The overall goal of my dissertation research was to identify the genetic components underlying the evolutionary divergence of complex, sexually dimorphic phenotypes. To address these questions, I have employed large scale sequencing, assessed gene expression, and identified population genetic divergence in a non-traditional model system, the dark-eyed junco (Junco hyemalis). These projects have combined field and aviary projects with laboratory and computational analysis to identify: sex differences in gene expression, transcriptional response to experimental elevation of hormone, and individual variation in gene expression. The knowledge, tools, and skills that I have learned provide a solid foundation on which to build a research program exploring the evolutionary divergence of gene expression mechanisms giving rise to sexually dimorphic phenotypes and hormonal responses.

Transcriptome sequencing: Creating genomic tools for a non-model system

Until recently, the only way to address genome level questions was to work in a model species. However, transcriptome sequencing (sequencing all expressed RNA) is rapidly opening the possibility of genome level studies in non-model systems. Working with the Center for Genomics and Bioinformatics (CGB) at Indiana University, I led a large research group as we sequenced 23,000 putative genes from the junco, covering approximately 90% of the expected genes (Peterson et al.,2012 BMC Genomics). From these sequence data, I designed a custom microarray to allow assessment of gene expression in the junco.

Sex differences and the role of testosterone: using microarrays to understand sexual conflict

Males and females share nearly identical genomes, yet often vary dramatically in appearance, physiology, and behavior. Differences in gene expression, often mediated by steroid hormones, account for much of this sexual dimorphism. Many studies have demonstrated the effect of elevated testosterone on physiology, behavior, and fitness in both males and females, including finding that species and sexes vary substantially in which traits are responsive to hormonal control. However, studies of gene expression in response to testosterone have not addressed these sex differences. To address this gap, I implanted male and female juncos with testosterone and identified the genes up- and down- regulated by the hormone. Additionally, I identified the genes that are differentially expressed between control- males and females.

The most interesting finding in this study was that testosterone masculinizes the gene expression pattern of females (that is, genes that are expressed higher in control males than females are turned up by testosterone in females), while testosterone appears to feminize gene expression in males (that is, genes that are higher in control males than females are turned down by testosterone in males; Peterson et al. 2013, PLoS One; Peterson et al., Online ahead of print, Journal of Experimental Biology). This appears to be occurring primarily because different genes are responding to testosterone in each sex: fewer than ten percent of genes regulated by testosterone are regulated in both sexes. This difference in transcriptional response to testosterone suggests that males and females may be interpreting a similar hormonal signal in a very different way and opens the door to questions about how this sex difference emerged and was modulated over evolutionary time.

Investigating individual variation: RNA-seq reveals the role of natural variation

Individual variation in testosterone-phenotype (the ability to elevate testosterone in response to a standardized hormonal challenge) is also related to fitness and to many of the same phenotypes as experimental changes in testosterone. However, to date, no studies have investigated the relationship between individual variation in testosterone-phenotype and gene expression in a natural population, an essential step in understanding how testosterone-sensitive sexually dimorphic phenotypes are mediated in the wild. This is especially important given the unexpected finding that experimentally elevated testosterone appears to feminize male gene expression.

Therefore, I sought to identify correlations between testosterone-phenotype and gene expression in the brain and peripheral tissue of dark-eyed juncos using RNA-seq. RNA collected from individuals with quantified ability to produce testosterone was sequenced at the Beijing Genomics Institute to estimate the number of copies of each gene expressed by each individual. I have identified many genes that are related to the ability to produce testosterone in each sex. Intriguingly, while many of these genes are also responsive to experimental elevation of testosterone, very few genes are related to ability to produce testosterone in both males and females (Peterson et al., In Prep).

Genetic Variation in relation to migratory behavior

The transcriptome project has also led to the development of junco-specific primers for several candidate gene projects. For example, I led a project to assess some of the potential genetic differences underlying the variation in migratory phenotypes across the genus Junco. In collaboration with Dr. Borja Milá, we genotyped individuals from 15 populations for two genes that were identified as related to migratory behavior in other species. We found that length variation in microsatellite repeats in Clock and ADCYAP1 do not explain differences in population migratory status across the genus Junco, though each marginally predicts migratory phenotype within smaller clusters of the genus (Peterson et al., 2013, F1000Research). This project, combined with others from my colleagues in the lab, demonstrate the value of large-scale sequencing for expanding the ability to do genetic research in non-model systems.

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