BACKGROUND Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Longan SE has been established and wildly used as model system for studying embryogenesis in woody plants, SE-related genes had been characterized. In spite of that, a comprehensive overview of SE at a molecular level is still absent. To understand the molecular mechanisms during longan SE, we examined the transcriptome changes by using Illumina HiSeq from the four distinct developmental stages, including non-embryogenic callus (NEC), embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE). RESULTS RNA-seq of the four samples generated a total of 243.78 million high quality reads, approximately 81.5% of the data were mapped to longan genome. The cDNA libraries of NEC, EC, ICpEC and GE, generated 22,743, 19,745, 21,144, 21,102 expressed transcripts, 1935, 1710, 1816, 1732 novel transcripts, 2645, 366, 505, 588 unique genes, respectively. Comparative transcriptome analysis showed that a total of 10,642, 4180, 5846 and 1785 genes were differentially expressed in the pairwise comparisons of NEC_vs_EC, EC_vs_ICpEC, EC_vs_GE, ICpEC_vs_GE, respectively. Among them, plant hormones signalling related genes were significantly enriched, especially the auxin and cytokinin signalling components. The transcripts of flavonoid biosynthesis related genes were mainly expressed in NEC, while fatty acid biosynthesis related genes mainly accumulated in early SE. In addition, the extracelluar protein encoding genes LTP, CHI, GLP, AGP, EP1 were related to longan SE. Combined with the FPKM value of longan nine tissues transcription, 27 SE specific or preferential genes (LEC1, LEC1-like, PDF1.3, GH3.6, AGL80, PIN1, BBM, WOX9, WOX2, ABI3, et al.) and 28 NEC preferential genes (LEA5, CNOT3, DC2.15, PR1-1, NsLTP2, DIR1, PIP1, PIP2.1, TIP2-1, POD-P7 and POD5 et al.) were characterized as molecular markers for longan early SE. qRT-PCR validation of SE-related genes showed a high correlation between RNA-seq and qRT-PCR data. CONCLUSION This study provides new insights into the role of the transcriptome during early SE in longan. Differentially expressed genes reveal that plant hormones signalling, flavonoid and fatty acid biosynthesis, and extracelluar protein related genes were involved in longan early SE. It could serve as a valuable platform resource for further functional studies addressing embryogenesis in woody plants.

中文翻译：

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全球规模转录组分析揭示了参与龙眼早期体细胞胚胎发生的差异表达基因。

背景技术体细胞胚胎发生(SE)是体细胞去分化为全能胚胎干细胞并在体外产生胚胎的过程。龙眼SE已建立并广泛用作研究木本植物胚胎发生的模型系统，SE相关基因已得到表征。尽管如此，仍然缺乏在分子水平上对SE的全面概述。为了了解龙眼 SE 过程中的分子机制，我们使用 Illumina HiSeq 检查了四个不同发育阶段的转录组变化，包括非胚性愈伤组织 (NEC)、胚性愈伤组织 (EC)、不完全致密前胚性培养物 (ICpEC)、球状胚胎（GE）。结果 4个样本的RNA-seq共生成24378万条高质量reads，约81.5%的数据映射到龙眼基因组。NEC、EC、ICpEC和GE的cDNA文库分别产生了22,743、19,745、21,144、21,102个表达转录本，1935、1710、1816、1732个新转录本，2645、366、505、588个独特基因。比较转录组分析显示，NEC_vs_EC、EC_vs_ICpEC、EC_vs_GE、ICpEC_vs_GE两两比较中分别有10,642、4180、5846和1785个基因存在差异表达。其中，植物激素信号相关基因显着富集，尤其是生长素和细胞分裂素信号成分。黄酮类生物合成相关基因的转录本主要在NEC中表达，而脂肪酸生物合成相关基因主要在SE早期积累。此外，细胞外蛋白编码基因LTP、CHI、GLP、AGP、EP1与龙眼SE相关。结合龙眼9个组织转录的FPKM值，27个SE特异性或优先基因（LEC1、LEC1-like、PDF1.3、GH3.6、AGL80、PIN1、BBM、WOX9、WOX2、ABI3等）和28个NEC优先基因（LEA5、CNOT3、DC2.15、PR1-1、NsLTP2、DIR1、PIP1、PIP2.1、TIP2-1、POD-P7和POD5等）被表征为龙眼早期SE的分子标记。SE 相关基因的 qRT-PCR 验证显示 RNA-seq 和 qRT-PCR 数据之间存在高度相关性。结论 这项研究为龙眼早期 SE 过程中转录组的作用提供了新的见解。差异表达基因揭示了龙眼早期SE涉及植物激素信号、类黄酮和脂肪酸生物合成以及细胞外蛋白相关基因。它可以作为解决木本植物胚胎发生的进一步功能研究的宝贵平台资源。