Global scale transcriptome analysis reveal differentially expressed genes during early somatic embryogenesis in Dimocarpus longan Lour

doi:10.21203/rs.2.12051/v1

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Global scale transcriptome analysis reveal differentially expressed genes during early somatic embryogenesis in Dimocarpus longan Lour

https://doi.org/10.21203/rs.2.12051/v1

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published 02 Jan, 2020

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Background

Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to the totipotent embryonic stem cells and generate embryos in vitro. Longan SE has been established and wildly used as a model system for studying embryogenesis in woody plants, and some SE-related genes had been characterized. In spite of that, a comprehensive overview of SE at a molecular level is still absent. With the aim of understanding the molecular mechanisms underlying SE in longan, we examined the transcriptome changes by using Illumina HiSeq platform 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 the reference genome. The cDNA libraries of NEC, EC, ICpEC and GE, generated 22743, 19745, 21144, 21102 expressed transcripts and 1935, 1710, 1816, 1732 novel transcripts, and 2645, 366, 505, 588 unique genes, respectively. Comparative transcriptome analysis revealed the important role of auxin and cytokinin during longan SE. The transcripts profiling of flavonoid and fatty acid biosynthesis related genes suggested that flavonoids were mainly accumulated in NEC, while fatty acid accumulated in early SE. In addition, the extracelluar protein encoding genes LTP, CHI, GLP, AGP, EP1 were related to longan SE. Transcript profiling combined with qRT-PCR performed on selected genes confirmed that 27 SE molecular markers (LEC1, LEC1-like, PDF1.3, GH3.6, AGL80, PIN1, BBM, WOX9, WOX2, ABI3, et al.) and 28 NEC markers (LEA5, CNOT3, DC2.15, PR1-1, NsLTP2, DIR1, PIP1, PIP2.1, TIP2-1, POD-P7 and POD5 et al.) were characterized as potential molecular markers for longan early SE, respectively.

Conclusion

Our transcriptome reveals the transcription regulation of auxin, cytokinin and other hormones signaling pathway, flavonoids biosynthesis, fatty acid biosynthesis, extracelluar protein encoding genes, and other SE-related genes during early SE. Furthermore, we characterizes the potential molecular markers to distinguish NEC and early SE of longan. The present work provides new insights into future functional studies, as a means of studying the molecular mechanisms in SE.

Epigenetics & Genomics

Dimocarpus longan

somatic embryogenesis

Illumina HiSeq

auxin and cytokinin

molecular marker gene

qRT-PCR

Longan (Dimocarpus longan Lour.), a tropical/subtropical evergreen fruit tree within the Sapindaceae family, native to South China and Southeast Asia, is now widely cultivated in Southeast Asia, South Asia, Australia and Hawaii [1]. Logan embryo development status was close association with the seed size, fruit-set rate, fruit production and quality. Base on the observation of histological and cytological, the change of endogenous hormones and polyamines, proteomics analysis of the isozymes and protein, and molecular biology researches on SE-related genes mRNA differential display, homologous cloning, expression pattern by qRT-PCR have been used to illuminate the potential regulation mechanism of longan SE [2]{Z．Z．Fang, 2010 #39;Lai, 2010 #1}. However, elucidating the embryo development mechanism at a molecular level remains a great challenge due to its highly genetic heterozygosity and difficulties in accessibility of early embryos in vivo [3]. Plant SE shares close similarities at almost all development stages to normal zygotic embryogenesis [4, 5], SE has been wildly used as a model system to study the molecular regulation mechanism of early embryogenesis in plants [6]. The longan SE system has been established and extensively used as a model system for investigating embryogenesis in woody plants, which revealed that the concentration of 2,4-D was the key factor in controlling longan high-consistency SE [1, 7, 8].

Over the last few years, the expression profiles of SE related genes and other differentially expressed genes during SE have been extensively excavated by RNA-seq sequencing in various species, including Gossypium hirsutum [1, 7, 8] [Yang, 2012 #142;Zhenzhen, 2013 #146］, Arabidopsis [13, 14], Maize [15], Norway spruce [16, 17], Coconut plam [18], Brazilian pine [18], Eleutherococcus senticosusk [20], Camphor tree [21], Strawberry [22], Rice [23], Lilium pumilum [24], Mangosteen [25], Papaya [26], Triticum aestivum [27]. Meanwhile, the comparative proteome analysis during SE also characterized numerous proteins that associated with SE in many plant species, such as Maize [28], Papaya [29], Cacao [30], Sugarcane [31], Musa. spp [32] and Gossypium hirsutum [33]. The transcriptome and proteome analysis of plant SE revealed several molecular regulation mechanisms of SE, and a large number of potential key factors of embryogenesis. Numerous genes and proteins that playing an important role in somatic embryogenesis have been reported such as Somatic embryogenesis receptor-like kinase (SERK) [34–36], Leafy Cotyledon [36–38], BABYBOOM [36, 39, 40], WUSCHEL [41, 42], WUSCHEL homeobox 2 [36, 43], AGAMOUS-like 15 [44, 45] and late embryogenesis abundant (LEA) protein [26].

To date, the transcript profiling of longan embryogenic callus (EC) has been illuminated by Lai and Lin [46], which revealed numerous embryogenesis-related and reproductive growth related unigenes in EC. Lin and Lai [47] had identified and profiled the conserved and novel miRNA during longan SE by using Solexa sequencing combined with computational, and qRT-PCR methods, and the potential roles of 20 conserved and 4 novel miRNA in longan SE was described by their tissue or stage-specific expression profiling. Recently, longan draft genome sequences become available [48], which provided the comprehensive genomic information for studying the molecular regulation of SE. Transition from NEC to EC, and from EC to somatic embryo are the key steps of SE. However, the molecular regulation mechanisms during longan SE remain largely unknown. The aim of this study was to elucidate the molecular mechanism in the transition from NEC to EC, and during early SE by investigating the expression profiling using Illumina RNA-seq technology, and to identify the molecular marker genes during SE. This RNA-seq of comparative transcriptome analysis will gain new insight into the molecular and developmental mechanisms of longan SE.

RNA-Seq analysis of longan early SE aligned with the Dimocarpus longan Draft Genome

To provide a comprehensive understanding of longan SE at a transcriptional level, we sequenced the four cDNA libraries constructed from the four in vitro embryo developmental stages (NEC, EC, ICpEC and GE, Figure 1). A total of 243,783,126 clean reads (comprising approximately 24.38 G of nucleotides) were obtained from all four RNA-seq datasets after data cleaning and quality checks. After aligned with longan draft genome sequence [48], 48,798,229 (81.62%), 52,623,741 (81.1%), 48,346,067 (81.14%) and 48,871,200 (82.08%) reads in four cDNA libraries were mapped to D.longan reference genome, respectively. Among these, 44,655,772 (74.69%), 48,333,703 (74.50%), 44,490,292 (74.67%) and 44,924,511 (74.45%) reads were uniquely mapped to one location, respectively. Meanwhile, 34,380,246 (57.51%), 35,386,494 (54.54%), 30,535,088 (51.25%) and 29,214,788 (49.07%) reads in four cDNA libraries were mapped to gene, respectively. A summary of mapping statistics obtained for each sample is given in Table 1.

The transcribed regions/units of four different stages samples were constructed independently, generated 22,743, 19,745, 21,144 and 21,102 expressed transcripts, showed 57.89%, 50.26%, 53.82% and 53.71% overlapped with longan genome (39,282 genes), respectively. After filtering out short sequences which less than 180 bp and low sequencing depth that lower than two, 1,935, 1,710, 1,816 and 1,732 novel transcripts in four samples were detected, respectively. Among these, 1,025, 819, 832 and 806 novel genes were identified as coding RNAs, and 910, 891, 984 and 926 novel genes were identified as non-coding RNAs in the longan genome.

