Project description:Restriction site Associated DNA (RAD) tags are a genome-wide representation of every site of a particular restriction enzyme by short DNA tags. Most organisms segregate large numbers of DNA sequence polymorphisms that disrupt restriction sites, which allow RAD tags to serve as genetic markers spread at a high-density throughout the genome. Here, we demonstrate the applicability of RAD markers for both individual and bulk-segregant genotyping. First, we show that these markers can be identified and typed on pre-existing microarray formats. Second, we present a method that uses RAD marker DNA to rapidly produce a low-cost microarray genotyping resource that can be used to efficiently identify and type thousands of RAD markers. We demonstrate the utility of the former approach by using a tiling path array for the fruit fly to map a recombination breakpoint, and the latter approach by creating and utilizing an enriched RAD marker array for the threespine stickleback. The high number of RAD markers enabled localization of a previously identified region, as well as a second novel region also associated with the lateral plate phenotype. Taken together, our results demonstrate that RAD markers, and the method to develop a RAD marker microarray resource, allow high-throughput, high-resolution genotyping in both model and non-model systems. Keywords: microarray genotyping
Project description:To investiage the ability of positve inotropism from myocardial Rad reduction we induced Rad knockout after onset of pressure overload to reverse or compensate progression of heart failure
Project description:Restriction-site Associated DNA sequencing (RAD-seq) of 47 males, females and hermaphrodites from one dioecious and one androdioecious population.
Project description:One of the most recognizable physiological phenomena is the adrenergic-induced fight-or-flight increase in heart rate and cardiac contraction. For the β-adenergic agonist-induced enhancement of calcium influx and transients, and contractility in the heart, we identify the dual requirement of a subpopulation of Rad-bound calcium channels under basal conditions and PKA phosphorylation of Rad. In mice expressing a non-phosphorylatable Rad mutant, basal cardiac contractility is reduced and adrenergic-augmentation of the calcium current and contractility are disabled. Expression of mutant calcium channel β-subunits that cannot bind the mutant Rad restored contractility, revealing a highly specific therapeutic approach to mimic the contractility imparted by adrenergic agonists. Our findings place Rad and its modulation of calcium channels at the nexus of adrenergic modulation of cardiac responses.
Project description:Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. Despite decades of investigations, predominantly focusing on ryanodine receptor and phospholamban phosphorylation, the molecular mechanisms underlying the sympathetic nervous system control of cardiac contractility remain controversial and incompletely elucidated. Here, we identify the calcium-channel inhibitor Rad as a critical component. In cardiomyocytes isolated from knock-in mice expressing Rad with alanine-substitutions of the four PKA-phosphorylated serine residues (4SA-Rad), calcium currents cannot be increased by adrenergic agonists or phosphatase inhibitor. In these mice, basal cardiac contractility, exercise capacity and heart rate are reduced, and the augmentation of contractile force by adrenergic agonists is severely blunted. Expression of mutant calcium-channel β-subunits that cannot bind Rad is sufficient to restore calcium influx and cardiac contractility in 4SA-Rad mice to levels induced by adrenergic agonists in wild-type mice, revealing a potential therapeutic approach to enhance cardiac contractility while bypassing stimulation of adrenergic receptors.
Project description:Normally, rice can elongate the coleoptile under submerged condition. However, reduced adh activity (rad) mutant cannot elongate the coleoptile under submergence. To investigate the change in gene expression, we performed microarray analysis. In this analysis, we used 1 day old seedling of rice. But it is difficult to isolate only coleoptile from rice embryo without any contamination in this stage. Therefore, we applied laser microdissection (LM) technique to this microarray. By use of LM, we isolated coleoptile from rice embryo and use for microarray analysis. As the results, we found that the differences in the gene expression profiles of coleoptile between wild type and rad mutant.
Project description:Normally, rice can elongate the coleoptile under submerged condition. However, reduced adh activity (rad) mutant cannot elongate the coleoptile under submergence. To investigate the change in gene expression, we performed microarray analysis. In this analysis, we used 1 day old seedling of rice. But it is difficult to isolate only coleoptile from rice embryo without any contamination in this stage. Therefore, we applied laser microdissection (LM) technique to this microarray. By use of LM, we isolated coleoptile from rice embryo and use for microarray analysis. As the results, we found that the differences in the gene expression profiles of coleoptile between wild type and rad mutant. Gene expression analysis in coleoptile of wild type and mutant.
Project description:We produced RNA-Seq reads from messenger RNA isolated from aerial seedling tissue for 9 hybrids (F1s) generated by crossing in a pairwise manner 18 of the founding accessions (inbred strains) of the Multiparent Advanced Generation Inter-Cross (MAGIC) genetic mapping resource for Arabidopsis thaliana (see Gan et al. 2011. Nature, 477:419-23 for a description of the MAGIC genetic mapping resource). The resulting RNA-Seq data provides a resource to assess allele-specific gene expression between A. thaliana accessions.
Project description:Restriction site Associated DNA Sequencing (RAD-Seq) is a technique characterized by the sequencing of specific loci along the genome that is widely employed in the field of evolutionary biology since it allows to exploit variants (mainly Single Nucleotide Polymorphism-SNPs) information from entire populations at a reduced cost. Common RAD dedicated tools, such as STACKS or IPyRAD, are based on all-vs-all read alignments, which require consequent time and computing resources. We present an original method, DiscoSnp-RAD, that avoids this pitfall since variants are detected by exploiting specific parts of the assembly graph built from the reads, hence preventing all-vs-all read alignments. We tested the implementation on simulated datasets of increasing size, up to 1,000 samples, and on real RAD-Seq data from 259 specimens of Chiastocheta flies, morphologically assigned to seven species. All individuals were successfully assigned to their species using both STRUCTURE and Maximum Likelihood phylogenetic reconstruction. Moreover, identified variants succeeded to reveal a within-species genetic structure linked to the geographic distribution. Furthermore, our results show that DiscoSnp-RAD is significantly faster than state-of-the-art tools. The overall results show that DiscoSnp-RAD is suitable to identify variants from RAD-Seq data, it does not require time-consuming parameterization steps and it stands out from other tools due to its completely different principle, making it substantially faster, in particular on large datasets.