Transcriptome Profiling in Syntrichia
We propose a complete transcriptomic profiling study for S. caninervis to fully document the genetic responses associated with sex, developmental state, hydration state, and temperature. These profiles will provide the necessary genomics resources to support our objectives throughout the remainder of the proposed project and help annotate the genomes. Using the same cultures that will be the source for the genomic DNA, we will construct a total of 48 barcoded HiSeq RNA libraries that will be multiplexed to run 10 samples per lane of Illumina HiSeq 2500, yielding approximately 200 M reads per sample. RNA will be isolated from the following S. caninervis triplicated samples for both male and female cultures: hydrated shoots, dehydrated shoots (50% RWC), desiccated shoots, desiccated shoots (polysomal fractions), rehydrated shoots, juvenile shoots, high temperature (slowly adapted to the upper limit of the natural temperature range) [shoots] and low temperature (slowly adapted to the lowest limit of the natural temperature range) [shoots] for a total of 8 treatments × 3 biological reps × 2 sexes = 48 libraries. The libraries for the polysomal fraction of dry shoots will enable us to sample the mRNP sequestered transcript pools in both the male and female gametophytes as described in Wood and Oliver (1999).
We do not expect the transcriptomes of S. caninervis and S. ruralis to differ in any major fashion but we will validate this assumption by sequencing duplicate barcoded S. ruralis HiSeq libraries for selected treatments: a total of ten libraries (hydrated (male and female), dry, high temperature adapted, low temperature adapted).
Transcripts will be assembled de novo for structural gene model annotations using SOAPdenovoTrans (http://soap.genomics.org.cn/SOAPdenovo-Trans.html), mapped to the assembled genome sequence using the GSNAP gapped aligner (http://www.gvst.co.uk/gsnap.htm), and expression quantified using Cufflinks (http://cufflinks.cbcb.umd.edu). The transcriptome analyses will allow us to develop qPCR-based phenotypic trait analyses more aptly described as “signature transcriptomic assays”: suites of genes whose transcript abundance patterns characterize the sex-specific responses of the moss to hydration level, temperature, and developmental cues. The assays will be developed from the transcriptomic differential expression analyses that define expression of clusters of genes whose transcript abundance patterns are specific to the particular treatment, developmental stage, and sex of the moss. The number of genes within each signature suite will be determined empirically in sufficient numbers to define the transcriptome in relation to treatment, developmental stage, and sex. We will develop gene specific qPCR primer sets for each signature gene suite, using PRIMEGENS (http://digbio.missouri.edu/primegens/), that we can use in a high-throughput qPCR based assay (384 well plate design) to determine the particular biological status of field collected material from our ecological, physiological, and community level inquiries. The qPCR analysis will be accomplished using our standard protocols for analysis with the ABI PRISM TM 7700 Sequence Detection System. The qPCRbased “signature transcriptomic assays” should allow for the rapid assessment of genome level responses to treatments and field conditions of large numbers of samples in a reasonable timeframe.