Alternative splicing (AS) events represented in our transcriptome were predicted by TopHat2. We analyzed the exon level of the four samples, 110,864, 103,200, 107,592 and 107,971 expressed exon were detected (Table 1). A total of 130,354 AS events were checked across the four stages, including exon skipping, intron retention, alternative 5’ splicing and alternative 3’ splicing. The largest number of AS events were detected in GE (39,768), followed by ICpEC (36,446), and NEC (35,084), and the smallest in EC (19,056). Exon skipping is the least type in all samples, and intron retention is the most popular type of AS events in NEC, ICpEC and GE (Figure 2).

Global analysis of gene expression across the four distinct developmental processes

There were 22,743, 19,745, 21,144, 21,102 expressed genes in NEC, EC, ICpEC and GE stage. Among these, more than 75.3% of the expressed genes were present in all four developmental stages. Significant numbers of genes were unique expressed in one process only, 2645 genes were only expressed in NEC, however, only 366, 505 and 588 genes were unique present in EC, ICpEC and GE stage, respectively (Figure 3a), which suggested that distinct spatial transcriptional patterns were present in the four developmental processes. To evaluate the differences of molecular response among four samples, gene expression were normalized to FPKM by using RSEM software. After filtering with FPKM>60, a total of 2961 (11.40%), 3445 (13.26%), 3445 (13.26%) and 3442 (13.25%) genes were highly expressed in NEC, EC, ICpEC and GE, respectively (Table 2). The Top10 most enriched (FPKM) genes were range from 5476 to 58812, 2766 to 15114, 2343 to 10330 and 2091 to 4004, respectively. The top 20 most expressed genes from the four libraries were shown in Tables 3, some SE-related genes such as leafy cotyledon 1 (LEC1), leafy cotyledon 1-like (L1L),, Protodermal factor 1 (PDF1), lipid transfer protein (LTP), Heat-Shock protein 90 (HSP90), chitinase (CHI),, Indole–3-acetic acid-amido synthetase GH3.6, glutathione S-transferase (GST),, root meristem growth factor 3 (RGF3) were highly expressed in EC, ICpEC or GE stage.

To reveal the potential key genetic factors involved in early SE, we filtered the significantly differentially expressed genes (DEGs) with |log₂fold change| ≥ 1 and FDR < 0.001 between these four pairwise comparisons among the four libraries as follow: NEC_vs_EC (Transition from NEC to EC), EC_vs_ICpEC, EC_vs_GE, ICpEC_vs_GE. Among these four comparisons (Figure 3b), a total of 10,642, 4,180, 5,846 and 1,785 DEGs were identified, respectively. Compared with NEC, EC had 4,887 up-regulated and 5,755 down-regulated genes. Compared with EC, ICpEC had 2,689 up-regulated and 1,491 down-regulated genes, GE had 3,451 up-regulated and 2,395 down-regulated genes. Compared with ICpEC, GE had 832 up-regulated and 953 down-regulated genes. DEGs analysis revealed that longan transcriptome undergoes significantly dynamic changes during SE, particularly during the transition period from NEC to EC. Therefore, the longan SE transcriptome datasets given here may serve as a valuable molecular resource for future studies.

Functional classification of DEGs base on GO and KEGG

To evaluate the potential functions of the DEGs, we used GO terms assignment to classify the functions of DEGs in pairwise comparisons under three GO main categories: biological process, cellular component and molecular function (Additional file 7: Fig. S1). In all pairwise comparisons mentioned above, the term with the largest proportion in “biological process” was ‘metabolic process’, followed by ‘cellular process’, ‘single-organism process’, ‘respond to stimulus’ and ‘localization’, the term with the largest proportion in “cellular component” were ‘cell’ and ‘cell part’, followed by ‘organelle’ and ‘membrane’, the term with the largest proportion in “molecular function” was ‘catalytic activity’, followed by ‘binding’, ‘transporter activity’, ‘molecular transducer activity’ and ‘nucleic acid binding transcription factor activity’.

To further investigate the biological pathways of the DEGs, we used the KEGG database to classify the DEGs function with emphasis on biological pathways (Additional file 8: Fig. S2). According to KEGG annotation, 6516 DEGs (NEC_vs_EC) were assigned to 128 pathways, 2514 DEGs (EC_vs_ICpEC) were assigned to 126 pathways, 3555 DEGs (EC_vs_GE) were assigned to 126 pathways, 1062 DEGs (ICpEC_vs_GE) were assigned to 111 pathways. The annotated changes in all comparisons were mainly enriched in ‘metabolic pathway’ (21.38%, 22.43%, 23.12% and 25.52%, respectively), ‘biosynthesis of secondary metabolites’ (11.97%, 11.46%, 11.70% and 14.52%, respectively), ‘plant–pathogen interaction’ (8.01%, 8.23%, 7.59% and 6.40%, respectively) and ‘plant hormone signal transduction’ (5.22%, 5.41%, 5.40% and 8.38%, respectively) pathway. Furthermore, dozens of genes were involved in ‘flavonoid biosynthesis’, ‘phenylpropanoid biosynthesis’, ‘zeatin biosynthesis’, ‘fatty acid biosynthesis’ and ‘biosynthesis of unsaturated fatty acids’.

Differential expression analysis of plant hormone signaling pathway related genes during longan SE

Based on the KEGG and other annotation, plant hormone signal transduction, zeatin biosynthesis and tryptophan metabolism were the representative pathways in our study. A large number of genes invovled in auxin (97 DEGs) and cytokinin (94 DEGs) biosynthesis and signal transduction pathway were differentially expressed when compared EC with NEC (Additional file 9: Fig. S3) and early SE. Transcript profiling showed that most of the genes associated with IAA and zeatin belonged to gene family, complex changes in transcript abundance of individual genes of all gene family were identified in NEC and early SE. For example, the expression level of PIN1, IAA (IAA6, IAA6-like, IAA9, IAA11, IAA14, IAA16, IAA29, IAA31 and IAA33),, ARFs (ARF1, ARF1-like, ARF2, ARF2-like, ARF5, ARF10, ARF16, ARF17, ARF18 ARF18–1 and ARF24),, GH3 (GH3.6, GH3.1, GH3.17),, and three SAUR, genes involved in auxin signal transduction, were significantly up-regulated from NEC to EC, most of them remained highly expression in EC, ICpEC and GE stages. Nevertheless, AUX1, TIR1, IAA (IAA1, IAA4, IAA13, IAA26, IAA26-like, IAA27),, ARFs (ARF4, ARF4-like, ARF10-like),, GH3.9 and GH3.17-like, and 12 SAUR were mainly expressed in NEC stage and down-regulated in EC. From EC to ICpEC and GE stages, AUX1 (Dlo_024286.1, Dlo_031956.2), IAA (IAA4, IAA14, IAA26-like, IAA27, IAA13),, ARFs (ARF4, ARF4-like, ARF10-like),, two SAUR showed noteworthy up-regulated expression (Figure 4a). In IAA biosynthesis, except PAI, Trp synthesis key genes ASA, IGS, TSA, TSB, were up-regulated in EC and remained high expression during early SE. CYP83B1, one ST5a, five YUCCAs, three CYP71A13 and NIT showed NEC-specific expression pattern. Three YUCCAs, three AAO1, one NIT, CYP71A13 and three ST5a were up-regulated in EC and remained high during early SE, and YUCCA_Dlo_013505.1 kept up-regulated during early SE (Figure 4b).

As showed in Figure 4c, TRIT1, a gene involved in cis-zeatin synthesis was up-regulated from NEC to GE. CisZOG family involved in Cis-zeatin O-glycosylation werehighly expressed in NEC, and significantly down-regulated from NEC to EC. During early SE, five CisZOG were up-regulated from EC to ICpEC, four CisZOG were down-regulated from ICpEC to GE. In trans-zeatin biosynthesis, six IPT1,4, five CYP735A, four CKX, three UGT76C were noteworthy down-regulated from NEC to EC; two IPT1,4, four CYP735A, one CKX, three UGT76C were up-regulated in EC. During early SE, IPT1,4 family, five CYP735A, two CKX, four UGT76C were up-regulated during early SE with minimal FPKM. Among the cytokinin signal pathway, two A_ARR, 10 B_ARR, 15 CRE1 were mainly expressed in NEC, and down-regulated in EC. One A_ARR, five B_ARR, seven CRE1 were up-regulated in EC. 13 CRE1, seven B_ARR and all A_ARR showed up-regulated expression during early SE, two B_ARR and five CRE1 were down-regulated during early SE (Figure 4d).

