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Gene regulation is tightly regulated to produce individual phenotypes appropriate to a animal’s placement in time and space. How specific genetic and environmental factors shape gene regulation has been extensively studied in laboratory model species. However, far less is known about sources of variance in gene regulation in the wild, in populations where natural selection operates. My dissertation research aimed to identify factors that influence individual variation in gene regulation by evaluating transcriptome-wide gene expression levels within and across populations. In my first chapter, I evaluated gene expression changes in captive Swainson’s thrushes (Catharus ustulatus) originating from two populations as they transitioned from the non-migratory to migratory condition. Birds exhibited significant seasonal differences in the expression of genes involved in cellular development in the brain, providing support for seasonal neural plasticity in migratory birds. In my second chapter, I focused on a single population, the North American gray wolves (Canis lupus) of Yellowstone National Park, to evaluate the relative impacts of intrinsic (age and sex) and environmental variables (social status and presence of sarcoptic mange) on gene expression variation in blood. I found that, unexpectedly, animal age, but not sex, social status, or sarcoptic mange infestation, impacted gene expression, suggesting that age may have pervasive, evolutionarily conserved effects on gene expression variation in natural populations of mammals. Finally, in my third chapter, I adopted an in vitro experimental approach to study how gene regulation is impacted by genotype at the CBD103 gene, which encodes a protein with antimicrobial properties, exists in two forms (alleles) in North American gray wolves, and is associated with lifetime reproductive success. I established keratinocyte lines from 24 gray wolves and utilized CRISPR/Cas9 to generate one heterozygous and homozygous line. To test whether the fitness effects of CBD103 genotype are explained by their roles in immune defense, I challenged the cells with synthetic antigens and live canine distemper virus. No effect of CBD103 genotype was detected, but immune challenge altered expression of thousands of genes. This work demonstrated the ability to establish a population panel of cell lines from a wild mammal, a potentially powerful method for studying gene regulation in natural populations.

BackgroundRNA-Seq analyses can benefit from performing a genome-guided and de novo assembly, in particular for species where the reference genome or the annotation is incomplete. However, tools for integrating assembled transcriptome with reference annotation are lacking.FindingsNecklace is a software pipeline that runs genome-guided and de novo assembly and combines the resulting transcriptomes with reference genome annotations. Necklace constructs a compact but comprehensive superTranscriptome out of the assembled and reference data. Reads are subsequently aligned and counted in preparation for differential expression testing.ConclusionsNecklace allows a comprehensive transcriptome to be built from a combination of assembled and annotated transcripts which results in a more comprehensive transcriptome for the majority of organisms. In addition RNA-seq data is mapped back to this newly created superTranscript reference to enable differential expression testing with standard methods. Necklace is available from https://github.com/Oshlack/necklace/wiki under GPL 3.0.

Non-model plants i.e., the species which have one or all of the characters such as long life cycle, difficulty to grow in the laboratory or poor fecundity, have been schemed out of sequencing projects earlier, due to high running cost of Sanger sequencing. Consequently, the information about their genomics and key biological processes are inadequate. However, the advent of fast and cost effective next generation sequencing (NGS) platforms in the recent past has enabled the unearthing of certain characteristic gene structures unique to these species. It has also aided in gaining insight about mechanisms underlying processes of gene expression and secondary metabolism as well as facilitated development of genomic resources for diversity characterization, evolutionary analysis and marker assisted breeding even without prior availability of genomic sequence information. In this review we explore how different Next Gen Sequencing platforms, as well as recent advances in NGS based high throughput genotyping technologies are rewarding efforts on de-novo whole genome/transcriptome sequencing, development of genome wide sequence based markers resources for improvement of non-model crops that are less costly than phenotyping.

