Single-molecule real-time sequencing of the full-length transcriptome of loquat under low-temperature stress.

Yunmiao Yang,Zhiwu Yang,Hui Zhang,Zhuoheng Chi,Maoteng Li,Qunxian Deng,Lian Tao,Cuiping Pan,Yongqing Wang

doi:10.1371/journal.pone.0238942

Abstract

In this study, third-generation full-length (FL) transcriptome sequencing was performed of loquat using single-molecule real-time(SMRT) sequencing from the pooled cDNA of embryos of young loquat fruit under different low temperatures (three biological replicates for treatments of 1°C, -1°C, and -3°C, for 12 h or 24 h) and the control group(three biological replicates for treatments of room temperature), Illumina sequencing was used to correct FL transcriptome sequences. A total of 3 PacBio Iso-Seq libraries (1–2 kb, 2–3 kb and 3–6 kb) and 21 Illumina transcriptome libraries were constructed, a total of 13.41 Gb of clean reads were generated, which included 215,636 reads of insert (ROIs) and 121,654 FL, non-chimaric (FLNC) reads. Transcript clustering analysis of the FLNC reads revealed 76,586 consensus isoforms, and a total of 12,520 high-quality transcript sequences corrected with non-FL sequences were used for subsequent analysis. After the redundant reads were removed, 38,435 transcripts were obtained. A total of 27,905 coding DNA sequences (CDSs) were identified, and 407 long non-coding RNAs (lncRNAs) were ultimately predicted. Additionally, 24,832 simple sequence repeats (SSRs) were identified, and a total of 1,295 alternative splicing (AS) events were predicted. Furthermore, 37,993 transcripts were annotated in eight functional databases. This is the first study to perform SMRT sequencing of the FL transcriptome of loquat. The obtained transcriptomic data are conducive for further exploration of the mechanism of loquat freezing injury and thus serve as an important theoretical basis for generating new loquat material and for identifying new ways to improve loquat cold resistance.

Highlights

A total of 13.41 Gb of clean data were generated via Pacific Biosciences single-molecule real-time (SMRT) sequencing technology
76,586 consensus isoforms were obtained by iterative clustering for error correction(ICE) (Table 3)
All the raw data were deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA623262 and are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA623262

Summary

Methods

Plant materials and treatmentsTwo-year-old grafted Ninghaibai loquat plants that were growing in pots and that had already produced fruit (with a diameter of approximately 1.5 cm) were used as the experimental materials, and the growth status of the plants was as uniform as possible. The treatments were applied in a low-temperature plant incubator with 60% relative humidity, a 3000 lx light intensity, and a 12-h/12-h light/dark cycle. The plants were removed and incubated at room temperature for 6 h to recover, after which the embryos of young loquat fruit were collected, immediately frozen in liquid nitrogen and stored at -80 ̊C. Plants that had been growing at room temperature were used as controls. A total of 21 samples of embryos of young loquat fruit (three biological replicates for treatments of 1 ̊C, -1 ̊C, and -3 ̊C, for 12 h or 24 h, including the control group) were collected for the following experiments

Results

Discussion

Conclusion