In addition, numerous genes involved in abscisic acid, gibberellin, ethylene, salicylic acid, jasmonic acid and brassinosteroid signal transduction pathway were differentially expressed during longan SE (Additional file 1: Table S1 a-h; Additional file 10: Fig. S4). Such an observation suggested an essential role of hormones and their complicated crosstalk during early SE. Therefore, the plant hormone signaling pathway may be a key regulator in longan early SE.

Flavonoids and fatty acid biosynthesis related genes were differential expressed during longan SE

Flavonoid biosynthesis and fatty acid biosynthesis were the representative KEGG pathways, a total of 125 significant DEGs were assigned to ‘flavonoid biosynthesis’ across the early SE processes (Figure 5). In the transition from NEC to EC, the flavonoid biosynthesis key genes, C4H, CHS, CHI, F3H, F3’5’H, DFR, LDOX/ANS, ANR, LAR, CCoAOMT were mainly expressed in NEC, drastic down-regulated from NEC to EC and remained very low expression level during early, except that F3H_Dlo_011012.1, F3’5’H_ Dlo_010496.1, LAR_ Dlo_022420.1, CCoAOMT_ Dlo_005144.2 were up-regulated in EC, but down-regulated during early SE. Besides, most of the FLS and F3’H family were mainly expressed in NEC, significantly down regulated in EC and kept low FPKM during early SE, especially, 15 F3’H and 9 FLS belonged to NEC specific genes. Only four FLS and six F3’H were first up-regulated from NEC to EC and then down-regulated or kept low expression level during early SE (Additional file 2: Table S2).

Several R2R3-MYB transcription factors are involve in the regulation of flavonoid biosynthesis in Arabidopsis [49–51]. For example, AtMYB11, –12, –111 regulated flavonol biosynthesis by up-regulated CHS, CHI, F3H, F3’H and FLS [49, 52]. AtMYB75, –90, –113, –114 controlled anthocyanin biosynthesis in vegetative [53]. AtMYB123 controlled the biosynthesis of proanthocyanidins in the seed coat [54]. MtMYB5, –14 played the key role in seed coat polymer biosynthesis [55]. AtMYB4 negative controlled sinapate ester biosynthesis through down-regulated C4H in a UV-dependent manner [55]. In our study, 11 R2R3-MYB transcripts were differential expressed. During longan SE, MYB12 and MYB111 were barely detected in NEC, significant up-regulated from NEC to EC and remained high during early SE. MYB75, MYB113, MYB4 and MYB123 were significant down-regulated in EC, and kept relative low expression during early SE.

The fatty acid composition rapidly changed during SE in Daucus carota [57], and Gossypium hirsutum [33]. In our study, a total of 35 fatty acid biosynthesis related genes were differently expressed during SE (Additional file 3: Table S3). From NEC to EC, except ACCase (Dlo_000360.1), three FabG, two FabZ, SAD (Dlo_031652.1), most of the ACCase, FabD, FabF, FabG, FabZ, FabI, FatB and SAD were significantly up-regulated in EC. During early SE, most of the DEGs remained high expression, part of them with slightly up/down-regulated expression. For example, ACCase (Dlo_023270.1) and SAD (Dlo_019646.1) were up-regulated from NEC to EC, and highly expressed during early SE. Our results indicated that flavonoids were mainly accumulated in NEC, while fatty acid were mainly accumulated in early SE, especially in EC.

Extracellular protein encoding genes effect on the transition from NEC to EC

It had been reported that extracellular protein germins and germin-like (GLPs), Arabinogalactan proteins (AGPs), chitinases (CHIs), lipid transfer proteins (LTPs) and glycoprotein were critical to SE, and can be served as protein marker during early SE [58]. In our study, 16 CHIs were differentially expressed, and most of them were preferential expressed in NEC, and remarkable down-regulated in EC, only seven CHIs were up-regulated during early SE with low FPKM. Among the 14 identified LTPs, only LTP (Dlo_013012.1, Dlo_013014.1) were highly and specific transcripts in early SE, most of them were mainly expressed in NEC and down-regulated from NEC to EC. Meanwhile, 12 GLPs and two secreted glycoprotein genes (EP1-like) were mainly expressed in NEC and kept very low FPKM during early SE. Except AGP10 was first up-regulated in EC and down-regulated during early SE, most of the AGPs were down-regulated in EC, and kept relative low expression level during early SE (Additional file 4: Table S4). The results indicated that most of the extracellular protein encoding genes were mainly expressed in NEC, they were predicted to take effect on the transition from NEC to EC.

Characterization of Molecular Markers for longan SE

Several genes have been reported to molecular marker of SE, such as somatic embryogenesis receptor-like kinase (SERK),, leafy cotyledon1 (LEC1),, BABYBOOM (BBM),, wuschel (WUS),, WUS-homeobox (WOX).. In order to characterize the full-scale of molecular markers for early SE, the comparative analysis of FPKM in nine tissues of longan including root, stem, leaf, flower, flower bud, young fruit, pericarp, pulp and seed which RNA-Seq at the same time [48] were employed to select the molecular marker genes during SE. For our purposes here, it is crucial to identify the reliable molecular marker genes for distinguishing NEC from the early SE stages. In our study, several embryogenesis-labeled genes that had been reported previously were differentially expressed in the four development processes (Additional file 5: Table S5). However, some of them showed down-regulated or slightly up-regulated in EC, and kept low expression level from NEC to GE, such as late embryogenesis abundant protein (LEA14A, LEAD34, LEA76), SERK1, SERK3, WUS, WOX5, WOX3, AIL6, AGL15, CLV1, EMB8,suggesting that they were unseemly markers for longan SE.

In our study, a total of 55 genes were identified as representative molecular markers, which were closely related to SE, can be classified as two main categories: NEC markers and SE molecular markers by their specific expression profiles in all test-samples (Table 4). The SE marker genes were barely or undetected in NEC, highly expressed during early SE, it also can be divided into SE-specific marker and SE-expressed marker. The SE-specific markers were highly transcribed only in somatic embryos, including LEC1, LEC2, WOX9, WOX2, Agamous-like 80 (AGL80),, PIN-FORMED1 (PIN1),, BBM, PLETHORA2 (PLT2),mannan endo 1,4-beta-mannosidase7 (MAN7),, Glycine-rich protein 5 (GRP–5),, GRF-interacting factor 2 (GIF2),, root meristem growth factor 3 (RGF3),, 60S ribosomal protein L17e (RPL17e),, zeta-carotene desaturase (ZDS),, 3-ketoacyl-CoA synthase (KCS),, CYP78A5, CYP87A3 and three unknown genes (DlU1, DlU2, DlU3) (Table 4). These SE-specific genes may played an important role in longan SE. The SE-expressed markers were similar to SE-specific markers, except that these genes also highly expressed in one or some tested tissues included in this study, including LEC1-like (L1L),, ABA-insensitive protein 3 (ABI3),, FUSCA3 (FUS3),, Indole–3-acetic acid-amido synthetase (GH3.6), Protodermal factor 1.3 (PDF1.3),, Lipid transfer protein (LTP, Dlo_013012.1)and Lipid binding protein (LBP).. For instance, L1L, FUS3 and ABI3 showed very strong transcription level not only in somatic embryos but also in seed, GH3.6 was high expressed in flower, PDF1.3 and LBP showed high expression level in pulp, LTP also highly transcribed in pulp, flower bud, flower and stem, suggesting their multifunctional on SE and other development processes (Table 4).