Understanding the genetic basis of adaptation is one of the central questions of evolutionary genetics. In the face of selective pressure, individuals with adaptive phenotypes survive and pass on their beneficial alleles that conferred this fitness advantage. Over sufficient time scales and under strong selection, differences accumulate between taxa in the form of physical and/or behavioral traits that have been shaped differently. By looking at the transcriptomes of related taxa we can identify sequence and regulatory differences that represent molecular signatures of these different selection regimes. When we study these differences between taxa that have evolved under opposing selective forces we can identify the genetic underpinnings of adaptation to those conditions. Species from the Heteromyidae form an exemplary system for studying the genetics of adaptation in a comparative framework. This new world family of rodents is comprised of three subfamilies (the Dipodomyinae, Perognathinae, and Heteromyinae) that range from the temperate/arid habitat of the southwestern United States down through Mexico and the mesic habitat of Central America into northern South America. Members of the Dipodomyinae and Perognathinae largely populate desert habitat and have developed numerous behavioral and physiological adaptations to combat the issue of water loss. This response to selection has manifested itself in extraordinarily efficient kidney function that forms a textbook example of adaptation. However, despite knowledge of their kidney physiology, the genetic basis of this ability to retain water during waste production is unknown. In contrast to the other Heteromyids, members of the Heteromyinae largely inhabit wet tropical and subtropical forest without the same need for efficient water use. This, coupled with a near simultaneous radiation into the three subfamilies provides a controlled framework to identify differences between species from dry and wet habitat as well as between temperate and tropical species. In this dissertation I utilize 2nd generation sequencing technologies to study these differences by characterizing the genes expressed in biologically interesting tissues of three Heteromyid species. In sequencing the kidney and spleen transcriptomes of adult Dipodomys spectabilis (banner-tailed kangaroo rat), Chaetodipus baileyi (Bailey's pocket mouse), and Heteromys desmarestianus (forest spiny pocket mouse), I have sampled a representative of the Dipodomyinae, Perognathinae, and Heteromyinae, respectively. In chapter 1, I took a first pass at characterizing the kidney transcriptome of D. spectabilis. I identified a priori candidate genes important to kidney function from genetic model rodent species in an effort to identify potential drivers of D. spectabilis' recorded ability to retain water during the production of highly concentrated waste. I hypothesized that those candidates identified in D. spectabilis coupled with genes upregulated in this adapted organ would contain signatures of selection and have drastically divergent patterns of expression as hallmarks of their role in adaptation to a desert lifestyle. In chapter 2, I sought to identify if these or other genes exhibited clear patterns of differential expression and differentiation from the mesic H. desmarestianus that were driven by the different selective regimes on desert and mesic species. I also compared both species to C. baileyi, hypothesizing that any consistent differences seen in both C. baileyi and D. spectabilis relative to H. desmarestianus would be attributable to adaptation to arid habitat. In this chapter I identified a large number of genes in each species (roughly 11,000 per species) and filtered this set down to a subset of genes that either due to expression patterns or positive selection have evidence to be possible drivers of efficient osmoregulation in desert Heteromyids. For chapter 3, I focused on the genes expressed in spleen as an important tissue for immune response. Again I utilized the comparative framework of the Heteromyid family history in comparing the tropical H. desmarestianus to the temperate D. spectabilis and C. baileyi. My hypothesis was that greater selective pressure on immune response from increased pathogen diversity and burden in the tropics would fashion sequence evolution and different levels of gene regulation for immunity genes in H. desmarestianus. As in chapter 2, I generated a large amount of sequence data while winnowing down to a small number of solid candidate genes displaying positive selection and differential gene expression between the tropical and temperate Heteromyids. Due to the consistent patterns seen in the comparisons of D. spectabilis and C. baileyi against H. desmarestianus I predict that a subset of the genes identified here from kidney and spleen RNA-seq will prove to play a significant adaptive role in other vertebrates. Namely, the genes identified in chapters 1 and 2 will play roles in adaptation to water scarcity and those identified in chapter 3 will be consistently affected by the immune challenge of increased pathogen exposure in tropical species.