A survey of the full-length transcriptome of Gracilariopsis lemaneiformis using single-molecule long-read sequencing

Abstract

Background Posttranscriptional processing of precursor mRNAs contributes to transcriptome and protein diversity and gene regulatory mechanisms in eukaryotes. However, this posttranscriptional mechanism has not been studied in the marine macroalgae Gracilariopsis lemaneiformis, which is the most cultivated red seaweed species in China. Results In the present study, third-generation sequencing (Pacific Biosciences single-molecule real-time long-read sequencing, SMRT-Seq) was used to sequence the full-length transcriptome of G. lemaneiformis to identify alternatively spliced transcripts and alternative polyadenylation (APA) sites in this species. RNAs were isolated from G. lemaneiformis under various treatments including abiotic stresses and exogenous phytohormones, and then equally pooled for SMRT-Seq. In summary, 346,544 full-length nonchimeric reads were generated, from which 13,630 unique full-length transcripts were obtained in G. lemaneiformis. Compared with the known splicing events in the gene models, more than 3000 new alternative splicing (AS) events were identified in the SMRT-Seq reads. Additionally, 810 genes were found to have poly (A) sites and 91 microRNAs (miRNAs), 961 long noncoding RNAs and 1721 novel genes were identified in G. lemaneiformis. Moreover, validation experiments showed that abiotic stresses and phytohormones could induce some specific AS events, especially intron retain isoforms, cause some alterations to the relative ratios of transcripts annotated to the same gene, and generate novel 3′ ends because of differential APA. The growth of G. lemaneiformis was inhibited by Cu stress, while this inhibition was alleviated by ACC treatment. RNA-Seq analysis further revealed that 211 differential alternative splicing (DAS) events and 142 DAS events was obtained in CK vs Cu and Cu vs Cu + ACC, respectively, suggesting that AS of functional genes could be regulated by Cu stress and ACC. Compared with Cu stress, the expression of transcripts with DAS events mainly involved in the carbon fixation in photosynthetic organisms and oxidative phosphorylation pathway was upregulated in Cu + ACC treatment, revealing that ACC alleviated the growth inhibition by Cu stress by increasing carbon fixation and oxidative phosphorylation. Conclusions Our results provide the first comprehensive picture of the full-length transcriptome and posttranscriptional mechanism in red macroalgae, including transcripts that appeared in the presence of common abiotic stresses and phytohormones, which will improve the gene annotations of Gracilariopsis and contribute to the study of gene regulation in this important cultivated seaweed.

Physiological, Transcriptome, and Metabolome Analyses Reveal the Tolerance to Cu Toxicity in Red Macroalgae Gracilariopsis lemaneiformis

“…RNA-seq was conducted on BGISEQ-500 by BGI company (Shenzhen, China). The reference transcripts were obtained through merging the annotation file from the reference genome [66] and the newly identified isoform annotation file by Iso-Seq [67]. We removed the low-quality reads (those exceeding 50% of low-quality bases with Q15) and reads that contained adaptor sequences or unidentified nucleotides more than 10% to the total read pool to obtain clean data.…”

Transcriptome analysis reveals ABA involved in the detoxification mechanism of macroalga Gracilariopsis lemaneiformis to cadmium toxicity

“…SOAPnuke (https:// github.com/BGI-flexlab/SOAPnuke) was used to remove reads containing adapter sequences or more than 10% unidentified nucleotides and low-quality reads to obtain clean read databases. The clean data were mapped to the reference transcripts, which combined the transcripts from genomic annotation and full-length transcripts obtained by SMRT-Seq (Chen et al, 2022b), by Bowtie2 (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Expectation Maximization (RSEM) tool was applied to calculate the transcript abundance of each gene in each sample based on fragments per kilobase per million reads (FPKM) (Li and Dewey, 2011).…”

Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera

“…It increases the chances for discovering new genes and detecting AS events, APA, as well as lncRNAs. The combined application of second- and third-generation sequencing technologies has emerged as a powerful tool in transcriptomics research, successfully revealing transcriptome and gene structure information in multiple plant species [ 22 – 25 ].…”