On the contrary, 28 representative NEC marker genes were highly and preferentially expressed in NEC, barely or undetected in EC, ICpEC and GE, including LEA5, CCR4-NOT transcription complex subunit 3 (CNOT3),, pathogenesis-related protein (PR1–1, PR1-like, PR4),, 14 kDa proline-rich protein DC2.15 (DC2.15),, chitinases (CHI: Dlo_030517.1, Dlo_024175.1), catalase (CAT),, Lipid transfer proteins (NsLTP2, DIR1),, aquaporins (PIP1, PIP2.1, TIP2–1), peroxidases (POD-P7, POD5),, osmotin-like protein 1 (OSM1),, expansin-like B1 (EXLB1),, Pectinesterase precursor (PME1),, chalcone synthase (CHS),, thaumatin-like protein (TLP1),, Gibberellic Acid Stimulated Transcript-like (GAST1),, ethylene-responsive transcription factor 114 (ERF114),, glutathione S-transferase (GST, Dlo_032871.1), germin-like protein 3 (GLP3),, and three unknown genes (DlU4, DlU5, DlU6) (Table 4). The NEC-specific marker genes maybe the key inhibitor of the transition from NEC to EC, while the SE markers may function as promoter in SE development.

qRT-PCR Verification of selected molecular markers

To experimentally confirm that the molecular markers were indeed expressed and played an important role during longan SE, 16 potential molecular markers, including 8 transcription factors DlLEC1_Dlo_017092.1, DlL1L_Dlo_020821.1, DlABI3_Dlo_012160.1, DlWOX9_ Dlo_022316.1, DlWOX2_Dlo_032045.1, DlAGL80_Dlo_017585.1, DlBBM_Dlo_011527.1 and DlPLT2_Dlo_004646.1, auxin metabolism gene DlGH3.6_ Dlo_020986.1, auxin polar transport gene DlPIN1_Dlo_020694.1, 3 meristem growth regulation genes DlPDF1.3_ Dlo_030812.1, DlRGF3_Dlo_026048.1, DlGIF2_Dlo_026819.1, 2 extracellular protein encoding genes DlLTP_Dlo_013012.1 and DlCHI_Dlo_030517.1, a late embryogenesis abundant protein gene DlLEA5_Dlo_019949.1, were selected for qRT-PCR identification in the synchronized cultures at distinct developmental stages during longan SE, including NEC, EC, ICpEC, GE, torpedo-shaped embryos (TE) and cotyledonary embryos (CE).

Base on the qRT-PCR results, all selected genes were expressed at varying levels at different development stages (Figure 6). The selected molecular markers DlLEC1, DlPDF1.3, DlGH3.6, DlPIN1, DlWOX9, DlWOX2, DlGIF2, DlRGF3, DlPLT2 and DlAGL80 were barely or undetected in NEC, while they mainly expressed during early SE, they all highest expressed in EC and then down-regulated during SE, showed relative low expression in TE and CE, indicated that those molecular markers played an important role in EC induction and maintainance. Meanwhile, DlL1L, DlBBM, DlABI3 and DlLTP were highly expressed or up-regulated during SE processes, and minimally or undiscovered expressed in NEC, suggested that those marker genes may positive regulated the longan SE development. In addition, the transcription level of DlLEA5 and DlCHI were highly and specific expressed in NCE, they may the inhibitor of the transition from NEC to EC. Consequently, the NEC and EC molecular markers can effectively used to distinguish the non-embryogenic and embryogenic somatic cells, and played an important role in longan SE.

Auxin and cytokinin play an important role in longan SE

It is well know that auxin and cytokinin (CTK) are key factors of plant cell division and differentiation, as well as SE induction [59]. Meanwhile, the level of endogenous IAA and CTK were influenced by the application of exogenous auxin and CTK [3, 10, 60–62]. Auxin is consider as a central regulator in SE, probably due to the establishment of auxin gradients during SE induction [9]. So far, the exogenous application of auxin during SE has been well documented [9, 10, 27, 60]. Among the auxin, synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) is most effective and widely used for induction of SE in several plants [63–65]. The level of endogenous IAA is correlated with pro-embryogenic mass formation and high-frequency SE competency [66]. Previous study have also proved that dynamic change of endogenous IAA was among the first signals leading to the induction of SE [67].

Over the past 20 years, longan SE has been established and widely used as model system for embryogenesis in woody plants, high concentration of 2,4-D in MS medium was require for inducing EC from immature zygotic embryo, while it suppressed further development of SE, moreover, 2,4-D and KT were the key factors in long term maintenance of longan EC [1, 7, 8]. Subsequent studies revealed that controlling the doses of 2,4-D could synchronized regulated the developmental processes of longan SE, withdrawal of 2,4-D from the medium triggered further embryo development [46, 68–70]. The level of endogenous IAA and CTK in early SE stages (EC, ICpEC and GE) were much higher than that in NEC, and IAA level reached the peak in GE and then significantly decreased at later stages which were cultured on 2,4-D free medium. In addition, the level of IAA higher than CTK at the same stage during early SE. The results indicated that high level of endogenous IAA and lower level of CTK were essential for early SE [3]. However, the molecular mechanism responsible for the endogenous IAA and CTK level changing during SE, and potential crosstalk with each other or other factors remains poorly-understood. Large numbers of DEGs associated with IAA and zeatin biosynthesis, transport, signal transduction and degradation were identified, the activities of these DEGs could be related to SE.

The increase of IAA during longan early SE might be due to the increased biosynthesis and transition of endogenous auxin precursor [9]. The tryptophan (Trp) dependent IAA biosynthesis is an important pathway in higher plants, exogenous applied the doses of Trp and IAA had similar enhancement during rice SE [9]. In our study, the expression level of ASA, IGS, TSA, TSB, the key genes in Trp synthesis, were drastic up-regulated in EC and remained high in early SE, only PAI showed NEC specific with low FPKM, suggested that the level of Trp during early SE was higher than NEC, high IAA level might be due to high level of auxin precursor during early SE. YUCCAs family encoding key enzymes in IAA biosynthesis, are required for SE induction in Arabidopsis [72], and three YUCCAs and AAO1, one NIT, CYP71A13 and three ST5a, showed up-regulated expression from NEC to EC, among them two YUCCAs, AAO1, ST5a were down-regulated during early SE, while YUCCA_Dlo_013505.1 kept up-regulated expression during early SE. Other IAA synthesis related genes were mainly expressed in NEC with minimal FPKM. The increase of IAA level may due to these differentially expressed genes during early SE. However, more evidences is needed to prove the relationship between these DEGs and increased IAA level.

During SE induction of C. canephora, the balance of free IAA and IAA conjugates is essential for embryogenic potential [73], the conjugation of auxin is synthesized by GH3 family [74], we found that GH3 genes were minimal expressed in NEC, most of them dramatic up-regulated in EC and then down-regulated during early SE, indicated that not only the level of free IAA but also the conjugated IAA is important during longan SE. Previous studies has revealed that auxin transports is complex and highly regulated for embryogenic development [75]. TIR1 mediates Aux/IAA proteins degradation and auxin-regulated transcription in the present of auxin [76], while TIR1 were down-regulated in EC and remained low during longan early SE. AUX1, which mediates influx of IAA into cells,were mainly expressed in NEC, and down-regulated during early SE. PIN1 plays a fundamental role in maintaining the embryonic auxin gradients [77], were up-regulated in EC and kept high in ICpEC and GE.

Auxin transcriptionally activates Aux/IAA, GH3 and SAUR family,the Aux/IAA family has 29 members in Arabidopsis, but not all members are induce by auxin [78]. SAUR is the most abundant family of early auxin-inducible genes, but only few members have been functional characterized, OsSAUR39 was reported to negatively regulate auxin biosynthesis and transport [79]. ARF show strongly disturbance during zygotic embryo development [80, 81], and ARF5 seems to be importance for SE [82]. Further transcript analysis during SE revealed that the components of auxin signaling: Aux/IAA, ARF, SAUR and other auxin-responsive genes were wildly modulated during SE [9, 13]. In our study, 11 ARF significantly up-regulated in EC and remained high during early SE, three ARF first down-regulated in EC and then up-regulated during SE. IAA family showed the similar expression pattern during SE, while most of SAUR were mainly expressed in NEC.

