Transcriptomes Analysis Reveals Novel Insight Into the Molecular Mechanisms of Somatic Embryogenesis in Hevea Brasiliensis

doi:10.21203/rs.3.rs-44131/v1

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Research article

Transcriptomes Analysis Reveals Novel Insight Into the Molecular Mechanisms of Somatic Embryogenesis in Hevea Brasiliensis

https://doi.org/10.21203/rs.3.rs-44131/v1

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published 12 Mar, 2021

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Background

Somatic embryogenesis (SE) is a promising technology for plant vegetative propagation, which has an important role in tree breeding. Though rubber tree (Hevea brasiliensis Muell. Arg.) SE has been founded, few late SE-related genes had been identified and the molecular regulation mechanisms of late SE still not well understood.

Results

In this study, the transcriptomes of embryogenic calluses (EC), primary embryo (PE), cotyledonary embryo (CE), abnormal embryo (AE), mature cotyledonary embryo (MCE) and withered abnormal embryo (WAE) were analyzed. A total of 887,852,416 clean reads were generated, more than 85.92% of the clean reads were mapped to the rubber tree genome. The de novo assembly generated 36937 unigenes. The differentially expressed genes (DEGs) were identified in the pairwise comparisons of CE vs. AE and MCE vs. WAE, respectively. The specific common DEGs mainly involved in phytohormones signaling pathway, biosynthesis of phenylpropanoid and starch and sucrose metabolism. Among them, hormone signal transduction related genes were significantly enriched, especially the auxin signaling factors (AUX-like1, GH3.1, SAUR32-like, IAA9-like, IAA14-like, IAA27-like, IAA28-like and ARF5-like). The transcript factors including WRKY40, WRKY70, MYBS3-like, MYB1R1-like, AIL6 and bHLH93-like were characterized as molecular markers for rubber tree late SE. CML13, CML36, CAM-7, SERK1 and LEAD-29-like were related to rubber tree late SE. In addition, histone modification had crucial roles during rubber tree late SE.

Conclusions

This study provides important information to elucidate the molecular regulation during rubber tree late SE.

Epigenetics & Genomics

Heveabrasiliensis

Somatic embryogenesis

RNA-seq

Hormone signal

Transcript factor

Histone modification

Rubber tree (Hevea brasiliensis Muell. Arg.), a tropical rubber-producing tree within the Euphorbiaceae family, native to the great Amazonian basin of South America, is now widely cultivated to product natural rubber in Southeast Asia [1]. Rubber tree is a perennial cross-pollination tree with a long juvenile period, which takes a long period of time and makes low efficiency of hybrid breeding [2]. Rubber tree is still propagated mostly by grafting, although the interaction between scion and rootstock of grafted tree affects growth, natural rubber yield [3, 4].

Somatic embryogenesis (SE) is a promising and rapid vegetative propagation technique for plant regeneration. Plant regeneration via SE process in rubber tree had been established using different kinds of explants including immature anthers, internal integuments of immature fruits, inflorescence, as well as root [5–8]. The regenerated plants have juvenile characters and their own roots, which are called self-rooted juvenile clones (SRJCs). Compared with donor clones, SRJCs is superior in growth, rubber yield and stress resistance [9–11], which is promising a new rubber tree planting material in the future. However, there are intrinsic limitations in SE of rubber tree, such as only a limited number of genotypes can obtain somatic embryos, different lines of friable calli from the same explant shows a different embryogenic potentials [12–14]. In addition, cotyledonary embryo can be germinate, while several abnormal embryos such as globular embryos are unable to germinate [14, 15].

To study the molecular regulation mechanism of plant SE, the transcriptomes analysis were carried out to identify SE related genes by RNA-seq in plant species, including herbaceous plants such as Arabidopsis [16], Gossypium hirsutum [17], Maize [18], Strawberry [19], Rice[20], etc., and woody plants such as norway spruce [21], coconut plam [22], brazilian pine [23], camphor tree [24], papaya [25], Dimocarpus longan [26] and so on. These study demonstrated regulation mechanisms of SE at molecular level, and several potential somatic embryogenesis key genes were identified, such as late embryogenesis abundant (LEA) protein [25], somatic embryogenesis receptor-like kinase (SERK) [27, 28], Leafy Cotyledon [28, 29], AGAMOUS-like 15 [30, 31], BABYBOOM [28, 32], WUSCHEL [33, 34], and WUSCHEL homeobox 2 [28, 35].

