SMRT sequencing of full-length transcriptome of seagrasses Zostera japonica

Abstract

Seagrass meadows are among the four most productive marine ecosystems in the world. Zostera japonica (Z. japonica) is the most widely distributed species of seagrass in China. However, there is no reference genome or transcriptome available for Z. japonica, impeding progress in functional genomic and molecular ecology studies in this species. Temperature is the main factor that controls the distribution and growth of seagrass around the world, yet how seagrass responds to heat stress remains poorly understood due to the lack of genomic and transcriptomic data. In this study, we applied a combination of second- and third-generation sequencing technologies to sequence full-length transcriptomes of Z. japonica. In total, we obtained 58,134 uniform transcripts, which included 46,070 high-quality full-length transcript sequences. We identified 15,411 simple sequence repeats, 258 long non-coding RNAs and 28,038 open reading frames. Exposure to heat elicited a complex transcriptional response in genes involved in posttranslational modification, protein turnover and chaperones. Overall, our study provides the first large-scale full-length trascriptome in Zostera japonica, allowing for structural, functional and comparative genomics studies in this important seagrass species. Although previous studies have focused specifically on heat shock proteins, we found that examination of other heat stress related genes is important for studying response to heat stress in seagrass. This study provides a genetic resource for the discovery of genes related to heat stress tolerance in this species. Our transcriptome can be further utilized in future studies to understand the molecular adaptation to heat stress in Zostera japonica.

Figure 1

Summary of PacBio RS II SMRT sequencing. (a) ROI length distribution in Z. japonica from different PacBio libraries with fractionated sizes of 1–2, 2–3, and 3–6 kb. (b) Proportion of different types of PacBio reads in Z. japonica. (c) Categorization of ROIs after error correction using Illumina short reads according to the locations of the erroneous fragments. (d) Number and length distributions of the 20,032 non-redundant full-length transcripts.

Figure 2

Venn diagram of the number of lncRNAs predicted by CPC, CNCI, CPAT and pfam.

Figure 3

The distribution of the coding sequence lengths of the complete open reading frames. The x-axis represents the coding sequence length and the y-axis represents the number of predicted open reading frames.

Figure 4

Homologous species distribution of Zostera japonica annotated in the NR database.

Figure 5

GO functional annotation and eggNOG annotation of Zostera japonica transcripts. (a) GO annotation, where blue represents cellular component, red represents molecular function, and green represents biological process. The x-axis represents GO categories, the y-axis (left) represents the percentage of genes, and the y-axis (right) represents the number of genes. (b) eggNOG annotations, where the x-axis represents eggNOG categories and the y-axis represents the number of transcripts.

Figure 6

GO functional annotation and COG annotation of Zostera japonica DEGs. (a) GO annotation, where red represents biological process, green represents cellular component, and blue represents molecular function. The x-axis represents GO categories, the y-axis (left) represents the percentage of genes, and the y-axis (right) represents the number of genes. (b) COG annotation, where the x-axis represents COG categories and the y-axis represents the number of transcripts.

Figure 7

qRT-PCR validation of differentially expressed genes in Z. japonica under heat stress. Expression levels were normalized to the reference gene actin. Error bars indicate standard deviations of the three biological replicates. The statistical difference between means is indicated as *P < 0.05 or **P < 0.01. Colors and numbers under the bar charts refer to the FPKM of genes.