Other than auxin being a main inducer of SE, exogenously supplied CTK to induce SE is well established in a lot of species [83–85]. Large numbers of transcripts involved in zeatin biosynthesis and signal transduction were differentially expressed during cotton SE [10]. Meanwhile, endogenous CTK level were higher in SE than in NEC [3]. From NEC to EC, a total of 40 DEGs implicated in cytokinin signal transduction, including 22 CRE1 (seven up-regulated and 15 down-regulated), 15 B-ARR (five up-regulated and 10 down-regulated), 3 A-ARR (one up-regulated and two down-regulated). During early SE, a total of 32 DEGs involved in cytokinin signaling pathway, most of them were up-regulated during early SE. In zeatin synthesis pathway, TRIT1 was up-regulated from NEC to ICpEC, most of CisZOG, IPT1,4, CYP735A, CKX, UGT76C were down-regulated in EC and remained low during early SE. However, IAA and zeatin biosynthesis and signal transduction related genes showed complex and integrated regulation during SE, further study of these genes is required in longan SE.

Molecular markers for longan early SE

The 55 molecular markers genes for longan SE belong to several distinct functional categories, they can be used to mark the embryogenic potential of plant cells and study various biochemical and physiological processes of plant embryogenesis and development. A number of transcription factors (TFs) have been reported to have a key role in SE induction. In Brassica napus, LEC1, LEC2, FUS3, ABI3, WOX9, WOX2, BBM, genes belonged to TFs, were identified as molecular markers for early microspore embryogenesis [86]. In our study, 10 of the molecular markers were TFs (DlLEC1, DlL1L, DlLEC2, DlABI3, DlFUS3, DlWOX9, DlWOX2, DlAGL80, DlBBM, DlPLT2),, most of them had been well characterized in various plants. Ectopic expression of LEC1 is sufficient to trigger embryogenic potential and to induce somatic embryo from Arabidopsis leaf surface [87]. ZmLEC1 can be used as a reliable marker for early SE in maize as its expression pattern during SE was similar to that of AtLEC1 during zygotic embryogenesis [87]. Mutational analyses in Arabidopsis showed that LEC genes were essential for induction of SE [37]. Ectopic expression of a carrot C-LEC1 that driven by AtLEC1 promoter, was able to rescue the defects of lec1–1 mutant [89]. Moreover, ectopic-expressed AtLEC1 in tobacco induce the start of embryogenic transition [90]. The LEC1-like (L1L) was most closely related to LEC1 and required for normal embryo development, ectopic-expressed L1L in Arabidopsis can complement LEC1 functions [91]. Meanwhile, L1L expression was mainly accumulated in the early stage SE of Theobroma cacao [92], Vitis vinifera [93], and Helianthus annuus [94].

LEC2, ABI3, FUS3 were B3 domain-containing transcription factors, ectopically expressed AtABI3 do not induce SE but endowed the embryo with traits to seedling [95]. BBM and PLT2 were clustered to AP2/ERF transcription factor family, their functions on embryogenesis and root meristem were overlap [96–98]. Over-expression of BBM trigger spontaneous somatic embryo formation in Arabidopsis thaliana and Brassica napus, BBM was server as a marker for embryogenesis cells in Brassica napus [96]. Recently study show that BBM and PLT2 induce SE in a quantitatively and context dependent manner by LEC1-ABI3-FUS3-LEC2 (LAFL) network, and LAFL/AGL15 are required for BBM mediated embryogenesis [40]. In this assay, DlLEC1, DlLEC2, DlFUS3 were early SE-specific genes, they can be used as remarkable markers for longan early SE. DlL1L, DlBBM, DlABI3, DlPLT2 were highly expressed during the SE processes, they can be used as molecular marker for longan SE development. To date, AGL15 is the only MADS-BOX member which preferentially expressed in developing embryos and promote the initiation of SE [44, 45], and AGL80 was essential for the central cell and endosperm development [99]. However, DlAGL15 was considered as poor marker. Firstly, we suggested another MADS-BOX gene DlAGL80,a SE-specific gene as a new marker for longan early SE.

WUS is a critical regulator for stem cell fate in the shoot apical meristem [100]. Over-expression of AtWUS can initiate the acquisition of embryogenic competence in Gossypium hirsutum [41, 42]. WUS was suggested as a useful gene marker for SE initiation [101]. Meanwhile, WOX genes mark cell fate during early embryogenesis in Arabidopsis [102], WOX2 was used as potential marker during early SE [103]. STIMPY/WOX9 plays an important role in promoting cell proliferation and preventing precocious differentiation in emerging seedlings [104]. WOX2 and WOX9 were highly expressed at the early stage of SE in Picea abies, they may function together on conifer embryo patterning105]. In addition, DlWUS was isolated from embryogenic callus and expressed in all the stage of SE, which consistent with our transcriptome date suggested that DlWUS genes are poor markers during longan SE. The qRT-PCR verification demonstrated that DlWOX2 and DlWOX9 were specific expressed in early SE and down-regulated during SE, they were suggested as reliable markers for longan early SE.

PIN1 was involved in auxin polar transport and cellular differentiation during embryogenesis [106, 107]. Antisense expression of PIN1 disrupted the formation of somatic embryos and reduced the expression of SE-related genes, indicated that PIN1 was essential for SE induction [108]. GH3.6 is contribute to maintain auxin homeostasis by converting excess IAA to IAA-amino acid conjugates, over-expression of GH3.6 significantly enhanced the accumulation of IAA-Asp [109]. DlPIN1 and DlGH3.6 were specific expressed in early SE and down-regulated during SE, they were suggested as reliable markers for longan early SE.

In our study, CYP78A5 and CYP87A3 were most abundant in EC and follow by ICpEC stage. In B. napus, CYP78A5 was identified as an early marker for microspore-derived embryos development [86]. PDF1.3 is closely related to Arabidopsis Protodermal factor 1, a gene exclusively expressed in L1 layer of vegetative, inflorescence, floral meristems and specific-expressed in protodermal cell during embryogenesis which related to cell fate determination [110]. In addition, AtGRP–5 was associated with somatic embryo formation in Arabidopsis and eggplant [111]. RGF3 and GIF2 were key genes of cell proliferation, showed SE-specific expression pattern during early SE. RGF3 belongs to root meristem growth factors family that played the redundant role in maintaining the post-embryonic root stem cell niche and by positive regulating cell proliferation [112]. GIF2 was required for cell proliferation and lateral organs grow [113]. Those SE-related genes DlPIN1, DlGH3.6, DlPDF1.3, DlGRP–1, DlRGF3 and DlGIF2 can be use to mark the early stage of longan early SE. Furthermore, DlLBP, DlKCS, DlZDS and DlRPL17, DlMAN7 and DlU1, DlU2, DlU3 were specific accumulated in early SE, despite that no functions on SE have been published yet for them, they also suggested as molecular markers for longan SE, but their functions on SE need further investigating.

SERK play a key role in the acquisition of embryogenic competence in plant cells, DcSERK was identified as a suitable marker for SE as it only abundant in embryogenic cultures and ceased after the globular stage, but not in any other tissues [34]. In Dactylis glomerata, SERK showed the similar expression pattern with DcSERK and used as a convenient marker for cells competent to form embryos in monocots [114]. AtSERK1 was highly expressed during Arabidopsis embryogenic cell formation and early embryogenesis, suggested that AtSERK1 is sufficient to mark embryogenic competence in culture [115]. However, ZmSERKs were detected in non-embryogenic callus [116], and the identification of SERK genes in rice [117], and wheat [118], suggest that their functions are not limit to embryogenesis. In our study, DlSERKs were expressed not only in SE stages, but also in non-embryogenic callus and other tissues, thus, DlSERK could not be used as a reliable marker for longan SE.

