Single nucleotide polymorphisms (SNPs) are single base-pair variations that exist between individuals. There are approximately a million or more SNPs located throughout the genome of each individual animal. Therefore, by taking advantage of these unique polymorphisms, SNPs can be used to resolve questions of unknown parentage in the livestock industry. Currently a panel of 88 SNPs, obtained from a panel of 121 SNPs originally created by USDA-MARC, is commercially available from Fluidigm®. The objective of this study was to determine whether the number of SNPs from the 88-SNP marker panel could be reduced to form a smaller, more cost-efficient parentage-testing SNP panel. A smaller panel would benefit farmers and researchers alike in reducing the time spent in running and analyzing the test, as well as reducing the overall cost for the procedure. Genotype data from over 3000 cattle samples containing offspring and potential parents were examined using two parentage calling software packages. Parentage assessment was analyzed using nine SNP panels of varying size. It was determined that a panel of 71 SNPs, chosen from the original 88 SNPs, was the minimum number required to maintain statistical accuracy and reliability.

Big sagebrush (Artemisia tridentata) is one of the ecologically most important shrub species in western North America. The species serves as a major source of food and habitat for the near-threatened sage grouse and various other fauna. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is considered as a serious threat to sustainability of the big sagebrush ecosystem. Because of its importance, restoration of this species is very crucial to those dependent on big sagebrush community. However, restoration of big sagebrush carried out by using diverse seed source can lead to imbalance and degradation in the native ecosystem. Therefore, restoration works aided by understanding of adaptive traits of big sagebrush using molecular markers will aid successful restoration. The major objective of this research was to create a substantial resource of nuclear sequence data and identify markers that can be used in future studies in big sagebrush. We report the development and annotation of the first expressed sequence tag (EST) collection for big sagebrush based on 454 sequencing of leaf tissue. Expressed genes of subspecies tridentata and vaseyana were sequenced using the 454 GS-FLX titanium platform, which produced 823,392 reads with an average read length of 404 bp and 702,001 reads with an average read length of 333 bp for sspp. tridentata and vaseyana, respectively. Assembly of the reads resulted in 212,102 consensus sequences in ssp. tridentata and 199,439 in ssp. vaseyana. A combined assembly of both subspecies sequences generated 29,541 contigs with an average length of 796 bp and 275,866 singletons with an average length of 370 bp. A BLASTx search against the non-redundant (NR) protein database using the contigs obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e-15). Gene Ontology (GO) IDs were assigned to 18,397 sequences. A total of 20,952 SNPs were detected between the two subspecies and 1,182 SNPs were confirmed in tetraploid ssp. wyomingensis. In addition, 1,003 and 507 SSRs were detected in ssp. tridentata contigs and ssp. vaseyana contigs, respectively.

To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.

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Vascular endothelial growth factor (VEGF) is a potent angiogenic agent. VEGF activates its biologic responses through two cell-surface receptors, Flt1 and Flk1. In addition to the transmembrane form of Flt1, the Flt1 gene also encodes a secreted, truncated form of the receptor (sFlt1) translated from an mRNA in which a portion of intron 13 is preserved. sFlt1 retains high affinity for VEGF and thereby inhibits its angiogenic activity. Intron 13 contains important cis elements involved in sFlt1 mRNA formation. Here, we test the hypothesis that polymorphisms in the human Flt1 gene, particularly SNPs at sites suspected to contain splicing or cleavage-polyadenylation signals, influence Flt1 pre-mRNA processing and rates of Flt1 and sFlt1 expression. The NCBI SNP database contained 23 SNPs in the region of interest, one each in exons 13 and 14. An independent human SNP screen confirmed several of the reported SNPs. The web-based ESEfinder software predicted that the exon 13/14 SNPs had reduced potential for recruitment of splicing components. To test effects of exonic SNPs on Flt1 pre-mRNA processing, wild type and mutant Flt1 minigene plasmids were transfected into NIH/3T3 cells. Both exonic SNPs were associated with ~40% decreases in Flt1:sFlt1 mRNA ratios determined by real-time PCR. To facilitate exploration of ESEs in regulated RNA splicing, a PERL computer program, "EXONerator" was written to silence predicted ESEs without altering polypeptide sequence. These results support the notion that small changes in exon composition can have significant effects on splicing activity and underscore the utility of software tools for hypothesis generation. / Ph. D.

