The evolutionary causes and consequences of sexual vs. asexual reproduction remain among the deepest mysteries in evolutionary genetics. Although the mechanisms responsible for the suppression of meiosis, which involve a loss of homologous recombination and segregation, appear to be widely available phylogenetically, the molecular basis for the conversion of meiotic haploid gamete production to mitotic diploid egg production remains unknown. The project will address this issue using the microcrustacean Daphnia pulex to elucidate the molecular and cell biological mechanisms underlying the transition to parthenogenetic egg production. The principal investigator's previous research has identified a key gene whose modification appears to be necessary to convert meiosis to mitosis, and as well as identifying chromosomal regions containing two to three others. To perform functional studies of these genes, it will be necessary to develop stable transgenic lines of D. pulex containing reporter gene constructs of the putative meiosis suppressor. If successful, the project will lead to an enhanced understanding of the mechanisms underlying the molecular basis of meiosis and meiosis suppression, which would in turn have considerable implications for applied breeding programs.
Broader Impact. The establishment of reliable transgenic methods will open up the Daphnia system to inquiry of a wide array of additional questions in molecular, cellular, and developmental biology, thereby greatly broadening the investigative capacity of this model system beyond the already substantial research community embodied in the Daphnia Genomics Consortium. Educational and training efforts will be focused on the career development of a postdoctoral researcher as well as on exposing undergraduate researchers to the full spectrum of scientific inquiry, from the development and implementation of new techniques to critical aspects of research design, hypothesis testing, and publication. Indiana University supports a number of summer programs for high-school and undergraduate minority students, and the principal investigator will continue to sponsor such students.
The goal of this project was to explore methods for developing transgenic Daphnia, i.e., methods for integrating novel gene variants that would be useful for a variety of downstream analyses associated with molecular, cellular, and developmental biology, as well as ecology and evolution. The study organism, Daphnia pulex, is a pond-dwelling crustacean that has become a major model system for studies in ecological genomics, population genetics, and evolution. The project was exploratory nature, with various phases involving attempts to utilize state-of-the-art technology to accomplish the primary goal of gene integration. We explored a number of vectors constructed out of mobile genetic elements, attempting to introduce these by injection and electroporation. Unfortunately, although this work led to the refinement of the latter approaches for work with Daphnia, the introduced vectors never established either in the genomes of the host animals or as simple extra-chromosomal plasmids. Towards the end of the project, an attempt was made to utilize another newly emerging system -- zinc-finger endonucleases, which have the capacity to cut DNA at pre-specified target sequences. Here again, despite our ability to develop this technology, it did not accomplish our original goals. Taken together, although this work led to the development of new technologies that could have other uses in this important model system, methods for the stable integration of exogenous DNA remain to be developed. As there is substantial variation among species in the receptivity to DNA introduction, it is possible that Daphnia is on the more recalcitrant end of the spectrum.
