Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration

Abstract

BackgroundThe naid annelids contain a number of species that vary in their ability to regenerate lost body parts, making them excellent candidates for evolution of regeneration studies. However, scant sequence data exists to facilitate such studies. We constructed a cDNA library from the naid Pristina leidyi, a species that is highly regenerative and also reproduces asexually by fission, using material from a range of regeneration and fission stages for our library. We then sequenced the transcriptome of P. leidyi using 454 technology.Results454 sequencing produced 1,550,174 reads with an average read length of 376 nucleotides. Assembly of 454 sequence reads resulted in 64,522 isogroups and 46,679 singletons for a total of 111,201 unigenes in this transcriptome. We estimate that over 95% of the transcripts in our library are present in our transcriptome. 17.7% of isogroups had significant BLAST hits to the UniProt database and these include putative homologs of a number of genes relevant to regeneration research. Although many sequences are incomplete, the mean sequence length of transcripts (isotigs) is 707 nucleotides. Thus, many sequences are large enough to be immediately useful for downstream applications such as gene expression analyses. Using in situ hybridization, we show that two Wnt/β-catenin pathway genes (homologs of frizzled and β-catenin) present in our transcriptome are expressed in the regeneration blastema of P. leidyi, demonstrating the usefulness of this resource for regeneration research.Conclusions454 sequencing is a rapid and efficient approach for identifying large numbers of genes in an organism that lacks a sequenced genome. This transcriptome dataset will be a valuable resource for molecular analyses of regeneration in P. leidyi and will serve as a starting point for comparisons to non-regenerating naids. It also contributes significantly to the still limited genomic resources available for annelids and lophotrochozoans more generally.

Characterization of Liaoning Cashmere Goat Transcriptome: Sequencing, De Novo Assembly, Functional Annotation and Comparative Analysis

“…Most isotigs (93.24%) were > 500 bp in length, and 40.64% of them were > 1000 bp. The average length of the Liaoning cashmere goat isotigs (1,011 bp) was longer than most of those assembled in other non-model organisms, such as Melitaea cinxia (197 bp) [30], Acropora millepora (440 bp) [31], P. contorta (500 bp) [32], Pristina leidyi (707 bp) [33], and Megalobrama amblycephala (730 bp) [19], but they were shorter than the average length in Junco hyemalis (1,248 bp) [34]. About 112,148 (18.70%) reads did not assembled into isotigs.…”

Large scale changes in the transcriptome of Eisenia fetida during regeneration

“…Gene expression profiling in anterior regenerating segments of Perionyx excavatus also yielded several novel ESTs, involved in regeneration[ 71 ]. Transcriptomics analysis of a mixed-stage sample of regeneration and fission in Pristina leidyi has also been recently attempted [ 72 ]. In our study, we found that 315 amongst the 9,645 genes upregulated during regeneration had no known ortholog.…”

Regeneration mechanisms in Syllidae (Annelida)

“…Emerging genomic methods will be useful to understand this process. By sequencing genomes and transcriptomes, genes involved in regeneration can be identified (Bhambri et al, 2017;Myohara et al, 2006;Nyberg et al, 2012), thereby opening up the possibility of a functional characterization, e.g., by CRISPR or RNAi methods (Boettcher & McManus, 2015). Different studies of annelid regeneration identified the expression of genes such as engrailed, wingless (Wnt), orthodenticle homologs, markers of germline cells (e.g.…”