Direct single-molecule sequencing of full-length transcripts allows efficient identification of gene isoforms, which is apt to alternative splicing (AS), polyadenylation, and long non-coding RNA analyses. However, the identification of gene isoforms and long non-coding RNAs with novel regulatory functions remains challenging, especially for species without a reference genome. Here, we present a comprehensive analysis of a combined long-read and short-read transcriptome sequencing in Camellia japonica. Through a novel bioinformatic pipeline of reverse-tracing the split-sites, we have uncovered 257,692 AS sites from 61,838 transcripts; and 13,068 AS isoforms have been validated by aligning the short reads. We have identified the tissue-specific AS isoforms along with 6,373 AS events that were found in all tissues. Furthermore, we have analysed the polyadenylation (polyA) patterns of transcripts, and found that the preference for polyA signals was different between the AS and non-AS transcripts. Moreover, we have predicted the phased small interfering RNA (phasiRNA) loci through integrative analyses of transcriptome and small RNA sequencing. We have shown that a newly evolved phasiRNA locus from lipoxygenases generated 12 consecutive 21 bp secondary RNAs, which were responsive to cold and heat stress in Camellia. Our studies of the isoform transcriptome provide insights into gene splicing and functions that may facilitate the mechanistic understanding of plants.

全长转录本的直接单分子测序可以有效鉴定基因同工型，易于进行选择性剪接（AS），聚腺苷酸化和长时间的非编码RNA分析。但是，鉴定具有新的调控功能的基因同工型和长非编码RNA仍然具有挑战性，特别是对于没有参考基因组的物种。在这里，我们对茶花的长，短读​​转录组测序相结合进行了全面的分析。通过新颖的反向追踪拆分位点的生物信息学途径，我们从61,838个转录本中发现了257,692个AS位点；和13068 AS异构体已经通过比对短读序列进行了验证。我们已经鉴定出组织特异性AS同工型以及在所有组织中发现的6,373例AS事件。此外，我们分析了转录物的聚腺苷酸化（polyA）模式，发现AS和非AS转录物对polyA信号的偏好不同。此外，我们已经通过转录组和小RNA测序的综合分析预测了分期的小干扰RNA（phasiRNA）基因座。我们已经表明，从脂氧合酶新进化的phasiRNA基因座产生了12个连续的21 bp二级RNA，它们对茶花的冷热胁迫响应。我们对同工型转录组的研究提供了对基因剪接和功能的见解，可促进对植物的机械理解。