LEA5 belonged to the fifth group of late embryogenesis abundant proteins gene, were abundant in late embryogenesis of mature seed, and involved in the abiotic stresses responses [119]. CNOT3, maybe a new CCR4-NOT complex gene in plant, which had proved in regulation of cell division in HeLa cell, while its functions on plant is poorly-understood. 14 kDa proline-rich protein DC2.15 was connected with the initiation of embryogenesis by the removal of auxin [120]. Aquaporins are the major channels of water transport pass through biological membranes, and involved in cell expansion, organ movement and elongation [121]. It is widely acceptable that the extracellular proteins (such as GLPs, LTPs, CHIs) are required for plant differentiation and morphogenesis, they can be used as protein markers for SE [58]. Our study revealed that a total of 28 transcripts were specific and extreme-highly expressed in NEC, while barely or undetected during early SE stages, we suggested these genes as NEC molecular markers, for example, the LEA5, CNOT3, DC2.15, PIP1;2, PIP2;1, GLP3, NsLPT, CAT, POD, GST, et al., these genes might played an important role in the transition from NEC to EC, and were helpfully for distinguishing NEC and early SE. However, some of these markers belonged to the certain gene family with distinct expression patterns during SE, further study of these genes function on longan SE is required.

In summary, our study generated a high resolution transcriptome datasets for longan SE. A comparative analysis of global gene expression patterns during early SE stages provided subsets of DEGs that regulated SE in longan. Our study revealed the expression profiles of genes involved in auxin and cytokinin signaling pathway, flavonoid and fatty acid biosynthesis pathway, extracellular protein, as well as the representative molecular marker genes, indicating their possible roles in longan SE. This transcriptomic data provides new insights into future functional studies, as a means of studying the molecular mechanisms in SE.

Plant Material and RNA Extraction

The synchronized cultures at different developmental stages, including non-embryogenic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE) of D. longan ‘Honghezi’, were obtained following previously methods [1, 7, 8], and stored at –80⁰C for RNA extraction. Total RNAs of the above samples were isolated using Trizol Reagent (Invitrogen, USA), then DNase I was used to digest any genomic DNA. Extracted RNAs were quantified by Agilent 2100 bioanalyzer (Agilent Technologies, USA) and evaluated the integrality by denaturing agarose gel electrophoresis and ethidium bromide staining. RNA samples with A260/A280 ratios between 1.9~2.1, 28S/18S ratios ≥1.0, and integrity numbers (RINs) more than 8.5 were selected to construct cDNA libraries.

Library Construction and RNA Sequencing

After enrichment with oligo(dT)₂₅-attached magnetic beads, the purification mRNA was interrupted into short fragments by the fragmentation buffer. Used these short fragments as templates and SuperScript III (Invitrogen, USA) as reverse transcriptase to synthesize first strand cDNA. The second strand cDNA was subsequently synthesized using random primers and end repaired, then adaptors were ligated by T4 DNA ligase after adenylation at the 3’-end. Finally, suitable adaptor-ligated fragments were selected as templates for PCR amplification. The four resulting cDNA libraries were quantified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and qRT-PCR (ABI StepOnePlus Real-Time PCR System, USA), and then RNA Sequencing (RNA-seq) was carried out with an Illumina HiSeq^TM 2000 system at The Beijing Genomics Institute (BGI, Shenzhen, China).

RNA-Seq Reads Mapping and Differential Expression

The raw reads were cleaned by removing adapter reads, reads containing poly-N larger than 10%, and low quality reads (Q_Phred < 20). Cleaned reads were then aligned to the longan reference genome using Bowtie software (http://bowtie-bio.sourceforge.net/index.shtml) and TopHat2 (http://ccb.jhu.edu/software/tophat/index.shtml), read count for each gene was then obtained after mapping. Gene expression levels for each sample were estimated by RSEM (RNA-Seq by Expectation Maximization). The expression levels of matched genes in each cDNA library were derived and normalized to FPKM (Fragments Per Kilobase of exon per Million fragments mapped).

The differentially expression analysis of the two varieties were performed using the ratio of their FPKM values, the False Discovery Rate (FDR) was used to determine the P-value threshold. The unique reads with the absolute value of log₂(Fold change of FPKM)≥ 1 and the FDR < 0.001 were used as the thresholds to identify and compare significantly DEGs in the study.

Expression Annotation and Functional analysis of DEGs.

Gene function was annotated based on the databases of Blast Nr (NCBI non-redundant protein sequences), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes database).The GO and KEGG functional enrichment analysis of DEGs were performed to identify which DEGs were significantly enriched in GO terms or KEGG pathways. GO terms with corrected P-value ≤ 0.05 were considered as significantly enriched terms. The KEGG enrichment was determined by Rich factor, Q-value, and the number of enriched genes in this pathway. Q-value ≤ 0.05 was defined as those with genes that showed significant differential expression.

Gene Validation and Expression Analysis

Sixteen genes with potential roles in longan SE were chosen for validation using quantitative real-time PCR (qRT-PCR) with gene specific primers designed using DNAMAN 7.0. Relative mRNA levels from each gene in RNA isolated as described above from six synchronized cultures during longan SE, including NEC, EC, ICpEC, GE, TE and CE were quantified with respect to internal standards. All reactions were performed with three replicates in a LightCycler 480 qRT-PCR instrument (Roche Applied Science, Switzerland). The expressive abundance of the sixteen selected genes were calculated relative to the expression of reference genes DlFSD, DlEF–1a, and Dlelf4a. Gene names, primer sequences, product sizes, and annealing temperatures are given in additional file 6: Table S6.

Table 1 Statistics of reads generated by transcriptome sequencing of longan SE

Sample Name	Total Clean reads	Total Reads Map to Genome	Genome Mapping Rate (%)	Total Reads Map to Gene	Gene Mapping Rate (%)	Expressed Transcripts	Expressed Exon	Novel Transcripts	Extend Gene	Alternative Splicing
NEC	59,785,854	48,798,229	81.62	34,380,246	57.51	22,743	110,864	1,935	10,281	35,084
EC	64,876,258	52,623,741	81.11	35,386,494	54.54	19,745	103,200	1,710	9,092	19,056
ICpEC	59,580,846	48,346,067	81.14	30,535,088	51.25	21,144	107,592	1,816	9,197	36,446
GE	59,540,168	48,871,200	82.08	29,214,788	49.07	21,102	107,971	1,732	9,638	39,768

Table 2 Gene expression levels given in FPKM during longan SE

FPKM Interval	NEC	EC	ICpEC	GE
≤0.1	3900(15.01%)	6910(26.60%)	5364(20.65%)	5391(20.75%)
0.11-1	3587(13.81%)	3075(11.84%)	3241(12.48%)	3384(13.03%)
1.01-3	2706(10.42%)	1957(7.53%)	2439(9.39%)	2362(9.09%)
3.01-15	6440(24.79%)	4774(18.38%)	5413(20.84%)	5208(20.05%)
15.01-60	6384(24.57%)	5817(22.39%)	6076(23.39%)	6191(23.83%)
60.01-100	1278(4.92%)	1431(5.51%)	1601(6.16%)	1573(6.06%)
≥100	1683(6.48%)	2014(7.75%)	1844(7.10%)	1869(7.19%)

Table 3 The top 20 most expressed genes in NEC, EC, ICpEC, GE library.

