Project description:Seven temperature sensitive Saccharomyces cerevisiae BY4741 mutants (rsc3-1, abf1-101, reb1-212, rap1-1, mcm1, tbf1 and cep3-1) were grown at restrictive temperatures until a difference in OD600 was observed relative to a wild-type control. Nucleosomal DNA, whole genomic DNA and total RNA were isolated and hybridized onto yeast whole-genome tiling arrays.<br>
Project description:Population samples of Drosophila melanogaster were hybridized to a genomic tiling array: separetely from 2 US localities, 3 Caribbean localities, 1 West Africa (Cameroon) locality and 1 Zimbabwe locality. We replicated each locality 3 times by pooling individual flies for each hybridization experiment replicate.
Project description:Understanding the mechanism of cadmium (Cd) accumulation in plants is important to help reduce its potential toxicity to both plants and humans through dietary and environmental exposure. Here, we report on a study to uncover the genetic basis underlying natural variation in Cd accumulation in a world-wide collection of 349 wild collected Arabidopsis thaliana accessions. We identified a 4-fold variation (0.5 M-bM-^@M-^S 2 M-NM-<g Cd g-1 dry weight) in leaf Cd accumulation when these accessions were grown in a controlled common garden. By combining genome-wide association mapping, linkage mapping in an experimental F2 population and transgenic complementation, we reveal that HMA3 is the sole major locus responsible for the variation in leaf Cd accumulation we observe in this diverse population of A. thaliana accessions. Analysis of the predicted amino acid sequence of HMA3 from 149 A. thaliana accessions reveals the existence of 10 major natural protein haplotypes. Association of these haplotypes with leaf Cd accumulation and genetics complementation experiments indicate that 5 of these haplotypes are active and 5 are inactive, and that elevated leaf Cd accumulation is associated with the reduced function of HMA3 caused by a nonsense mutation and polymorphisms that change two specific amino acids. Genomic DNA from two F2 progeny pools (low leaf Cd pool and high leaf Cd pool) of CS28181 x Col-0 was labelled and hybridized separately to the Affymetrix SNP-tiling array AtSNPtile. The signal intensity difference between the two pools for all probes were analyzed using R scripts that are available at http://ars.usda.gov/mwa/bsasnp .
Project description:Background:; Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomics strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis (BSA) and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na+ from the soil. Methodology/Principal Findings:; Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that Ts-1 and Tsu-1 accumulate higher shoot levels of Na+ than Col-0 and other accessions. We identify AtHKT1, known to encode a Na+ transporter, as being the causal locus driving elevated shoot Na+ in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence ~5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na+. Interestingly, and in contrast to the hkt1-1 null mutant, under NaCl stress conditions this novel AtHKT1 allele not only does not confer NaCl sensitivity, but co-segregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; www.purdue.edu/dp/ionomics). Conclusions/Significance:; Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the causal locus driving the natural variation in shoot Na+ accumulation we observed in Ts-1 and Tsu-1, two coastal populations of Arabidopsis. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions, and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes. Experiment Overall Design: Hybridizations from two sets of Bulk Segregant analysis. F2 populations from col-0 crossed to two High Na accessions: Ts-1 and Tsu-1 were analyzed. This series contains the 3 hybs from each accession that were used to identify Single Feature Polymorphisms, the 3 hybs of Col-0 they were compared to, and 1 hyb for each pool from the BSA mapping(High Na pool, low Na Pool).
Project description:ABSTRACT; Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 -MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence, and expression of MOT1 tagged with GFP in protoplasts, and transgenic plants, establishes the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants, and shows that novel loci can be detected by association mapping. Experiment Overall Design: Hybridizations from a set of Bulk Segregant analysis. An F2 population from 14501 in the col-0 background crossed to Ler-0 was analyzed. This series contains the 3 hybs from Ler used to identify Single Feature Polymorphisms, the 3 hybs of Col-0 they were compared to, and 1 hyb for each pool from the BSA mapping(High Mo pool, low Mo Pool).
Project description:There is abundant transcription from eukaryotic genomes unaccounted for by protein coding genes. A high-resolution genome-wide survey of transcription in a well annotated genome will help relate transcriptional complexity to function. By quantifying RNA expression on both strands of the complete genome of Saccharomyces cerevisiae using a high-density oligonucleotide tiling array, this study identifies the boundary, structure, and level of coding and noncoding transcripts. A total of 85% of the genome is expressed in rich media. Apart from expected transcripts, we found operon-like transcripts, transcripts from neighboring genes not separated by intergenic regions, and genes with complex transcriptional architecture where different parts of the same gene are expressed at different levels. We mapped the positions of 3' and 5' UTRs of coding genes and identified hundreds of RNA transcripts distinct from annotated genes. These nonannotated transcripts, on average, have lower sequence conservation and lower rates of deletion phenotype than protein coding genes. Many other transcripts overlap known genes in antisense orientation, and for these pairs global correlations were discovered: UTR lengths correlated with gene function, localization, and requirements for regulation; antisense transcripts overlapped 3' UTRs more than 5' UTRs; UTRs with overlapping antisense tended to be longer; and the presence of antisense associated with gene function. These findings may suggest a regulatory role of antisense transcription in S. cerevisiae. Moreover, the data show that even this well studied genome has transcriptional complexity far beyond current annotation.