<p>Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.</p><p>In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.</p> / Dissertation

No description available.

Hypoxia regulates many hundreds of genes with important roles in ischemic and neoplastic disorders. Central to this response are the hypoxia inducible transcription factors (HIF). This work aimed to better understand the direct transcriptional response to HIF by mapping HIF-binding sites across the genome using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). ChIP-seq for HIF in MCF-7 breast cancer cells under hypoxic conditions revealed more than 400 high-stringency HIF-binding sites genome-wide. Each member of the HIF heterodimer was present with near complete concordance. Binding of the two principle isoforms revealed a high degree of overlap with no differences in the DNA-binding motif. HIF-binding was associated with upregulation, but not downregulation of genes indicating that it functions as a transcriptional activator but not as a repressor. HIF-binding occurred preferentially at gene promoters, but was also present at promoter-distant sites, which were also associated with gene regulation, implicating long-range interactions in hypoxic gene activation. HIF-binding was associated with markers of open chromatin and active enhancers that were present in normoxia, indicating that HIF-binding sites are already “prepared” to bind HIF before the hypoxic stimulus. Analysis of normoxic and hypoxic RNA pol2 and H3K4me3 signals revealed distinctive hypoxia-inducible changes unique to HIF-binding genes. Comparable numbers of HIF-binding sites were observed in a second cell line (von Hippel-Lindau defective 786-O renal cancer cells) as in MCF-7 breast cancer cells, although approximately 65% were unique to 786-O cells. These unique sites were more frequently promoter-distant. Correlation with expression analyses from renal tumours indicated that many HIF-binding genes were upregulated in renal cancer. One such RCC unique promoter-distant HIF-binding site was identified at an intergenic locus on chromosome 11q13.3 that has been associated with renal cancer in Genome-Wide Association Studies. The HIF-binding site was in high linkage disequilibrium with the disease associated SNP and had the epigenetic hallmarks of an enhancer. Analysis of pan-genomic expression analyses identified the cell-cycle regulator cyclin D1 as highly HIF-regulated, and a physical association between the HIF-binding site and the CCND1 promoter could be determined. Furthermore, in a renal cancer cell line heterozygous at this locus, the RCC-protective allele disrupted HIF-binding leading to an allelic imbalance in cyclin D1 expression.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries. Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

La symbiose mycorhizienne est une interaction mutualiste formée entre les racines des plantes terrestres et des champignons du sol. Les changements morphoanatomiques associés au développement de cette symbiose sont accompagnés de modifications dans la régulation de l'expression génique. L'étude des profils transcriptomiques est donc fondamentale afin de caractériser les mécanismes moléculaires gouvernant la symbiose mycorhizienne. Le développement récent des approches de transcriptomique à haut débit offre de nouvelles perspectives pour la compréhension de ces mécanismes. Le travail entrepris dans le cadre de ce projet de thèse visait à caractériser in silico le transcriptome symbiotique du champignon ectomycorhizien Tuber melanosporum et du champignon endomycorhizien Glomus intraradices. Il s'agissait de mettre en place les outils et les protocoles bioinformatiques permettant l'exploitation des données transcriptomiques issues des nouvelles technologies de séquençage, afin de caractériser les transcrits exprimés par les symbiotes et d'identifier les gènes régulés au cours de la symbiose. Ce travail original a permis de souligner l'existence de traits communs aux profils d'expression des champignons mycorhiziens. De plus, la caractérisation du transcriptome de G. intraradices a permis d'établir le premier répertoire de gènes à l'échelle du génome pour un champignon endomycorhizien. Cette étude de génomique contribue à l'amélioration des connaissances sur les processus moléculaires qui sous-tendent la symbiose mycorhizienne et constitue une ressource unique pour de futures recherches sur les réseaux de gènes contrôlant la symbiose / Mycorrhizal symbiosis is a mutualistic interaction involving roots of terrestrial plants and soil fungi. Morphological changes associated with the development of this symbiosis are accompanied by changes in gene expression. The study of transcriptomic profiles is thus essential to characterize the molecular mechanisms that govern the mycorrhizal symbiosis. The recent development of high-throughput transcriptomic approaches provides new insights for the understanding of these mechanisms. The work undertaken during this thesis aimed to characterize in silico the transcriptome of the ectomycorrhizal fungus Tuber melanosporum and the endomycorrhizal fungus Glomus intraradices. In order to characterize transcripts expressed by the symbionts and to identify genes regulated during symbiosis, bioinformatic tools and protocols were implemented to process transcriptomic data derived from new sequencing technologies. This work has allowed to highlight common features in the expression profiles of mycorrhizal fungi. In addition, characterization of the G. intraradices transcriptome has allowed to establish the first genome-wide repertoire of genes for an endomycorrhizal fungus. The study helps to improve knowledge about the molecular processes underlying the mycorrhizal symbiosis and provides a unique resource for future research on the gene networks controlling symbiosis

