Longan (Dimocarpus longan Lour.), an important subtropical fruit in the family Sapindaceae, is grown in more than 10 countries. Longan is an edible drupe fruit and a source of traditional medicine with polyphenol-rich traits. Tree size, alternate bearing, and witches' broom disease still pose serious problems. To gain insights into the genomic basis of longan traits, a draft genome sequence was assembled. The draft genome (about 471.88 Mb) of a Chinese longan cultivar, “Honghezi,” was estimated to contain 31 007 genes and 261.88 Mb of repetitive sequences. No recent whole-genome-wide duplication event was detected in the genome. Whole-genome resequencing and analysis of 13 cultivated D. longan accessions revealed the extent of genetic diversity. Comparative transcriptome studies combined with genome-wide analysis revealed polyphenol-rich and pathogen resistance characteristics. Genes involved in secondary metabolism, especially those from significantly expanded (DHS, SDH, F3΄H, ANR, and UFGT) and contracted (PAL, CHS, and F3΄5΄H) gene families with tissue-specific expression, may be important contributors to the high accumulation levels of polyphenolic compounds observed in longan fruit. The high number of genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) and leucine-rich repeat receptor-like kinase proteins, as well as the recent expansion and contraction of the NBS-LRR family, suggested a genomic basis for resistance to insects, fungus, and bacteria in this fruit tree. These data provide insights into the evolution and diversity of the longan genome. The comparative genomic and transcriptome analyses provided information about longan-specific traits, particularly genes involved in its polyphenol-rich and pathogen resistance characteristics.

MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16, and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs), and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE) in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid (SA) and heat stress. The pri-miR160 was down-regulated in response to SA but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a∗ and -d∗ reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in friable-embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a∗ expression level. This suggests that they may function to abolish the binding between dlo-miR160a∗ and its targets. These two eTMs also participated in 2,4-D and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The eTMs, miR160, and ARF10, -16, and -17 exhibited tissue specificity in ‘Sijimi’ longan vegetative and reproductive organs, but were not significant negatively correlated. These results provide insights into the possible role of the eTM-miR160-ARF10-16-17 pathway in longan somatic embryo development.

BackgroundSomatic 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).ResultsRNA-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.ConclusionThis 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.

The changes in betacyanin levels among various tissues and following phytohormone treatments were related to the differences in betalain biosynthesis gene expression levels.

Light is an important factor that affects the synthesis of functional metabolites in longan embryogenic calli (ECs). However, analysis of the effect of light on functional metabolites in longan ECs via RNA sequencing has rarely been reported and their light regulation network is unclear. The contents of various functional metabolites as well as the enzymatic activities of superoxide dismutase and peroxidase and the level of H2O2 in longan ECs were significantly higher under blue light treatment than under the other treatments (dark, white). In this study, we sequenced three mRNA libraries constructed from longan ECs subjected to different treatments. A total of 4463, 1639 and 1806 genes were differentially expressed in the dark versus blue (DB), dark versus white (DW) and white versus blue (WB) combinations, respectively. According to GO and KEGG analyses, most of the differentially expressed genes (DEGs) identified were involved in transmembrane transport, taurine and hypotaurine metabolism, calcium transport and so forth. Mapman analysis revealed that more DEGs were identified in each DB combination pathway than in DW combination pathways, indicating that blue light exerts a significantly stronger regulatory effect on longan EC metabolism than the other treatments. Based on previous research and transcriptome data mining, a blue light signaling network of genes that affect longan functional metabolites was constructed and HY5, PIF4 and MYC2 were shown to be the key regulatory genes in the network. The results of this study demonstrate that the expression levels of phase-specific genes vary with changes in longan EC functional metabolites.