Genetic Interaction Score (S-Score) Calculation, Clustering, and Visualization of Genetic Interaction Profiles for Yeast.

This protocol describes computational analysis of genetic interaction screens, ranging from data capture (plate imaging) to downstream analyses. Plate imaging approaches using both digital camera and office flatbed scanners are included, along with a protocol for the extraction of colony size measurements from the resulting images. A commonly used genetic interaction scoring method, calculation of the S-score, is discussed. These methods require minimal computer skills, but some familiarity with MATLAB and Linux/Unix is a plus. Finally, an outline for using clustering and visualization software for analysis of resulting data sets is provided.

Genetic Interaction Screens in Schizosaccharomyces pombe Using the Pombe Epistasis Mapper (PEM) System and a Manual Colony Replicator.

In laboratories in which a colony-replicating robot is not available, manual replication provides a good, low-cost alternative for genetic interaction screening using the Pombe Epistasis Mapper (PEM) system. The protocol presented here describes the minimum number of steps required to identify genetic interactions. First, a query deletion is introduced to a library of deletion mutants by mating. Through a series of subsequent selection steps, single and double mutants are isolated and analyzed.

Local adaptation of sex induction in a facultative sexual crustacean: insights from QTL mapping and natural populations of Daphnia magna.

Dormancy is a common adaptation in invertebrates to survive harsh conditions. Triggered by environmental cues, populations produce resting eggs that allow them to survive temporally unsuitable conditions. Daphnia magna is a crustacean that reproduces by cyclical parthenogenesis, alternating between the production of asexual offspring and the sexual reproduction of diapausing eggs (ephippia). Prior to ephippia production, males (necessary to ensure ephippia fertilization) are produced parthenogenetically. Both the production of ephippia and the parthenogenetic production of males are induced by environmental factors. Here, we test the hypothesis that the induction of D. magna resting egg production shows a signature of local adaptation. We postulated that Daphnia from permanent ponds would produce fewer ephippia and males than Daphnia from intermittent ponds and that the frequency and season of habitat deterioration would correlate with the timing and amount of male and ephippia production. To test this, we quantified the production of males and ephippia in clonal D. magna populations in several different controlled environments. We found that the production of both ephippia and males varies strongly among populations in a way that suggests local adaptation. By performing quantitative trait locus mapping with parent clones from contrasting pond environments, we identified nonoverlapping genomic regions associated with male and ephippia production. As the traits are influenced by two different genomic regions, and both are necessary for successful resting egg production, we suggest that the genes for their induction co-evolve.

The first-generation Daphnia magna linkage map.

BACKGROUND: Daphnia magna is a well-established model species in ecotoxicology, ecology and evolution. Several new genomics tools are presently under development for this species; among them, a linkage map is a first requirement for estimating the genetic background of phenotypic traits in quantitative trait loci (QTL) studies and is also very useful in assembling the genome. It also enables comparative studies between D. magna and D. pulex, for which a linkage map already exists. RESULTS: Here we describe the first genetic linkage map of D. magna. We generated 214 F2 (intercross) clonal lines as the foundation of the linkage analysis. The linkage map itself is based on 109 microsatellite markers, which produced ten major linkage groups ranging in size from 31.1 cM to 288.5 cM. The total size of this linkage map extends to 1211.6 Kosambi cM, and the average interval for the markers within linkage groups is 15.1 cM. The F2 clones can be used to map QTLs for traits that differ between the parental clones. We successfully mapped the location of two loci with infertility alleles, one inherited from the paternal clone (Iinb1) and the other from the maternal clone (Xinb3). CONCLUSIONS: The D. magna linkage map presented here provides extensive coverage of the genome and a given density of markers that enable us to detect QTLs of moderate to strong effects. It is similar in size to the linkage map of D. pulex.

Intragenic tandem repeats in Daphnia magna: structure, function and distribution.

BACKGROUND: Expressed sequence tag (EST) databases provide a valuable source of genetic data in organisms whose genome sequence information is not yet compiled. We used a published EST database for the waterflea Daphnia magna (Crustacea:Cladocera) to isolate variable number of tandem repeat (VNTR) markers for linkage mapping, Quantitative Trait Loci (QTL), and functional studies. FINDINGS: Seventy-four polymorphic markers were isolated and characterised. Analyses of repeat structure, putative gene function and polymorphism indicated that intragenic tandem repeats are not distributed randomly in the mRNA sequences; instead, dinucleotides are more frequent in non-coding regions, whereas trinucleotides (and longer motifs involving multiple-of-three nucleotide repeats) are preferentially situated in coding regions. We also observed differential distribution of repeat motifs across putative genetic functions. This indicates differential selective constraints and possible functional significance of VNTR polymorphism in at least some genes. CONCLUSION: Databases of VNTR markers situated in genes whose putative function can be inferred from homology searches will be a valuable resource for the genetic study of functional variation and selection.