Master of Science / Department of Plant Pathology / John P. Fellers / Allan K. Fritz / Leaf rust is caused by Puccinia triticina and is one of the most widespread diseases of wheat worldwide. Breeding for resistance is one of the most effective methods of control. Lr16 is a leaf rust resistance gene that provides partial resistance at the seedling stage. One objective of this study was to use RNA-seq and in silico subtraction to develop new resistance gene analog (RGA) markers linked to Lr16. RNA was isolated from the susceptible wheat cultivar Thatcher (Tc) and the resistant Thatcher isolines TcLr10, TcLr16, and TcLr21. Using in silico subtraction, Tc isoline ESTs that did not align to the Tc reference were assembled into contigs and analyzed using BLAST. Primers were designed from 137 resistance gene analog sequences not found in Tc. A population of 260 F[subscript]2 lines derived from a cross between the rust-susceptible cultivar Chinese Spring (CS) and a Thatcher isoline containing Lr16 (TcLr16) was developed for mapping these markers. Two RGA markers XRGA266585 and XRGA22128 were identified that mapped 1.1 cM and 23.8 cM from Lr16, respectively. Three SSR markers Xwmc764, Xwmc661, and Xbarc35 mapped between these two RGA markers at distances of 4.1 cM, 10.7 cM, and 16.1 cM from Lr16, respectively. Another objective of this study was to use genotyping-by-sequencing (GBS) to develop single nucleotide polymorphism (SNP) markers closely linked to Lr16. DNA from 22 resistant and 22 susceptible F[subscript]2 plants from a cross between CS and TcLr16 was used for GBS analysis. A total of 39 Kompetitive Allele Specific PCR (KASP) markers were designed from SNPs identified using the UNEAK and Tassel pipelines. The KASP marker XSNP16_TP1456 mapped 0.7 cM proximal to Lr16 in a TcxTcLr16 population consisting of 129 F[subscript]2 plants. These results indicate that both techniques are viable methods to develop new molecular markers. RNA-seq and in silico subtraction were successfully used to develop two new RGA markers linked to Lr16, one of which was more closely linked than known SSR markers. GBS was also successfully used on an F[subscript]2 population to develop a KASP marker that is the most closely linked marker to Lr16 to date.

RNA-Sequencing (RNA-Seq) has become one of the most widely used techniques to interrogate the transcriptome of an organism since the advent of next generation sequencing technologies [1]. A plethora of tools have been developed to analyze and visualize the transcriptome data from RNA-Seq experiments, solving the problem of mapping reads back to the host organism's genome [2] [3]. This allows for analysis of most reads produced by the experiments, but these tools typically discard reads that do not match well with the reference genome. This additional information could reveal important insight into the experiment and possible contributing factors to the condition under consideration. We introduce PARSES, a pipeline constructed from existing sequence analysis tools, which allows the user to interrogate RNA-Sequencing experiments for possible biological contamination or the presence of exogenous sequences that may shed light on other factors influencing an organism's condition.

The main theme of this dissertation is to develop a distributed computational pipeline for processing next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate hundreds of millions of short reads for each DNA/RNA sample. There are many existing bioinformatics tools developed for the analysis and visualization of this data, but very large studies present computational and organizational challenges that are difficult to overcome manually. We designed a comprehensive pipeline for the analysis of RNA sequencing which leverages many existing tools and parallel computing technology to facilitate the analysis of extremely large studies. RNA CoMPASS provides a web-based graphical user interface and distributed computational pipeline including endogenous transcriptome quantification and additionally the investigation of exogenous sequences.

No description available.

