Unknown,Transcriptomics,Genomics,Proteomics

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Identification of sex-biased genes in Drosophila using gonad RNAseq data

ABSTRACT: Purpose: Accurate identification of sex-biased genes requires precise measurement of gene expression levels in gonads. This study is designed to provide such data for various Drosophila species to enhance studies of sex-biased gene expression and evolution across the genus. Methods: Virgin flies were collected and aged 6-10 days before dissecting 2-3 replicates of testes or ovaries. Total RNA was extracted using the Arcturus® PicoPure® kit . Illumina® TruSeq® RNA library kits were used to poly-A+ select and reverse-transcribe mRNA, shear cDNA into ~120 bp fragments, and produce libraries for 1x50 bp sequencing on an Illumina GAIIx or HiSeq2000. Illumina®’s Real Time Analysis v1.13 module processed images, called bases, and provided base qualities. Reads were mapped to the current reference genomes using Bowtie v2.1.0 (Langmead and Salzberg, 2012, Nat Meth) with default settings. Differentially expressed genes were detected using Cufflinks v 2.1.0 (Trapnell et al., 2010, Nat Biotech; default settings) or edgeR (Robinson et al., 2010, Bioinformatics; full-quantile GC-content normalization and full-quantile between-sample normalization). Genes were called differentially expressed at a Benjamini-Hochberg false discovery rate of 0.01. Results: Thousands of male- and female-biased genes were detected for each species using both DE detection methods. These results provide a significant improvement in sensitivity of sex-biased gene detection relative to using whole-body RNA-sequencing data. These data provide a foundation for accurate identification of sex-biased genes throughout the Drosophila genus. Testis and ovary samples from Drosophila species were sequenced 1 x 50 bp in duplicate from 6-10 day old virgin, Wolbachia-free adult flies on an Illumina GAIIx or HiSeq2000.

ORGANISM(S): Drosophila yakuba

SUBMITTER: Maria Vibranovski

PROVIDER: E-GEOD-52058 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:African clawed frog Xenopus sp. is used extensively for developmental biology and toxicology research. Amid concerns of environmental pollutants disrupting endocrine systems and causing altered reproductive development in wildlife, eco-toxicology research has led to a focus on linking molecular initiating events to population-level effects. As such, efforts to better understand reproductive development at the molecular level in these model species are warranted. To that end, transcriptomes were characterized in differentiating Xenopus tropicalis gonad tissues at Nieuwkoop and Faber (NF) stage 58 (pro-metamorphosis), NF66 (completion of metamorphosis), one week post-metamorphosis (1WPM), and two weeks post-metamorphosis (2WPM). Differential expression analysis between tissue types at each developmental stage revealed a substantial divergence of ovary and testis transcriptomes starting between NF58 and NF66; transcriptomes continued to diverge through 2WPM. Generally, testis-enriched transcripts were expressed at relatively constant levels, while ovary-enriched transcripts were up-regulated within this developmental period. Functional analyses of differentially expressed transcripts allowed linkages to be made between their putative human orthologues and specific cellular processes associated with differentiating gonad tissues. In ovary tissue, genetic programs direct germ cells through meiosis to the diplotene stage when maternal mRNAs are transcribed and trafficked to oocytes for translation following fertilization. In the testis, gene expression is consistent with connective tissue development, tubule formation, and germ cell support (Leydig and Sertoli cells). This dataset exhibited remarkable consistency with transcript profiles previously described in gonad tissues across species, and emphasizes the universal importance of certain transcripts for germ cell development and preparation of these tissues for reproduction. Four developmental time-points were considered for global expression analysis: NF58 (pro-metamorphosis), NF66 (completion of metamorphosis), one week post-metamorphosis (1WPM), and two weeks post-metamorphosis (2WPM). Gonad tissues were time-matched and stage-matched, so collection occurred on a single day for each developmental time-point and all animals used were from the same spawn. For the 1WPM and 2WPM time-points, all animals reached NF66 on the same day and were separated from the rest of the spawn until sample collection one and two weeks later respectively. On the day of tissue collection, animals were developmentally staged (Nieuwkoop and Faber, 1994) and euthanized by submersion in a lethal concentration of tricane methanesulfonate ([MS-222], Argent Chemical Laboratories, Redmond, WA, USA) buffered with sodium bicarbonate. Gonad tissues were then excised independently of any other tissues (i.e. “pure” gonad tissue) and transferred to a separate vial for each individual, homogenized in lysis buffer from an RNeasy micro kit (Qiagen Inc., Valencia, CA, USA), and immediately frozen on dry ice until samples could be moved to -80oC storage. Tail or toe tissue was also collected from each individual for genetic sex determination using methods previously described by Olmstead et al. (2010). Once genetic sex was determined for each individual, testes and ovaries were randomly assigned to pools of four individuals (eight gonads per pool) to make a total of five pools of testis tissue and five pools of ovary tissue at each developmental time-point, with the exception of NF58 which had four pools of testis tissue and four pools of ovary tissue (38 total sample pools).

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Identification of sex-biased genes in Drosophila using gonad RNAseq data

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