Yes / Single nucleotide polymorphisms (SNPs) in PLCE1 and MICB genes increase risk for the development of dengue shock syndrome (DSS). We used Bioinformatics tools to predict alterations at the transcriptional and posttranslational levels driven by PLCE1 and MICB SNPs associated with DSS. Functional and phenotypic analysis conducted to determine deleterious SNPs and impact of amino acid substitution on the structure and function of proteins identified rs2274223 (H1619R) as deleterious to protein coding as it induces structural change in the C2 domain of PLCε, with the mutant residue more positively charged than the wild-type residue (RMSD score, 1.75 Å).Moreover, rs2274223 condenses the chromatinrepressing PLCε expression in DSS. Briefly, this study presents the impact of a single nucleotide transition at SNPs associated with DSS on differential protein binding patterns with PLCE1 and MICB genes and on protein structure modification and their possible role in the pathogenesis of DSS.

Fusarium verticillioides è l’agente responsabile della Fusariosi della Spiga del mais, contamina la granella con fumonisine, micotossine responsabili di diverse patologie umane e animali. Per la resistenza alla fusariosi e all’accumulo di fumonisine esiste variabilità tra genotipi diversi ma non sono ancora disponibili ibridi immuni. L’obiettivo di questo lavoro è stato quello di individuare marcatori associati alla resistenza a F. verticillioides. Mediante un bioassay è stato testato un association panel per la resistenza a F. verticillioides. Al fine di identificare i marcatori di resistenza sono stati applicati un approccio GWAS e uno per geni candidati. L’analisi GWAS è stata eseguita con 227K SNPs restituendo 206 marcatori significativi. Da un lavoro di RNASequencing sono stati individuati i geni coinvolti nella risposta a F. verticillioides mentre i geni R sono stati recuperati della letteratura scientifica. Genotipi resistenti (CO433 e CO441) e suscettibili (CO354 e CO389) sono stati scelti per individuare polimorfismi nei geni candidati da associare ai fenotipi rilevati mediante il bioassay. Quattro marcatori sono risultati significativi. Infine, la correlazione tra l’incidenza della fusariosi rilevata in campo e mediante bioassay è stata analizzata in una popolazione di 172 RIL derivanti da CO441 x CO354, tuttavia, non è stata individuata alcuna corrispondenza. / Fusarium verticillioides is the causal agent of Fusarium ear rot (FER) in maize and contaminates grains with fumonisin, a family of mycotoxins involved in several human and animal diseases. Quantitative genetic variation exists for resistance to FER and fumonisin contamination among genotypes, however, resistant maize hybrids are currently not available. The aim of this work was the identification of genetic markers associated to resistance against F. verticillioides. A bioassay was used to screen inbred lines of the maize association population for FER resistance, GWAS and candidate gene approaches were applied to identify markers. GWAS was performed using a 227K SNP matrix and resulting in 206 significant markers. Genes involved in F. verticillioides response in developing maize kernels were retrieved from a previous RNASequencing study while maize R genes were retrieved from scientific literature. Resistant (CO433 and CO441) and susceptible genotypes (CO389 and CO354) were selected to amplify and sequence candidate genes. Polymorphisms detected were used to find association with phenotypes scored using the bioassay. Four significant markers were found. Finally, the correlation between FER phenotypes scored in field experiments and bioassay phenotypes was investigated. A population of 172 RILs (CO441 x CO354), was tested. No correlation was found.

