TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

SummaryWe have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled¯uorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one¯uorescence channel can be veri®ed by appearance of the opposite cut strand in the other channel. We demonstrated the ef®ciency of this technology, called Ecotilling, by the discovery in 150 individuals of 55 haplotypes in ®ve genes, ranging from sequences differing by a single nucleotide polymorphism to those representing complex haplotypes. The discovered polymorphisms were con®rmed by sequencing and included base-pair changes, small insertions and deletions, and variation in microsatellite repeat number. Ecotilling allows the rapid detection of variation in many individuals and is cost effective because only one individual for each haplotype needs to be sequenced. The technology is applicable to any organism including those that are heterozygous and polyploid.

Meta-analysis based techniques are emerging as powerful, robust tools for developing models of connectivity in functional neuroimaging. Here, we apply meta-analytic connectivity modeling to the human caudate to 1) develop a model of functional connectivity, 2) determine if meta-analytic methods are sufficiently sensitive to detect behavioral domain specificity within region-specific functional connectivity networks, and 3) compare meta-analytic driven segmentation to structural connectivity parcellation using diffusion tensor imaging. Results demonstrate strong coherence between meta-analytic and data-driven methods. Specifically, we found that behavioral filtering resulted in cognition and emotion related structures and networks primarily localized to the head of the caudate nucleus, while perceptual and action specific regions localized to the body of the caudate, consistent with early models of nonhuman primate histological studies and postmortem studies in humans. Diffusion tensor imaging (DTI) revealed support for meta-analytic connectivity modeling's (MACM) utility in identifying both direct and indirect connectivity. Our results provide further validation of meta-analytic connectivity modeling, while also highlighting an additional potential, namely the extraction of behavioral domain specific functional connectivity.