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Colleen A Fisher, Eric K Bhattarai,
Jason B Osterstock,
Scot E Dowd,
Paul M Seabury,
Meenu Vikram,
Robert H Whitlock,
Ynte H Schukken,
Robert D Schnabel,
Jeremy F Taylor,
James E Womack,
Christopher M Seabury
PLoS ONE 01/2012; 7(2). · 4.09 Impact Factor
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ABSTRACT: The white-tailed deer (Odocoileus virginianus) represents one of the most successful and widely distributed large mammal species within North America, yet very little nucleotide sequence information is available. We utilized massively parallel pyrosequencing of a reduced representation library (RRL) and a random shotgun library (RSL) to generate a complete mitochondrial genome sequence and identify a large number of putative single nucleotide polymorphisms (SNPs) distributed throughout the white-tailed deer nuclear and mitochondrial genomes. A SNP validation study designed to test specific classes of putative SNPs provides evidence for as many as 10,476 genome-wide SNPs in the current dataset. Based on cytogenetic evidence for homology between cow (Bos taurus) and white-tailed deer chromosomes, we demonstrate that a divergent genome may be used for estimating the relative distribution and density of de novo sequence contigs as well as putative SNPs for species without draft genome assemblies. Our approach demonstrates that bioinformatic tools developed for model or agriculturally important species may be leveraged to support next-generation research programs for species of biological, ecological and evolutionary importance. We also provide a functional annotation analysis for the de novo sequence contigs assembled from white-tailed deer pyrosequencing reads, a mitochondrial phylogeny involving 13,722 nucleotide positions for 10 unique species of Cervidae, and a median joining haplotype network as a putative representation of mitochondrial evolution in O. virginianus. The results of this study are expected to provide a detailed template enabling genome-wide sequence-based studies of threatened, endangered or conservationally important non-model organisms.
PLoS ONE 01/2011; 6(1):e15811. · 4.09 Impact Factor
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PLoS ONE 01/2011; 6(2). · 4.09 Impact Factor
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Colleen A Fisher, Eric K Bhattarai,
Jason B Osterstock,
Scot E Dowd,
Paul M Seabury,
Meenu Vikram,
Robert H Whitlock,
Ynte H Schukken,
Robert D Schnabel,
Jeremy F Taylor,
James E Womack,
Christopher M Seabury
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ABSTRACT: Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination.
PLoS ONE 01/2011; 6(11):e27744. · 4.09 Impact Factor
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ABSTRACT: Forward genetic analysis is the most broadly applicable approach to discern gene functions. However, for some organisms like the filamentous ascomycete Neurospora crassa, genetic mapping frequently represents a limiting step in forward genetic approaches. We describe an efficient method for genetic mapping in N. crassa that makes use of a modified bulked segregant analysis and PCR-based molecular markers. This method enables mapping with progeny from a single cross and requires only 90 PCR amplifications. Genetic distances between syntenic markers have been determined to ensure complete coverage of the genome and to allow interpolation of linkage data. As a result, most mutations should be mapped in less than one month to within 1-5 map units, a level of resolution sufficient to initiate map-based cloning efforts. This system also will facilitate analyses of recombination at a genome-wide level and is applicable to other perfect fungi when suitable markers are available.
Fungal Genetics and Biology 07/2007; 44(6):455-65. · 3.74 Impact Factor
