Characterization and analysis of the transcriptome in Gymnocypris selincuoensis on the Qinghai-Tibetan Plateau using single-molecule long-read sequencing and RNA-seq

Abstract

The lakes on the Qinghai-Tibet Plateau (QTP) are the largest and highest lake group in the world. Gymnocypris selincuoensis is the only cyprinid fish living in lake Selincuo, the largest lake on QTP. However, its genetic resource is still blank, limiting studies on molecular and genetic analysis. In this study, the transcriptome of G. selincuoensis was first generated by using PacBio Iso-Seq and Illumina RNA-seq. A full-length (FL) transcriptome with 75,435 transcripts was obtained by Iso-Seq with N50 length of 3,870 bp. Among all transcripts, 75,016 were annotated to public databases, 64,710 contain complete open reading frames and 2,811 were long non-coding RNAs. Based on all- vs.-all BLAST, 2,069 alternative splicing events were detected, and 80% of them were validated by reverse transcription polymerase chain reaction (RT-PCR). Tissue gene expression atlas showed that the number of detected expressed transcripts ranged from 37,397 in brain to 19,914 in muscle, with 10,488 transcripts detected in all seven tissues. Comparative genomic analysis with other cyprinid fishes identified 77 orthologous genes with potential positive selection (Ka/Ks > 0.3). A total of 56,696 perfect simple sequence repeats were identified from FL transcripts. Our results provide valuable genetic resources for further studies on adaptive evolution, gene expression and population genetics in G. selincuoensis and other congeneric fishes.

Keywords: Gymnocypris selincuoensis; alternative splicing; gene expression; single-molecule sequencing; transcriptome.

Figure 1

The length distribution of transcripts obtained by Iso-Seq and RNA-seq.

Figure 2

The number of full-length transcripts annotated with Nt, Nr, Swissprot, KOG, KEGG, GO and Pfam databases.

Figure 3

The features of long non-coding RNAs (lncRNAs) in G. selincuoensis. (A) Venn graph of lncRNA transcripts from PLEK, CNCI, CPC and Pfam analysis. (B) The length distribution of lncRNA transcripts.

Figure 4

RT-PCR validation of 20 alternative splicing events identified by Iso-Seq.

Figure 5

Analysis of gene expression in seven tissues of G. selincuoensis. (A) The number of transcripts with different expression abundances in various tissues based on FPKM values. (B) Venn diagram of detected transcripts (with a cut-off of 1 FPKM) in each tissue. (C) The number of tissue-specific transcripts in each tissue. The tissue-specific transcripts are represented by 50-fold higher FPKM level in one tissue compared with all other tissues. (D) Heat map showing the pairwise Spearman correlations among various tissues.

Figure 6

Quantitative real-time PCR confirmation of the transcript expression obtained by high-throughput sequencing. K: kidney; L: liver; O: ovary; G: gill; B: brain; H: heart; M: muscle.

Figure 7

Comparative analysis of the one-to-one orthologous genes between G. selincuoensis and other cyprinid fishes. (A) Distribution of Ka/Ks ratio. (B) Venn diagram of orthologous genes with Ka/Ks >0.3.

Figure 8

Summary of SSRs isolated from the full-length transcripts of G. selincuoensis. (A) The number of SSRs with different repeat times and motifs. (B) The dominant motifs of dinucleotide, trinucleotide and tetranucleotide SSRs.