A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species

Association and Linkage Analysis of Aluminum Tolerance Genes in Maize

Aluminum (Al) toxicity is a major worldwide constraint to crop productivity on acidic soils. Al becomes soluble at low pH, inhibiting root growth and severely reducing yields. Maize is an important staple food and commodity crop in acidic soil regions, especially in South America and Africa where these soils are very common. Al exclusion and intracellular tolerance have been suggested as two important mechanisms for Al tolerance in maize, but little is known about the underlying genetics. linkage populations with approximately 200 individuals each were used to study genetic variation in this complex trait. Al tolerance was measured as net root growth in nutrient solution under Al stress, which exhibited a wide range of variation between lines. Comparative and physiological genomics-based approaches were used to select 21 candidate genes for evaluation by association analysis.). These four candidate genes are high priority subjects for follow-up biochemical and physiological studies on the mechanisms of Al tolerance in maize. Immediately, elite haplotype-specific molecular markers can be developed for these four genes and used for efficient marker-assisted selection of superior alleles in Al tolerance maize breeding programs

An 11-bp Insertion in Zea mays fatb Reduces the Palmitic Acid Content of Fatty Acids in Maize Grain

The ratio of saturated to unsaturated fatty acids in maize kernels strongly impacts human and livestock health, but is a complex trait that is difficult to select based on phenotype. Map-based cloning of quantitative trait loci (QTL) is a powerful but time-consuming method for the dissection of complex traits. Here, we combine linkage and association analyses to fine map QTL-Pal9, a QTL influencing levels of palmitic acid, an important class of saturated fatty acid. QTL-Pal9 was mapped to a 90-kb region, in which we identified a candidate gene, Zea mays fatb (Zmfatb), which encodes acyl-ACP thioesterase. An 11-bp insertion in the last exon of Zmfatb decreases palmitic acid content and concentration, leading to an optimization of the ratio of saturated to unsaturated fatty acids while having no effect on total oil content. We used three-dimensional structure analysis to explain the functional mechanism of the ZmFATB protein and confirmed the proposed model in vitro and in vivo. We measured the genetic effect of the functional site in 15 different genetic backgrounds and found a maximum change of 4.57 mg/g palmitic acid content, which accounts for ∼20–60% of the variation in the ratio of saturated to unsaturated fatty acids. A PCR-based marker for QTL-Pal9 was developed for marker-assisted selection of nutritionally healthier maize lines. The method presented here provides a new, efficient way to clone QTL, and the cloned palmitic acid QTL sheds lights on the genetic mechanism of oil biosynthesis and targeted maize molecular breeding

The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells

Abstract Introduction There is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein. Methods Using full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture. Results Transfection with WT1–ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1–ZF form lacking the lysine–threonine–serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1–ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1–ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells. Conclusion Our observations strongly argue that the WT1–ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1–ZF - KTS provides a potential candidate of gene therapy for breast cancer

Carotid Body AT4 Receptor Expression and its Upregulation in Chronic Hypoxia

Hypoxia regulates the local expression of angiotensin-generating system in the rat carotid body and the me-tabolite angiotensin IV (Ang IV) may be involved in the modulation of carotid body function. We tested the hypothesis that Ang IV-binding angiotensin AT4 receptors play a role in the adaptive change of the carotid body in hypoxia. The expression and localization of Ang IV-binding sites and AT4 receptors in the rat carotid bodies were studied with histochemistry. Specific fluorescein-labeled Ang IV binding sites and positive staining of AT4 immunoreactivity were mainly found in lobules in the carotid body. Double-labeling study showed the AT4 receptor was localized in glomus cells containing tyrosine hydroxylase, suggesting the expression in the chemosensitive cells. Intriguingly, the Ang IV-binding and AT4 immunoreactivity were more intense in the carotid body of chronically hypoxic (CH) rats (breathing 10% oxygen for 4 weeks) than the normoxic (Nx) control. Also, the protein level of AT4 receptor was doubled in the CH comparing with the Nx group, supporting an upregulation of the expression in hypoxia. To examine if Ang IV induces intracellular Ca2+ response in the carotid body, cytosolic calcium ([Ca2+]i) was measured by spectrofluorimetry in fura-2-loaded glomus cells dissociated from CH and Nx carotid bodies. Exogenous Ang IV elevated [Ca2+]i in the glomus cells and the Ang IV response was significantly greater in the CH than the Nx group. Hence, hypoxia induces an upregulation of the expression of AT4 receptors in the glomus cells of the carotid body with an increase in the Ang IV-induced [Ca2+]i elevation. This may be an additional pathway enhancing the Ang II action for the activation of chemoreflex in the hypoxic response during chronic hypoxia

Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis

<p>Abstract</p> <p>Background</p> <p>The identification of mutations that confer unique properties to a pathogen, such as host range, is of fundamental importance in the fight against disease. This paper describes a novel method for identifying amino acid sites that distinguish specific sets of protein sequences, by comparative analysis of matched alignments. The use of mutual information to identify distinctive residues responsible for functional variants makes this approach highly suitable for analyzing large sets of sequences. To support mutual information analysis, we developed the AVANA software, which utilizes sequence annotations to select sets for comparison, according to user-specified criteria. The method presented was applied to an analysis of influenza A PB2 protein sequences, with the objective of identifying the components of adaptation to human-to-human transmission, and reconstructing the mutation history of these components.</p> <p>Results</p> <p>We compared over 3,000 PB2 protein sequences of human-transmissible and avian isolates, to produce a catalogue of sites involved in adaptation to human-to-human transmission. This analysis identified 17 characteristic sites, five of which have been present in human-transmissible strains since the 1918 Spanish flu pandemic. Sixteen of these sites are located in functional domains, suggesting they may play functional roles in host-range specificity. The catalogue of characteristic sites was used to derive sequence signatures from historical isolates. These signatures, arranged in chronological order, reveal an evolutionary timeline for the adaptation of the PB2 protein to human hosts.</p> <p>Conclusion</p> <p>By providing the most complete elucidation to date of the functional components participating in PB2 protein adaptation to humans, this study demonstrates that mutual information is a powerful tool for comparative characterization of sequence sets. In addition to confirming previously reported findings, several novel characteristic sites within PB2 are reported. Sequence signatures generated using the characteristic sites catalogue characterize concisely the adaptation characteristics of individual isolates. Evolutionary timelines derived from signatures of early human influenza isolates suggest that characteristic variants emerged rapidly, and remained remarkably stable through subsequent pandemics. In addition, the signatures of human-infecting H5N1 isolates suggest that this avian subtype has low pandemic potential at present, although it presents more human adaptation components than most avian subtypes.</p

Vagueness and Imprecise Credence

In this paper I investigate an alternative to imprecise probabilism. Imprecise probabilism is a popular revision of orthodox Bayesianism: while the orthodox Bayesian claims that a rational agent’s belief-state can be represented by a single credence function, the imprecise probabilist claims instead that a rational agent’s belief-state can be represented by a set of such functions. The alternative that I put forward in this paper is to claim that the expression ‘credence’ is vague, and then apply the theory of supervaluationism to sentences containing this expression. This gives us a viable alternative to imprecise probabilism, and I end by comparing the two accounts. I show that supervaluationism has a simpler way of handling sentences relating the belief-states of two different people, or of the same person at two different times; that both accounts may have the resources to develop plausible decision theories; and finally that the supervaluationist can accommodate higher-order vagueness in a way that is not available to the imprecise probabilist