TECHNICAL ADVANCE: Indel arrays: an affordable alternative for genotyping

SummaryNatural variation and induced mutations are important resources for gene discovery and the elucidation of genetic circuits. Mapping such polymorphisms requires rapid and cost‐efficient methods for genome‐wide genotyping. Here we report the development of a microarray‐based method that assesses 240 unique markers in a single hybridization experiment at a cost of less than US$50 in materials per line. Our genotyping array is built with 70‐mer oligonucleotide elements representing insertion/deletion (indel) polymorphisms between the Arabidopsis thaliana accessions Columbia‐0 (Col) and Landsberg erecta (Ler). These indel polymorphisms are recognized with great precision by comparative genomic hybridization, eliminating the need for array replicates and complex statistical analysis. Markers are present genome‐wide, with an average spacing of approximately 500 kb. PCR primer information is provided for all array indels, allowing rapid single‐locus inquiries. Multi‐well chips allow groups of 16 lines to be genotyped in a single experiment. We demonstrate the utility of the array for accurately mapping recessive mutations, RIL populations and mixed genetic backgrounds from accessions other than Col and Ler. Given the ease of use of shotgun sequencing to generate partial genomic sequences of unsequenced species, this approach is readily transferable to non‐model organisms

High-Resolution Genotyping via Whole Genome Hybridizations to Microarrays Containing Long Oligonucleotide Probes

To date, microarray-based genotyping of large, complex plant genomes has been complicated by the need to perform genome complexity reduction to obtain sufficiently strong hybridization signals. Genome complexity reduction techniques are, however, tedious and can introduce unwanted variables into genotyping assays. Here, we report a microarray-based genotyping technology for complex genomes (such as the 2.3 GB maize genome) that does not require genome complexity reduction prior to hybridization. Approximately 200,000 long oligonucleotide probes were identified as being polymorphic between the inbred parents of a mapping population and used to genotype two recombinant inbred lines. While multiple hybridization replicates provided ∼97% accuracy, even a single replicate provided ∼95% accuracy. Genotyping accuracy was further increased to >99% by utilizing information from adjacent probes. This microarray-based method provides a simple, high-density genotyping approach for large, complex genomes

HSP90-buffered genetic variation is common in Arabidopsis thaliana

HSP90 is a protein chaperone particularly important in the maturation of a diverse set of proteins that regulate key steps in a multitude of biological processes. Alterations in HSP90 function produce altered phenotypes at low penetrance in natural populations. Previous work has shown that at least some of these phenotypes are due to genetic variation that remains phenotypically cryptic until it is revealed by the impairment of HSP90 function. Exposure of such “buffered” genetic polymorphisms can also be accomplished by environmental stress, linking the appearance of new phenotypes to defects in protein homeostasis. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. In evaluating this hypothesis, a key unknown factor is the frequency with which HSP90-buffered polymorphisms occur in natural populations. Here, we present Arabidopsis thaliana populations suitable for genetic mapping that have constitutively reduced HSP90 levels. We employ quantitative genetic techniques to examine the HSP90-dependent polymorphisms affecting a host of plastic plant life-history traits. Our results demonstrate that HSP90-dependent natural variation is present at high frequencies in A. thaliana, with an expectation that at least one HSP90-dependent polymorphism will affect nearly every quantitative trait in progeny of two different wild lines. Hence, HSP90 is likely to occupy a central position in the translation of genotypic variation into phenotypic differences

Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from post-mortem brain, generating 3,227 sets of single neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish novel and orthologous neuronal subtypes as well as regional identity within the human brain

Detection and resolution of genetic loci affecting circadian period in Brassica oleracea

Circadian rhythms regulate many aspects of plant growth, Wtness and vigour. The components and detailed mechanism of circadian regulation to date have been dissected in the reference species Arabidopsis thaliana. To determine the genetic basis and range of natural allelic variation for intrinsic circadian period in the closest crop relatives, we used an accurate and high throughput data capture system to record rhythmic cotyledon movement in two immortal segregating populations of Brassica oleracea, derived from parent lines representing diVerent crop types. Periods varied between 24.4 and 26.1 h between the parent lines, with transgressive segregation between extreme recombinant lines in both populations of »3.5 h. The additive eVect of individual QTL identiWed in each population varied from 0.17 to 0.36 h. QTL detected in one doubled haploid population were veriWed and the mapping intervals further resolved by determining circadian period in genomic substitution lines derived from the parental lines. Comparative genomic analysis based on collinearity between Brassica and Arabidopsis also allowed identiWcation of candidate orthologous genes known to regulate period in Arabidopsis, that may account for the additive circadian eVects of speciWc QTL. The distinct QTL positions detected in the two populations, and the extent of transgressive segregation suggest that there is likely to be considerable scope for modulating the range of available circadian periods in natural populations and crop species of Brassica. This may provide adaptive advantage for optimising growth and development in diVerent latitudes, seasons or climate conditions