En plus des facteurs environnementaux et démographiques, les propriétés génétiques des populations sont devenues une préoccupation majeure pour préserver les populations en déclin de l'extinction. Afin d’acquérir des informations pertinentes pour la planification et la mise en œuvre des stratégies de conservation, les biologistes de la conservation ont réalisé le besoin d’avoir des connaissances en génétique des populations. Grace à l'acquisition de plus en plus rapide et de moins en moins chère d'une large gamme de marqueurs moléculaires, le recours a l’usage de l’outil moléculaire se répand de plus en plus. Ainsi, la génétique de la conservation se confirme comme une discipline à part entière qui est donc l’utilisation de la génétique dans la préservation des espèces comme entités dynamiques capables d'évoluer pour faire face aux changements environnementaux et afin de minimiser leur risque d'extinction. Par le biais de l’utilisation d’un large panel de marqueurs moléculaires (gènes mitochondriaux et nucléaires, microsatellites et SNPs), nous nous sommes intéresse à l’histoire évolutive à différentes échelles spatio-temporelles de la sous-espèce ouest méditerranéenne Testudo. hermanni hermanni (THH), qui présente une distribution insulaire et continentale très fragmentée. Le but de ce travail consiste à 1) comprendre les processus qui expliqueraient la distribution actuelle de la diversité génétique des populations et leur structure, 2) identifier l'origine des populations introduites (à Minorque et au Delta de l’Ebre), et 3) dater l’origine de la sous espèce THH. A l’échelle des populations, il s’agit d’identifier le nombre de groupes génétiques homogènes chez la tortue d’Hermann et le degré de différentiation génétique entre ces groupes afin de définir des unités de conservation évolutivement significatives (ESU) et des unités de gestion (MU). Enfin, nous nous sommes intéresses à l’étude des derniers noyaux de populations de THH dans le Var par des approches de génétique du paysage. Nos résultats ont révélé qu’une divergence par vicariance est à l’ origine de l’apparition de la sous-espèce T.h. hermanni. Ce scenario biogéographique s’expliquerait par les successions d’évènements glaciaires et interglaciaires qu’a connu le Pléistocène depuis plus de 2 MA provoquant un mouvement de retrait de l’espèce vers des zones refuges sur la frange côtière nord-méditerranéenne. Par ailleurs, le patron de différentiation mitochondriale Ile-continent observe et confirme par les microsatellites est très original par rapport à ce qui est connu chez d’autres espèces de reptiles partageant la même aire de distribution. Au vue de l’analyse phylogénétique confirmée par les microsatellites, on peut affirmer que la tortue d’Hermann n’est pas native sur Minorque et qu’elle a une double origine : la première, résultant d’une introduction à partir d’une seule source, probablement d’une population continentale génétiquement proche des Albères. La seconde d'origine insulaire, serait le résultat d’apports multiples, à partir de la Corse, de la Sardaigne ou de la Sicile. Enfin, l’isolement des populations de THH au sein de chaque région géographique reflète une structure génétique très forte. Par conséquent, six unités de gestion (MUs) sont proposées comme unités de conservation et de suivi sur le terrain. / In addition to environmental and demographic factors, the study of genetic properties of populations became inevitable issues in the conservation of declining populations. To acquire relevant information for conservation planning and implementing conservation strategies, conservationists have realized the need of population genetics tools. Moreover, this discipline has become more efficient with the development of a wide range of effective and relatively cheap methods for the characterization of a huge number of molecular markers. This led to define the conservation genetics as a separate discipline, which is the use of genetics in species preservation as dynamic entities evolving to cope with environmental changes and to minimize their extinction risk. Using a broad panel of molecular markers (mitochondrial and nuclear genes, microsatellites and SNPs), we interested in the evolutionary history at different spatial and temporal scales of the Mediterranean western subspecies Testudo hermanni hermanni (THH), which presents a very fragmented insular and continental distribution. The aim of this study is to 1) understand the processes that explain the current distribution of the structure and genetic diversity of populations, 2) identify the origin of introduced populations (Menorca and Ebro Delta) and 3) Dating the origin of the subspecies THH. At the population level, our study aimed to identify the number of homogeneous genetic groups of THH tortoise and the degree of genetic differentiation between these groups in order to identify evolutionarily significant units (ESU) and management units (MU). Finally, we were interested in the study of the last core populations of THH in the Var by landscape genetics approach. Our results revealed that a divergence by vicariance pattern explains the origin of the appearance of the subspecies THH. This biogeographic scenario is explained by the succession of glacial and interglacial events of the Pleistocene causing a withdrawal of the species toward refugia on the northern Mediterranean fringe. Moreover, the observed differentiation pattern (island vs continent) is very original compared to the reported diversity patterns of other reptiles sharing the same distribution range. According to our results, we may conclude that the Hermann’s tortoise is not native in Menorca and has a double origin: the first, is an introduction resulting from a unique source, probably from a continental lineage genetically close to Albera. The second, from an island origin, is the result of multiple contributions, from Corsica, Sardinia or Sicily. Lastly,the isolation of THH populations within each geographic region reflects a very strong genetic structure, therefor the six most relevant management units forconservation purposes are proposed on the basis that they represent a significant part of the evolutionary legacy of the species.