NO.	Gene_id	FPKM_NEC	Description
1	Dlo_008315.1	58812.43	repetitive proline-rich cell wall protein 2
2	Dlo_019949.1	36215.45	Late embryogenesis abundant protein Lea5
3	Dlo_008311.1	11187.75	unknow protein
4	Dlo_028175.1	10885	unknow protein
5	Dlo_011615.1	10317.69	extensin-2-like
6	Dlo_030517.1	8931.79	chitinase CHI
7	Dlo_004649.1	7800.33	metallothionein
8	Dlo_024177.1	6055.72	chitinase
9	Dlo_017033.1	5645.16	pathogenesis-related protein 1
10	Dlo_008997.3	5476.22	unknow protein
11	Dlo_009172.1	5469.19	osmotin-like protein I
12	Dlo_021620.1	4483.38	peroxidase 4
13	Dlo_003142.1	4116.17	unknow protein
14	Dlo_030075.1	3732.29	Wound-induced protein WIN1 precursor
15	Dlo_030519.1	3587.55	chitinase CHI
16	Dlo_022694.1	3546.1	14 KDa proline-rich protein DC2.15-like
17	Dlo_030516.1	3288.7	chitinase
18	Dlo_030074.1	3170.89	PR-4 protein
19	Dlo_011076.1	2572.89	ubiquitin C
20	Dlo_011004.1	2367.71	non-specific lipid-transfer protein 2-like
NO.	Gene_id	FPKM_EC	Description
1	Dlo_030812.1	15114.88	Protodermal factor 1.3 PDF1.3
2	Dlo_013012.1	7392.37	lipid transfer protein
3	Dlo_030517.1	5117.6	chitinase CHI
4	Dlo_025725.1	4537.79	Pollen-specific protein C13 precursor
5	Dlo_020986.1	4136.45	Indole-3-acetic acid-amido synthetase GH3.6
6	Dlo_011615.1	4031.23	extensin-2-like
7	Dlo_021620.1	3793.82	peroxidase 4
8	Dlo_032035.1	2962.58	histone H4-like
9	Dlo_031913.1	2959.02	lipid binding protein
10	Dlo_003789.1	2766.68	EXORDIUM-like 2 EXL2
11	Dlo_005176.1	2363.28	omega-6 fatty acid desaturase
12	Dlo_026048.1	2219.4	root meristem growth factor 3 RGF3
13	Dlo_033433.1	2202.74	unknow
14	Dlo_019526.1	2145.11	unknow
15	Dlo_017203.1	2144.67	Hsp90
16	Dlo_026351.1	2131.12	peptidase
17	Dlo_017092.1	2118.54	transcription factor leafy cotyledon1
18	Dlo_020190.1	2035.05	cysteine protease
19	Dlo_007905.1	2000.4	small ubiquitin-related modifier 2-like
20	Dlo_012332.1	1967.95	26S proteasome complex subunit DSS1

Table 3-5 Continue.

NO.	Gene_id	FPKM_ICpEC	Description
1	Dlo_030812.1	10330.16	protodermal factor 1.3
2	Dlo_026048.1	6620.49	root meristem growth factor 3 RGF3
3	Dlo_031913.1	4694.68	lipid binding protein
4	Dlo_013012.1	4301.99	lipid transfer protein
5	Dlo_008315.1	4140.34	proline-rich cell wall protein 2-like PRP2
6	Dlo_032146.1	3401.87	NADH dehydrogenase (ubiquinone)1 beta subcomplex 7
7	Dlo_028379.1	2815.75	dehydrin 1
8	Dlo_025725.1	2772.74	Pollen-specific protein C13 precursor
9	Dlo_020986.1	2439.09	Indole-3-acetic acid-amido synthetase GH3.6
10	Dlo_021620.1	2343.85	peroxidase 4
11	Dlo_019476.1	2300.16	unknow
12	Dlo_028328.1	2106.48	high mobility group box 1
13	Dlo_017203.1	1958.55	Hsp90
14	Dlo_019638.1	1958.53	elongation factor 1-alpha
15	Dlo_030608.1	1823.94	unknow
16	dlo_034323.1	1818.75	histone H1
17	Dlo_011615.1	1789.67	extensin-2-like
18	Dlo_010406.1	1773.3	transcription factor BTF3
19	Dlo_017539.1	1684.81	histone H2B.1-like
20	Dlo_030517.1	1598.6	chitinase CHI
NO.	Gene_id	FPKM_GE	Description
1	Dlo_013012.1	4004.61	lipid transfer protein
2	Dlo_026048.1	3762.46	root meristem growth factor 3 RGF3
3	Dlo_021620.1	3304.97	peroxidase 4
4	Dlo_031913.1	2957.57	lipid binding protein
5	Dlo_032146.1	2784.3	NADH dehydrogenase (ubiquinone)1 beta subcomplex 7
6	Dlo_028379.1	2676	dehydrin 1
7	Dlo_014867.1	2300.44	argonaute 4
8	Dlo_008315.1	2218.56	proline-rich cell wall protein 2-like PRP2
9	Dlo_030608.1	2100.52	unknow
10	Dlo_012964.1	2091.04	extensin, proline-rich protein
11	Dlo_032870.1	2072.74	glutathione S-transferase parC-like
12	Dlo_025725.1	2018.61	Pollen-specific protein C13 precursor
13	Dlo_019476.1	1997.45	unknow
14	Dlo_015927.1	1907.93	unknow
15	Dlo_030812.1	1899.07	protodermal factor 1.3
16	Dlo_028328.1	1848.42	high mobility group box 1
17	Dlo_018634.1	1743.25	60S ribosomal protein L27Ae
18	Dlo_019638.1	1709.96	elongation factor 1-alpha
19	Dlo_017203.1	1696.73	Hsp90
20	Dlo_020821.1	1665.7	leafy cotyledon1-like

Table 4 The expression profile of candidate markers during SE and in nine tissues of longan.

Gene_id	Gene name	FPKM value													Putative protein function/homology
Gene_id	Gene name	NEC	EC	ICpEC	GE	Root	Stem	Leaf	Flower	Flower bud	Young fruit	Pericarp	Pulp	Seed	Putative protein function/homology
Dlo_017092.1	LEC1	0	2118.54	1262.61	727.62	0	0	0	0	0	0	0	0	1.52	leafy cotyledon 1 transcription factor
Dlo_020821.1	L1L	0.21	511.04	393.39	1665.7	0	0	0	0	0	0	0	0	209.01	leafy cotyledon 1-like protein
Dlo_022316.1	WOX9	1.34	1602.12	1424.69	665.41	0.3	0	0	0	0.52	1.5	0	5.98	0.67	WUSCHEL-related homeobox 9
Dlo_032045.1	WOX2	0.48	141.61	83.91	59.36	0	0	0	0	0	0	0	0	0.17	WUSCHEL-related homeobox 2
Dlo_011527.1	BBM	6.28	367.26	484.12	511.04	9.86	0.28	0.1	0.12	0.19	0.34	0.14	0	0.67	AP2/ERF, BABYBOOM
Dlo_004646.1	PLT2	3.94	491.37	610.1	759.4	0.41	0	0	0	0	0	0	0	0	AP2/ERF, PLT2
Dlo_012160.1	ABI3	0	82.94	177.78	374.51	0	0	0	0.13	13.28	0	0	0	119.83	ABA-insensitive protein 3
Dlo_022372.1	LEC2	0	45.35	55.63	51.22	0.11	0	0	0	0	0	0	0	0.4	leafy cotyledon 2 transcription factor
Dlo_024008.1	FUS3	0	5.75	37.93	260.7	0	0	0	0	0.35	0	0	0	60.65	FUSCA3
Dlo_017585.1	AGL80	0	829.45	590.14	278.12	0.62	0.58	0.82	0.49	0	0.29	0.84	0.58	1.56	Agamous-like MADS-box protein 80
Dlo_020986.1	GH3.6	0.06	4136.45	2439.09	1630.99	0	1.2	0.06	843.22	0.32	0	0.25	0.2	0.14	IAA-amido synthetase GH3.6
Dlo_020694.1	PIN1	3.23	216.9	96.91	100.29	0	0	0	0.07	0	0	0	0	3.04	Auxin efflux carrier component 1
Dlo_030812.1	PDF1.3	15.81	15114.88	10330.16	1899.07	0.68	0	0.16	2.4	60.18	76.88	21.19	680.91	16.67	Protodermal factor 1.3
Dlo_027182.2	GRP-5	0	1699.47	1094.49	874.66	0	0.35	0	0	0.32	0	0	0	8.03	glycine-rich protein 5
Dlo_032565.1	CYP78A5	0.97	320.4	102.08	12.72	31.26	0	1.85	0.09	6.93	1.78	0.4	0.47	4.39	Cytochrome P450 family 78A5
Dlo_017331.1	CYP87A3	0	274.33	130.99	25.01	0	0.11	0	5.98	0.63	0.22	0.22	4.42	0.18	Cytochrome P450 family 87A3
Dlo_026048.1	RGF3	0	2219.4	6620.49	3762.46	0	0	0	3.27	1.96	0	0	0	0	root meristem growth factor 3
Dlo_026819.1	GIF2	0	726.78	454.72	339.9	0	0.94	0	1.08	6.39	0.45	0.3	1.79	12.93	GRF1-interacting factor 2, GIF2
Dlo_013012.1	LTP	6.56	7392.37	4301.99	4004.61	3.92	1182.18	5.16	1241.54	1954.47	354.17	62.65	4225.76	67.83	Lipid transfer protein
Dlo_031913.1	LBP	3.92	2959.02	4694.68	2957.57	1.19	25.49	12.3	28.54	27.78	32.46	4.93	428.03	5.16	Lipid binding protein
Dlo_025851.1	MAM7	3.43	1112.42	436.33	119.05	0	0	0	0	0	0	0	0	0	mannan endo-1,4-beta-mannosidase 7
Dlo_029005.1	ZDS	0	1819.85	1213.68	932.22	0.15	0.15	0.11	0.76	0.81	20.69	0.42	14.54	2.2	zeta-carotene desaturase
Dlo_023272.1	KCS	0	511.05	371.46	205.05	0	0	0.08	0	0.1	1.36	0	1.49	0.09	3-ketoacyl-CoA synthase (fatty acid)
Dlo_032570.1	RPL17	0	1081.06	1123.65	990.78	48.99	40.71	0	0	3.48	3.17	0	0	0	60S ribosomal protein L17

