Comment[ArrayExpressAccession] E-GEOD-54819 MAGE-TAB Version 1.1 Public Release Date 2014-02-15 Investigation Title De novo transcriptome analysis profiles gene expression underlying seasonal polyphenism in butterfly wing patterns Comment[Submitted Name] De novo transcriptome analysis profiles gene expression underlying seasonal polyphenism in butterfly wing patterns Experiment Description In the eastern United States the buckeye butterfly, Junonia coenia, shows a seasonal wing color polyphenism where adults emerging in the spring are pale brown, while those emerging in the autumn are dark red. This variation can be artificially induced in laboratory colonies, thus making J. coenia a useful model system to examine the developmental basis of phenotypic plasticity. We used RNA-seq to generate the first set of assembled transcripts for this species while simultaneously quantifying relative gene expression associated with development of alternative seasonal color morphs. The assembled consolidated wing transcriptome was 77.55 Mb. 16,251 contigs of over 1000bp in length were assembled, of which 3,145 were differentially expressed between stages and/or color morphs. Depending on the developmental stage, between 547 and 1420 transcripts were significantly differentially expressed between brown and red wing morphs. These extensive differences in gene expression stand in stark contrast to the much smaller numbers found in previous studies on genetic wing pattern variation, and suggest that environmentally induced phenotypic shifts may arise from very broad systemic processes. Overall gene ontology (GO) analyses revealed that genes associated with structural constituents of ribosomes and oxygen transport were significantly upregulated in the pale brown morph, while genes associated with peptidase activity were very significantly upregulated in the dark red morph. Focused analyses of candidate endocrine and pigmentation pathways revealed a number of notable genes upregulated in the red morph, including several ecdysone-related genes and cinnabar, an ommochrome pigment gene implicated in color pattern variation in other butterflies. Surprisingly, we found numerous melanin-related transcripts, including tan and yellow-family genes, strongly upregulated in the red morph, leading us to speculate that red pigmentation in autumn J. coenia may include red or brown melanins in addition to ommochromes. While we identified several endocrine and pigmentation genes as obvious candidates for color morph differentiation, we speculate that the majority of gene expression differences we observed were due to thermal stress response. The buckeye transcriptome provides a basis for further developmental studies of phenotypic plasticity. mRNA profiling of hind wings from 4 developmental stages of two color morphs (Rosa and Linea) of the buckeye butterfly (J. coenia), generated by deep sequencing, in triplicate, using Illumina GAII or HiSeq 2000. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Mortazavi Daniels Murad Mortazavi Reed Person First Name Ali Emily Rabi Ali Robert Person Mid Initials V D Person Email ali.mortazavi@uci.edu Person Affiliation University of California Irvine Person Address Developmental & Cell Biology, University of California Irvine, 2300 Biological Sciences III, Irvine, California, USA Person Roles submitter Protocol Name P-GSE54819-4 P-GSE54819-1 P-GSE54819-3 P-GSE54819-2 Protocol Description Each dataset was filtered with Khmer (parameters: -C 20 -k 20 -N 4 -x 2e9 ) to even out coverage, remove redundant reads and to remove reads with sequencing errors. Replicates of each developmental stage of each color morph were pooled and assembled into developmental stage transcriptomes using Oases v.1.2.03 with hash lengths ranging from 29 to 61 bp. The developmental stage transcriptomes were consolidated using CD-HIT-EST of CD-HIT package v.4.5.7 (parameters: ) into a reference transcriptome. All transcripts longer than 1 Kb were kept for subsequent analyses. #1 mate of the paired-end reads were mapped to the reference transcriptome using Bowtie v.0.12.7 (parameters: -aS --offrate 1 -e 200) and transcript expression levels were obtained using eXpress v.1.0.0 (default options). Supplementary_files_format_and_content: tab-delimited text file with RPKM values for the indicated samples (no measurements for samples 1,8,11,17) Hindwings from four developmental stages were sampled for both color morphs.The four stages sampled include imaginal disks from fifth instar larvae, pre-pigment pupa, early (yellow) ommochrome development, and late (orange/red) ommochrome development). To preserve mRNA quality, wings were rapidly dissected into RNAlater (Life Technologies, Grand Island, NY), incubated for ~24h at 4oC to allow the solution to permeate tissues, and then stored at -80oC until mRNA extraction. Total RNA was extracted from wing tissues using RNeasy Mini Kit - spin column protocol (QIAGEN, Valencia, CA). TURBOtm DNase treatment (Life Technologies, Grand Island, NY) was then used to degrade contaminating DNA and mRNA was purified from the total RNA using the Oligotex mRNA Mini Kit (QIAGEN, Valencia, CA). The OvationM-. RNA-seq system (NuGEN, San Carlos, CA) was used to prepare double stranded cDNA from mRNA and this cDNA product was then purified with QIAquick PCR purification columns (QIAGEN, Valencia, CA). The purified double stranded cDNA was sheared by a Covaris Ultrasonicator to 300bp lengths. The EncoreTM NGS Multiplex System I (NuGEN, San Carlos, CA) was used for final library preparation. Multiplexed libraries were sequenced as 75 bp paired-end Illumina reads or 100 bp paired-end Illumina reads. A population of J. coenia from Durham, North Carolina (35M-059M-bM-^@M-^Y19M-bM-^@M-^]N, 78M-054M-bM-^@M-^Y26M-bM-^@M-^]W) was reared in incubators, and fed a standard artificial diet (Nijhout 1980). Two sets of conditions known to induce alternative color morphs in North Carolina populations (Smith 1991) were applied: 21M-0C with 8 hr light per day for short/cool day conditions, and 27M-0C with 16 hr light for long/warm day conditions. Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name DEVELOPMENTAL STAGE Experimental Factor Type developmental stage Comment[SecondaryAccession] GSE54819 Comment[GEOReleaseDate] 2014-02-15 Comment[ArrayExpressSubmissionDate] 2014-02-10 Comment[GEOLastUpdateDate] 2014-02-15 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE54819_JC_RPKM.txt Comment[SecondaryAccession] SRP037531 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1166407-SRR1166426 SDRF File E-GEOD-54819.sdrf.txt