CUR - The Council on Undergraduate Research

Sequencing of the Sf1 Gene in Apalone spinifera Softshell Turtles

Katherine Flatt, Thea Gessler, and Dr. Nicole Valenzuela, Department of Ecology, Evolutional, and Organismal Biology, 239 Bessey Hall, Ames IA 50011

Turtles exhibit high variability in sex determination, with many species exhibiting environmental sex determination (ESD), more specifically temperature-dependent sex determination (TSD). Under ESD, the sexual fate of developing embryos is controlled by external factors rather than by sex chromosomes. An exception is the softshell turtle Apalone spinifera, a species which exhibits genotypic sex determination (GSD) with ZZ/ZW sex chromosomes. The Steroidogenic factor 1, also known as the Sf1 gene, functions to regulate the transcription of several other genes contributing to the individual’s reproduction and male sex differentiation. Our lab discovered that Sf1 moved to the ZW chromosomes from its ancestral location in a pair of autosomes, but it is unclear if the Sf1 copy on the W has mutated during the divergence of this chromosome from the Z over evolutionary time. Here we test the hypothesis that the Sf1 sequence differs in males and females, and that mutations in the W-Sf1 may impair its function. Polymerase chain reactions (PCR) with varying conditions were run to amplify the segment of DNA encompassing the Sf1 gene from DNA template, extracted from tissue of males and females. PCR reactions varied in their annealing temperatures, extension times, mastermix reagent amounts (MgCl2 or template DNA), and primer sets. Amplified segments (amplicons) were separated via agarose gel electrophoresis to identify clean products for Sanger sequencing. Results will be discussed and will provide a greater understanding of the contributing factors to the evolution of GSD in this species.

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