based on homology assessment with Oryzias latipes (Adrianichthyidae), is about 95 kb upstream of exon 1. Standard molecular protocols are being used to generate DNA sequences however, as amplicons will be of large size relative to what can generally be Sanger sequenced, and as we have previously discovered evidence of length variation within introns, molecular cloning using competent E. coli will be used to improve sequence reads. Sequences will be verified as PitX1 via alignment to teleost orthologs that are publicly available. We will here note that early attempts at primer design were frustrated by the fact that exon 1 of the Fundulus reference sequence was found to have been misidentified in the reference assembly. We hope to take the first steps in understanding the molecular mechanisms that explain a novel, vertebrate loss of a complex anatomical feature.

Acknowledgements

Funding was provided by the University of North Georgia Department of Biology.

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