Sex is determined by genetic constitution (GSD) in most animals, such as by the sex chromosome pairs XX or XY in humans, or ZZ or ZW in birds. In certain other animals, however, sex is determined by environmental temperature (TSD). Among turtle species, however, examples of both types of sex determination can be found. It remains unclear, however, how and why different sex determining mechanisms exist. This collaborative project will help fill this gap by comparing the chromosomes and sex-related genes of turtles using XX/XY, ZZ/ZW, or TSD mechanisms for sex determination. Particularly, this research will test whether (1) all sex chromosomes in turtles derive from a common ancestral pair of non-sex chromosomes (or "autosomes"), and whether (2) the molecular evolution of genes located on turtle sex chromosomes differs from that of the same genes in TSD turtles, in which there are no sex chromosomes- i.e., whether the sequences of genes located on sex chromosomes change differently over evolutionary time than do the sequences of the same genes when they are located on autosomes.
This project will integrate undergraduate discovery-based learning for undergraduates into the field and lab modules of this project and will broaden the participation of underrepresented groups in biology by providing direct training opportunities for students and their mentors, by engaging women and minorities through outreach activities, and by providing further opportunities for both the investigators to serve as role models for minorities.
Reptiles such as turtles possess several types of mechanisms that determine the gender of offspring. Genetic sex determination (GSD) consists of an chromosomal factors (genes) that determine sex, either in an X/Y chromosome system (such as is found in humans) or a W/Z system, in which females have two types of sex chromosomes (W & Z) and males have one (Z, unlike humans). Finally, in many turtle species gender is determined not by genetics but by the environment, specifically the temperature at which embryos developed. Such â€˜temperature-dependent sex determinationâ€™ (TSD) is quite common in turtles and other reptiles. The goals of this project were to understand how many times GSD has given rise to TSD and vice versa during the evolutionary history of turtles, and to understand where the sex chromosomes come from in those species that have GSD. Do these sex chromosomes come from particular sets of autosomes in different turtle species? These questions are important because they will help us understand how quickly sex-determining mechanisms can change during evolution. This in turn is important because many sex determination abnormalities in humans involve sex chromosomes, and an understanding of how these chromosomes evolve can help us understand the frequency and nature of genetic aberrations in humans. We have so far identified several genes in turtles that are known to determine gender in mammals, and these genes are important for understanding the nature of sex determination in turtles. Many of these genes are actually on non-sex determining chromosomes (autosomes), despite their role in sex determination. Additionally we have found several genes that we know are located on sex chromosomes in turtles. Both of these sets of genes are proving useful for determining the origin of sex chromosomes in turtles and the number of switches between TSD and GSD. The precise number of switches is an index of how rapidly sex chromosomes change and will be determined in the final months of the grant. This research trained several undergraduates, graduate students and postdoctoral fellows in basic molecular biology research that will allow them to explore careers in many fields, including biotechnology, zoology, genetics and evolutionary biology. We also trained high school teachers and high school students as part of this project. We helped a high school teacher from Buckingham, Brown and Nichols School in Cambridge, MA develop a genetics curriculum using principles employed as part of this project. The high school student and teacher participated in many aspects of the research, including isolation of RNA and DNA from turtle cells, conducting the polymerase chain reaction (PCR) and cloning DNA in bacterial cells. Our research helped a Puerto Rican undergraduate receive crucial research experience that allowed him to apply and gain entry to graduate school at the University of California Berkeley, thereby diversifying the American scientific research work force. Finally, the research provided training for a postdoctoral fellow who is now a grants administrator for the NIH. Thus the research has had an impact beyond the individual laboratory and into the classroom and general society.