A teca é uma árvore de grande importância comercial pelas características de cor e durabilidade de sua madeira. Devido a sua rusticidade e fácil adaptação ao clima, plantios de teca tornam-se cada vez mais atrativos ao redor do mundo. Contudo, esta espécie apresenta escassez de estudos genéticos moleculares a respeito tanto de sua madeira, quanto de sua tolerância às variações ambientais. Uma vez que o transcriptoma pode apresentar grande quantidade de informação a respeito dos genes expressos por um conjunto celular, neste trabalho foi realizado o primeiro transcriptoma de teca, onde foram sequenciadas flores, folhas, raízes e seedlings pela tecnologia Illumina. A montagem do transcriptoma foi realizada com o programa Trinity acima de 100 milhões de reads e gerou mais de 400 mil contigs, os quais tiveram as anotações funcionais adquiridas com o programa Blas2GO. 51% dos contigs foram anotados, mostrando alta similaridade com as espécies Vitis vinifera e Solanum licopersicum; destes, 78% obtiveram anotações funcionais com o Gene Ontology, totalizando 5.165 termos para Processo Biológico, 2.846 termos para Função Molecular e 742 para Componente Celular. A expressão diferencial foi obtida com o programa edgeR a 5% de probabilidade de erro e mostrou que, para 187.315 contigs montados através da fusão de todas as bibliotecas sequenciadas, 18 mostraram expressão diferencial para flor, 14 para folha, 13 para raiz e 29 para seedling. Após a etapa de caracterização do transcriptoma, foi realizado um experimento de estresse por déficit hídrico em casa-de-vegetação, onde plantas de teca foram submetidas a estresse Moderado (40% de água no substrato por 20 dias), estresse Severo (20 a 40% de água por 30 dias) e tratamento controle (substrato saturado). As medições através de analisador de gases por infravermelho (IRGA) mostraram queda na fotossíntese (até 70% a menos do que o controle), na transpiração (até 77%) e na condutância estomática (até 85%) entre os tratamentos; além disto, o conteúdo relativo de água foliar caiu 13% entre o tratamento severo e o controle, e níveis de prolina livre foram até 3,5 vezes mais altos nos tratamentos de estresse. A temperatura foliar aumentou significativamente com o aumento da irradiância de fótons aplicada. A busca por genes relacionados ao estresse por déficit hídrico na biblioteca de transcritos de Raiz retornou 1.145 sequências, e destas, 4 foram caracterizadas: TgTPS (trealose 6-fosfato sintase), TgPIP (aquaporina, proteína intrínseca de membrana plasmática), TgDREB2 (proteína de ligação a elemento responsivo a desidratação) e TgAREB (proteína de ligação a elemento responsivo a ácido abscísico). Apenas TgTPS, TgPIP e TgDREB2 mostraram alto grau de conservação entre as espécies, podendo ser corretamente amplificadas via PCR e validadas por sequenciamento. Assim, com o banco de dados de transcritos obtido pelo RNA-seq, foi possível identificar genes candidatos ao estudo de características vegetativas e reprodutivas de teca, contribuindo para entender os mecanismos moleculares desta espécie florestal. / Teak is a tree of great commercial importance by the characteristics of color and durability of its wood. Due to its hardiness and easy adaptation to climate, teak plantations become increasingly attractive around the world. However, this species has a lack of molecular genetic studies on both of its wood, as their tolerance to environmental variations. Once the transcriptome can provide lots of information about the genes expressed by a cell group, this work represents the first transcriptome teak, which were sequenced flowers, leaves, roots and seedlings by Illumina technology. The transcriptome assembly was performed with Trinity program above 100 million reads and generated more than 400,000 contigs, which have acquired the functional annotations with Blas2GO program. 51% of the contigs were annotaded, showing high similarity to Vitis vinifera and Solanum licopersicum; of these, 78% had functional annotations with the Gene Ontology, totaling 5,165 terms for Biological Process, 2846 terms for Molecular Function and 742 for Cell Component. The differential expression was obtained with the edgeR program at 5% probability of error and showed that for 187,315 contigs assembled by merging all sequenced libraries, 18 showed differential expression to flower, 14 to leaf, 13 to root and 29 for seedling. After this step of characterization of the transcriptome, we performed a stress experiment by water deficit at greenhouse, where teak plants were subjected to Moderate stress (40% of water in the substrate for 20 days), Severe stress (20 to 40% water for 30 days) and control treatment (saturated substrate). Measurements by infrared gas analyzer (IRGA) showed a decrease in photosynthesis (up to 70% less than the control), transpiration (up 77%) and stomatal conductance (up 85%) between treatments; furthermore, leaf relative water content dropped 13% between the treatment control and severe, and free proline levels were up to 3.5 fold greater in stress treatments. The leaf temperature increased significantly with increasing irradiance of photons applied. The search for genes related to stress by water deficit in the root transcripts library returned 1,145 sequences, and these, 4 were characterized: TgTPS (trehalose 6-phosphate synthase), TgPIP (aquaporin, protein intrinsic of plasma membrane), TgDREB2 (dehydration responsive element binding protein) and TgAREB (abscisic acid responsive element binding protein). Only TgTPS, TgPIP and TgDREB2 showed a high degree of conservation between species, and can be properly amplified by PCR and validated by sequencing. Thus, with the database of transcripts obtained by RNA-seq, candidate genes were identified for the study of vegetative and reproductive characteristics teak, helping to understand the molecular mechanisms of this forest species.