Sequencing and De Novo Assembly of the Toxicodendron radicans (Poison Ivy) Transcriptome

Abstract

Contact with poison ivy plants is widely dreaded because they produce a natural product called urushiol that is responsible for allergenic contact delayed-dermatitis symptoms lasting for weeks. For this reason, the catchphrase most associated with poison ivy is "leaves of three, let it be", which serves the purpose of both identification and an appeal for avoidance. Ironically, despite this notoriety, there is a dearth of specific knowledge about nearly all other aspects of poison ivy physiology and ecology. As a means of gaining a more molecular-oriented understanding of poison ivy physiology and ecology, Next Generation DNA sequencing technology was used to develop poison ivy root and leaf RNA-seq transcriptome resources. De novo assembled transcriptomes were analyzed to generate a core set of high quality expressed transcripts present in poison ivy tissue. The predicted protein sequences were evaluated for similarity to SwissProt homologs and InterProScan domains, as well as assigned both GO terms and KEGG annotations. Over 23,000 simple sequence repeats were identified in the transcriptome, and corresponding oligo nucleotide primer pairs were designed. A pan-transcriptome analysis of existing Anacardiaceae transcriptomes revealed conserved and unique transcripts among these species.

Keywords: Anacardiaceae; Toxicodendron radicans; contact dermatitis; poison ivy; skin rash; transcriptome; urushiol.

Figure 1

Process for de novo RNA-seq assembly and annotation. RNA from two replicates each of roots and leaves was sequenced with an Illumina HiSeq 2000. Trinity RNA seq was used to de novo assemble a reference transcriptome from the combined paired reads from all four T. radicans leaves and roots samples. Assembled transcripts were then annotated using a variety of programs.

Figure 2

Transcript taxonomy annotation summary as determined by blobtools and BLASTX searches of the NCBI nr database. Transcript taxonomy annotation summary as determined by blobtools and BLASTX searches of the NCBI nr database for the four-sample de novo assembly. Annotated transcripts are displayed as percent of paired reads mapped to assembled transcripts of a given taxonomic grouping. (A) Leaves A sample summary; (B) Leaves B sample summary; (C) Roots A sample summary; (D) Roots B sample summary.

Figure 3

Assembly transcript length distribution and estimation of expressed transcripts. a. Assembly transcript length distribution. (A) Histogram shows the binned counts of the lengths of individual transcripts represented by 50 bp bins. Sequences with length greater than 5000 were binned together. The minimum transcript length reported by Trinity is 200 bp. (B) Estimation of expressed number of transcripts supported by the sequenced dataset. Plotted as circles are the number of transcripts with a minimum transcripts per million (TPM) value in any one sample at each threshold (plotted as negative values). A linear regression model fit to values between –100 and –10 neg. min. TPM is shown as a green line. The y-intercept (7753.52) indicates the estimated number of expressed transcripts supported by the sequenced dataset.

Figure 4

BLAST search results and species distribution. (A) E-value distribution for best BLASTX hit of transcripts against the SwissProt database. (B) Distribution of species for best BLASTX hit of transcripts against the SwissProt database.

Figure 5

Distribution of top GO term categories. Distribution of most abundant GO terms for each of three major GO term hierarchies from GO terms assigned to T. radicans transcripts.

Figure 6

Anacardiaceae pan-transcriptome Venn diagram. Counts of orthologous transcripts shared by one or more members of Anacardiaceae are represented in each segment. The count of orthologous transcripts shared by all (core) is colored red. Regions are not to scale.

Figure 7

Most abundant SSR sequence motifs. Distribution of the most abundant SSR repeat motif sequences identified in assembled transcripts.