Table 4 (continued)

Gene_id	Gene name	FPKM value													Putative protein function/homology
Gene_id	Gene name	NEC	EC	ICpEC	GE	Root	Stem	Leaf	Flower	Flower bud	Young fruit	Pericarp	Pulp	Seed	Putative protein function/homology
Dlo_033433.1	DlU1	3.52	2202.74	1163.64	830.54	14.46	1.61	0	0	0	0	0	0	0	Unknown 1
Dlo_026772.1	DlU2	0	50.68	397.92	1516.17	0.97	0	0	0	0	0	0	0	0.24	Unknown 2
Dlo_028569.1	DlU3	0	558.23	418.54	121.81	0.78	7.07	0	3.14	4.43	3.26	3.67	0	0	Unknown 3
Dlo_019949.1	LEA5	36215.45	27.67	145.54	256.16	3165.31	6822.69	861.14	7685.55	11712.56	3205.39	24022.25	1205.56	3737.57	Late embryogenesis abundant protein 5
Dlo_008311.1	CNOT3	11187.75	12.96	27.95	44.39	306.61	11545.36	933.45	1611.48	393.14	1560.72	2650.38	7249.71	1296.04	CCR4-NOT transcription complex 3
Dlo_028175.1	DlU4	10885	0.31	2.94	3.3	5220.16	576.03	3.61	217.29	35.02	4434.9	1942.11	5575.02	396.85	Unknown 4
Dlo_017033.1	PR1-1	5645.16	40.36	26.32	10.4	2601.85	122.2	1.45	86.79	22.85	0.96	361.66	577.38	17.99	Pathogenesis-related protein 1-1
Dlo_022694.1	DC2.15	3546.1	0.29	0	0	965.82	92.92	9.21	930.75	46.71	788.54	400.61	1323.15	301.29	14 kDa proline-rich protein DC2.15
Dlo_030517.1	CHI	3288.7	12.16	54.25	8.31	2.21	1.5	0.7	0.62	0.45	0	0.46	1.65	0.19	Chitinase
Dlo_030074.1	PR-4	3170.89	0.31	2.48	1.62	517.32	105.29	0.59	90.21	85.99	0.79	10.68	4.68	25.33	PR-4 protein
Dlo_028350.1	CAT	2411.98	15.42	41.81	55.28	4254.18	1219.17	398.73	293.83	464.27	651.57	3884.71	264.3	319.86	Catalase
Dlo_011004.1	NsLTP2	2367.71	0	0	0	1410.22	836.82	1.46	171.89	732.06	69.9	148.79	31.47	45.31	Non-specific lipid-transfer protein 2
Dlo_032927.1	PIP2.1	2191.16	8.96	7.95	4.38	2253.88	162.85	81.62	45.29	21.99	140.46	842.74	1733.29	217.06	Aquaporin PIP2.1
Dlo_011314.1	POD P7	2007.14	3.73	4.04	2.59	67.72	32.71	1.25	33.7	10.84	1.82	24.34	4.33	1.23	Peroxidase P7-like
Dlo_020889.1	DlU5	1534.05	0.27	18.34	62.55	1224.15	79.36	8.05	83.74	14.28	2100.25	1136.71	29080.32	892.17	Unknown 5
Dlo_009170.1	OSM1	1459.85	8.09	7.15	4.64	1450.79	3031.81	58.61	3028.02	3208.04	144.45	3184.56	2337.21	430.64	Osmotin-like protein 1
Dlo_006330.1	EXLB1	1145.83	1.14	4.4	3.19	303.02	2258.39	7.29	32.98	200.08	12.5	543.39	0.26	111.72	Expansin-like B1
Dlo_003365.1	DIR1	1049.5	0	0	0	614.07	12.2	5.3	35.6	37.19	284.21	88.68	0.7	24.18	Lipid-transfer protein DIR1
Dlo_024175.1	CHI	983.75	0.14	0	1.5	3965.12	6.77	5.9	46.39	47.66	1.53	10.77	5.23	5.17	class I chitinase
Dlo_011438.1	PIP1	812.41	0.56	1.69	2.71	1138.83	1183.76	441.46	491.94	345.78	1651.58	806.49	239.44	651.72	Aquaporin PIP1
Dlo_014725.1	PME1	753.54	0	1.19	0.57	501.17	0.62	0.96	0.53	0	16.39	1.39	21.11	8.61	Pectinesterase precursor
Dlo_027164.1	CHS	610.74	0.1	1.76	0.91	1988.95	12.84	204.09	222.61	19.12	40.45	5.95	0.96	122.71	chalcone synthase
Dlo_023321.1	TLP1	573.07	0.8	0.14	0.26	39.55	75.91	0.5	162.54	45.36	2.27	2.42	0.79	0.99	thaumatin-like protein

Table 4 (continued)

Gene_id	Gene name	FPKM value													Putative protein function/homology
Gene_id	Gene name	NEC	EC	ICpEC	GE	Root	Stem	Leaf	Flower	Flower bud	Young fruit	Pericarp	Pulp	Seed	Putative protein function/homology
Dlo_000377.1	POD5	566.61	1.32	1.3	1.41	79.76	728.88	601.84	3319.92	905.76	380.12	225.83	1440.49	117.02	Peroxidase 5
Dlo_004275.1	TIP2-1	546.09	0	0	0	55.57	139.05	71.7	147.25	132.03	102.82	84.76	0.26	45.97	aquaporin MIP family TIP2-1
Dlo_017039.1	PR1-like	462.02	7.87	3.65	2.14	52.13	26.21	0	121.87	14.14	137.08	3.17	0.42	0	Pathogenesis-related protein 1
Dlo_017003.1	GAST-like	459.7	0.24	5.45	5.45	28.63	219.33	1.56	1181.5	449.48	0.89	3.56	67.29	4.4	Gibberellic Acid Stimulated Transcript-like
Dlo_012593.1	DlU6	433.86	0	0	0	0	0	0.64	0	0.73	1.88	0.74	0	0	Unknown 6
Dlo_005429.1	ERF114	352.05	0	2.51	1.98	9.51	137.66	32.36	98.04	49.11	1.79	2.92	0.53	3.31	ethylene-responsive transcription factor
Dlo_032871.1	GST	293.99	0	0	0	0	0	0	0	0	0	2.07	0	0	glutathione S-transferase
Dlo_014570.1	GLP3	259.25	0.18	4.45	4.26	107.7	0.72	4.66	2.57	1.99	0	0	0	0.23	germin-like protein 3

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Global scale transcriptome analysis reveal differentially expressed genes during early somatic embryogenesis in Dimocarpus longan Lour

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