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PacBio single-molecule long-read sequencing shed new light on the transcripts and splice isoforms of the perennial ryegrass

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Perennial ryegrass (Lolium perenne), one of the most widely used forage and cool-season turfgrass worldwide, has a breeding history of more than 100 years. However, the current draft genome annotation and transcriptome characterization are incomplete mainly because of the enormous difficulty in obtaining full-length transcripts. To explore the complete structure of the mRNA and improve the current draft genome, we performed PacBio single-molecule long-read sequencing for full-length transcriptome sequencing in perennial ryegrass. We generated 29,175 high-confidence non-redundant transcripts from 15,893 genetic loci, among which more than 66.88% of transcripts and 24.99% of genetic loci were not previously annotated in the current reference genome. The re-annotated 18,327 transcripts enriched the reference transcriptome. Particularly, 6709 alternative splicing events and 23,789 alternative polyadenylation sites were detected, providing a comprehensive landscape of the post-transcriptional regulation network. Furthermore, we identified 218 long non-coding RNAs and 478 fusion genes. Finally, the transcriptional regulation mechanism of perennial ryegrass in response to drought stress based on the newly updated reference transcriptome sequences was explored, providing new information on the underlying transcriptional regulation network. Taken together, we analyzed the full-length transcriptome of perennial ryegrass by PacBio single-molecule long-read sequencing. These results improve our understanding of the perennial ryegrass transcriptomes and refined the annotation of the reference genome.

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Data availability

The PacBio sequencing reads (accession number PRJNA549115) and the Illumina SGS reads (accession number PRJNA566226) generated in this study have been submitted to the BioProject database of National Center for Biotechnology Information.



Polyadenylation sites


Alternative splicing events


Coding sequences


Coding potential assessment tool


Coding potential calculator


Coding–non-coding index


Full-length non-chimeric reads


Gene ontology


High-quality isoforms


Iterative isoform-clustering program


Kyoto Encyclopedia of Genes and Genomes


EuKaryotic orthologous groups


Long non-coding RNA


Low-quality isoforms




Next-generation sequencing


NCBI non-redundant proteins


Reads of insert


Open reading frames

PacBio sequencing:

The PacBio single-molecule long-read sequencing technology


A database of conserved Protein families or domains


Reverse transcription polymerase chain reaction


A manually annotated, non-redundant protein database


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We are very grateful to Prof. Luis A. J. Mur from Institute of Biological, Environmental and Rural Sciences, Aberystwyth University for critically discussion with the manuscript. We also thank Biomarker Technology Corporation (Beijing, China) for the facilities and expertise of PacBio platform for libraries construction and sequencing and the Editage Company ( for language editing.


This research was supported by the Scientific Technology Plan Program of Shenzhen (No. JCYJ20160331151245672)‚ the National Natural Science Foundation of China (No. 31971770 and No. 31901397) and Beijing Natural Science Foundation (No.6204039).

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Conceived and designed the experiments: LH and YC. Performed the experiments: LX, KT and PT. Data analysis and draft of the manuscript were performed by KT, YL and WG. All authors approved the final version of the manuscript for submission.

Corresponding author

Correspondence to Liebao Han.

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This study does not contain any studies with human participants or animals performed by any of the authors.

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Communicated by Stefan Hohmann.

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Fig.S1 Soil water content measurement. (JPG 211 kb)


Fig.S2 Physiological measurement of perennial ryegrass seedlings under different drought stresses conditions. (A) Leaf relative water content. (B) MDA content. (C) Proline content. (D) Total sugar content. Means ± SDs (n = 4). Different letters indicate significant differences at 5% level of probability. (JPG 469 kb)


Fig.S3 GO annotation of the biological processes identified in Drought_0-3d (A), Drought_0-8d (B) and Drought_3-8d (C). (TIFF 334 kb)

Table S1 Summary of AS events. (XLSX 800 kb)

Table S2 Summary of APA sites. (XLS 1417 kb)

Table S3 Summary of lncRNAs in perennial ryegrass. (XLSX 12 kb)

Table S4 Summary of the target genes of lncRNAs. (XLSX 16 kb)

Table S5 Summary of fusion genes in perennial ryegrass. (XLSX 176 kb)

Table S6 Sequence of primers used for alternative splicing events verification. (DOCX 15 kb)

Table S7 Summary of the DEGs identified in Drought_0-3d, Drought_0-8d and Drought_3-8d groups. (XLS 1433 kb)

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Xie, L., Teng, K., Tan, P. et al. PacBio single-molecule long-read sequencing shed new light on the transcripts and splice isoforms of the perennial ryegrass. Mol Genet Genomics 295, 475–489 (2020).

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