Caenorhabditis briggsae Recombinant Inbred Line Genotypes Reveal Inter-Strain Incompatibility and the Evolution of Recombination

The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes

Characterization of the Photoconversion on Reaction of the Fluorescent Protein Kaede on the Single-Molecule Level

Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with λ = 350–410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (λ = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at λ = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s(−1) for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed

Rapid and Inexpensive Whole-Genome Genotyping-by-Sequencing for Crossover Localization and Fine-Scale Genetic Mapping

The reshuffling of existing genetic variation during meiosis is important both during evolution and in breeding. The reassortment of genetic variants relies on the formation of crossovers (COs) between homologous chromosomes. The pattern of genome-wide CO distributions can be rapidly and precisely established by the short-read sequencing of individuals from F(2) populations, which in turn are useful for quantitative trait locus (QTL) mapping. Although sequencing costs have decreased precipitously in recent years, the costs of library preparation for hundreds of individuals have remained high. To enable rapid and inexpensive CO detection and QTL mapping using low-coverage whole-genome sequencing of large mapping populations, we have developed a new method for library preparation along with Trained Individual GenomE Reconstruction, a probabilistic method for genotype and CO predictions for recombinant individuals. In an example case with hundreds of F(2) individuals from two Arabidopsis thaliana accessions, we resolved most CO breakpoints to within 2 kb and reduced a major flowering time QTL to a 9-kb interval. In addition, an extended region of unusually low recombination revealed a 1.8-Mb inversion polymorphism on the long arm of chromosome 4. We observed no significant differences in the frequency and distribution of COs between F(2) individuals with and without a functional copy of the DNA helicase gene RECQ4A. In summary, we present a new, cost-efficient method for large-scale, high-precision genotyping-by-sequencing