The Dscam homologue of the crustacean Daphnia is diversified by alternative splicing like in insects.

In insects, the homologue of the Down syndrome cell adhesion molecule (Dscam) is a unique case of a single-locus gene whose expression has extensive somatic diversification in both the nervous and immune systems. How this situation evolved is best understood through comparative studies. We describe structural, expression, and evolutionary aspects of a Dscam homolog in 2 species of the crustacean Daphnia. The Dscam of Daphnia generates up to 13,000 different transcripts by the alternative splicing of variable exons. This extends the taxonomic range of a highly diversified Dscam beyond the insects. Additionally, we have identified 4 alternative forms of the cytoplasmic tail that generate isoforms with or without inhibitory or activating immunoreceptor tyrosine-based motifs (ITIM and ITAM respectively), something not previously reported in insect's Dscam. In Daphnia, we detected exon usage variability in both the brain and hemocytes (the effector cells of immunity), suggesting that Dscam plays a role in the nervous and immune systems of crustaceans, as it does in insects. Phylogenetic analysis shows a high degree of amino acid conservation between Daphnia and insects except in the alternative exons, which diverge greatly between these taxa. Our analysis shows that the variable exons diverged before the split of the 2 Daphnia species and is in agreement with the nearest-neighbor model for the evolution of the alternative exons. The genealogy of the Dscam gene family from vertebrates and invertebrates confirmed that the highly diversified form of the gene evolved from a nondiversified form before the split of insects and crustaceans.

Using molecular and quantitative variation for assessing genetic impacts on Nucella lapillus populations after local extinction and recolonization.

The dogwhelk Nucella lapillus is a predatory marine gastropod living on rocky shores in the North Atlantic. As with many other gastropod species, Nucella was affected by tributyltin (TBT) pollution during the 1970s and 1980s, and local populations underwent extinction. After a partial ban on TBT in the UK in 1987, vacant sites have been recolonized. Levels of genetic diversity and quantitative genetic variation in shell form were compared between recolonized sites and sites that showed continuous population at three localities across the British Isles. Overall, estimates of genetic diversity were only slightly lower in recolonized populations, suggesting that populations have recovered from previous impacts due to the relatively high levels of migration from non-impacted sites. Molecular and quantitative analyses are broadly concordant and a positive correlation was observed (although not statistically significant) between molecular and quantitative estimates of genetic diversity, indicating the potential usefulness of quantitative methods to complement molecular population genetics analyses.

Effects of reciprocal chromosomal translocations on the fitness of Saccharomyces cerevisiae.

Yeast species have undergone extensive genome reorganization in their evolutionary history, including variations in chromosome number and large chromosomal rearrangements, such as translocations. To determine directly the contribution of chromosomal translocations to the whole organism's fitness, we devised a strategy to construct in Saccharomyces cerevisiae collinear "evolutionary mimics" of other species originally differing by the presence of reciprocal translocations in their genome. A modification of the Cre/loxP system was used to create in S. cerevisiae the translocations detected in the sibling species Saccharomyces mikatae IFO 1815 and 1816. Competition experiments under different physiological conditions showed that the translocated strains of S. cerevisiae consistently outcompeted the reference S. cerevisiae strain with no translocation, both in batch and chemostat culture, especially under glucose limitation. These results indicate that chromosomal translocations in Saccharomyces may have an adaptive significance, and lend support to a model of fixation by natural selection of reciprocal translocations in Saccharomyces species.

Engineering evolution to study speciation in yeasts.

The Saccharomyces 'sensu stricto' yeasts are a group of species that will mate with one another, but interspecific pairings produce sterile hybrids. A retrospective analysis of their genomes revealed that translocations between the chromosomes of these species do not correlate with the group's sequence-based phylogeny (that is, translocations do not drive the process of speciation). However, that analysis was unable to infer what contribution such rearrangements make to reproductive isolation between these organisms. Here, we report experiments that take an interventionist, rather than a retrospective approach to studying speciation, by reconfiguring the Saccharomyces cerevisiae genome so that it is collinear with that of Saccharomyces mikatae. We demonstrate that this imposed genomic collinearity allows the generation of interspecific hybrids that produce a large proportion of spores that are viable, but extensively aneuploid. We obtained similar results in crosses between wild-type S. cerevisiae and the naturally collinear species Saccharomyces paradoxus, but not with non-collinear crosses. This controlled comparison of the effect of chromosomal translocation on species barriers suggests a mechanism for the generation of redundancy in the S. cerevisiae genome.