Project description:In order to test the hypothesis that adult hybrid misexpression results from the cascading effect of earlier-expressed developmentally important improperly regulated genes, as well as address whether Von Baer’s 3rd law (suggesting that earlier stages of development should be more similar between species than later stages) holds at the level of gene expression, we conducted whole-transcriptome Drosophila melanogaster cDNA microarray-based expression profiling of males of D. melanogaster, D. sechellia, and D. simulans, at four synchronized developmental time-points (3rd instar larval [larval], early pupal, late pupal, and newly-emerged adult [adult]). D. simulans and D. sechellia shared a most recent common ancestor (MRCA) ~0.5 to 1.0 million years ago (MYA) and form a clade that shared an MRCA with D. melanogaster approximately 5.4 MYA. In addition, we also performed the same analysis on the male interspecific F1 hybrids of the D. simulans (♀) × D. sechellia (♂) cross.
Project description:Curration of small RNAs from four melanogaster-subgroup species (Drosophila simulans, Drosophila sechellia, Drosophila erecta, and Drosophila yakuba) for the purpose of non-coding RNA annotation and comparative genomics assessment.
Project description:Y chromosome from different Drosophila simulans were introgressed into the same genetic background. Strains showing distinct sex-ratio distortion were clustered according to the ratio of males and females observed in the progeny. Strains showing disproportionally high number of female flies in the progeny were contrasted with strains displaying the proportion of male and females flies close to 1:1. In addition, interspecific Y chromosome from Drosophila sechellia (Ysec) was compared with D. simulans Y chromosomes. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). A minimum of 2 biological replicas was used, however this number varied according to the hybridization design (See 'Branco et. al, Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans' for details of the hybridization design). Microarrays were ~18,000-feature cDNA arrays spotted with D. melanogaster cDNA PCR products. We have recently annotated these probes using the D. simulans genome. Possible biases introduced by using a D. melanogaster platform for a D. simulans genome are minimized because the probed genomes are essentially identical except for the Y chromosomes. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma.
Project description:We designed this experiment to investigate the transcriptional changes in gonads as a result of sex transformation. Here we performed transcriptional profiling of the ovary transformed into testis from the tra loss of function (XX_tra_lof), testis transformed into ovary from the tra gain of function (XY_tra_gof) and ovary transformed into testis in dsxM gain of function (XX_DsxM_gof/lof) Drosophila melanogaster third instar larvae in biological quadruplicates. In addition, as controls we sequenced ovaries and testes from the female and male wildtype larvae respectively. We constructed polyA+ libraries of the gonads, cleaned off the fatbody and performed 50 bp, stranded single-end RNA-Seq.
Project description:Y chromosome from different Drosophila simulans were introgressed into the same genetic background. Strains showing distinct sex-ratio distortion were clustered according to the ratio of males and females observed in the progeny. Strains showing disproportionally high number of female flies in the progeny were contrasted with strains displaying the proportion of male and females flies close to 1:1. In addition, interspecific Y chromosome from Drosophila sechellia (Ysec) was compared with D. simulans Y chromosomes. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). A minimum of 2 biological replicas was used, however this number varied according to the hybridization design (See 'Branco et. al, Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans' for details of the hybridization design). Microarrays were ~18,000-feature cDNA arrays spotted with D. melanogaster cDNA PCR products. We have recently annotated these probes using the D. simulans genome. Possible biases introduced by using a D. melanogaster platform for a D. simulans genome are minimized because the probed genomes are essentially identical except for the Y chromosomes. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma. Dye "swaps," loop design.
Project description:In order to characterize duplication polymorphisms in Drosophila simulans, we applied comparative genome hybridization (CGH) using tiling arrays originally designed to cover the full euchromatic genome of its sister species D. melanogaster. We only used the ~900,000 probes in the tiling arrays that had a perfect and unique match to the D. simulans genome (droSim1). We inferred copy number changes with a Hidden Markov Model (HMM) that returned the posterior probabilities for copy number by comparing DNA hybridization intensities between natural isolates. The probabilities of mutation were parsed to make duplication calls. The supplementary file linked to each Sample record contains for each probe, its location in the D. simulans genome and its posterior probability of being duplicated (output from the Hiddem Markov Model)
Project description:High-throughput sequencing of Drosophila pseudoobscura and Drosophila simulans small RNAs. ~18-26nt RNAs were isolated from total RNA using PAGE, ligation to adapters requires 5' monophosphate and 3' OH.