In rubber tree, a total of 28 differentially expressed cDNAs were identified during induction in the embryogenic regenerating line using the differential display method and five cDNAs could be enable an early diagnosis of friable rubber tree callus embryogenic potential [12]. Three MADS-box genes were differentially expressed during rubber tree SE [36]. The transcript profiling of AP2/ERF genes and ethylene biosynthesis genes were analyzed in different regeneration potential callus lines [14]. These studies about rubber tree SE-related genes mainly focused on early embryogenesis. However, the molecular regulation mechanisms of the late stage of rubber tree SE still not well understood. To clarifying the mechanism of the regenerate competence of different embryos depended on the potential genes during late SE, we investigated the expression profiling using RNA-seq technology. This study will offer valuable information for the molecular regulation mechanisms of rubber tree late SE.

Induction of somatic embryogenesis

An efficient H. brasiliensis SE system was established (Fig. 1). The immature anthers were cultured in solid MS medium supplemented with 2, 4-D, KT and NAA for 50 days. At the end of the period, ECs were obtained. The ECs were placed in the MS medium containing IAA and GA3 for embryo induction. After 40 days, PEs were collected. The PEs were transferred to MS medium containing BA and AgNO₃ for growing. After 40 days, there were two different embryos (CEs, AEs) in the culture medium. We observed significant difference between CEs and AEs in phenotype. The CEs and AEs were placed on half-strength MS medium containing IAA and BA. The CEs turned stronger into MCE 20 days later, whereas the AEs turned brown and hardly grown up into WAE. After 30 days, the MCEs grew into complete seedlings, whereas the WAEs turned black and died. Based on the above phenotypic observation, six different samples during SE were selected for further study.

Transcriptome analysis of rubber tree SE

To provide the transcriptional analysis of H. brasiliensis SE, the cDNA libraries of EC, PE, CE, AE, MCE and WAE (three biological replicates for each sample) were constructed and sequenced by using the Illumina HiSeq™ 2500 platform. In total, 915,535,874 sequence reads were obtained from all 18 cDNA libraries. A total of 887,852,416 clean reads were retained by filtering the reads with adaptor sequences and ambiguous “N” base more than 1% and base quality less than Q15. A quality score above 30 (Q30) percentage was over 97.92% and the GC percentage was over 43% (Table 1). On average more than 85.92% of the clean reads were mapped to H. brasiliensis genome.

All unigenes were annotated by the blast search against the public databases of using BLASTx (E-value–5 ≤ 10). All 36937 unigenes were annotated in 4 databases involved in COG, GO, KOG and eggNOG (Table 2). Among 36937 unigenes, 19619, 20954 and 36362 unigenes were successfully annotated in GO, KOG, eggNOG, respectively (Fig. S1, S2, S3).

According to the COG functional classification, the 13421 unigenes were categorized into 25 COG categories. The four most highly represented COG categories were “general function prediction only” (20.57%), “transcription” (11.75%), “replication, recombination and repair” (11.53%) and “signal transduction mechanisms” (10.51%)(Fig. 2).

Global analysis of gene expression during rubber tree

To confirm the overlap existing, statistical analysis was performed on the expressed genes in the four different developmental stages of H. brasiliensis SE (Fig. 3a). A total of 25841 genes overlapped in the four stages. 155 genes overlapped between EC and PE. 290 genes overlapped between PE and CE. 193 genes overlapped between CE and MCE; 388, 297, 152 and 582 genes were preferentially expressed in EC, PE, CE and MCE respectively. Statistical analysis was performed on the expressed genes in the comparisons of PE, AE and CE (Fig. 3b), 662 genes were exclusive to PE vs. AE.1369 genes were exclusive to PE vs. CE. Moreover, 365 genes were found in AE vs. CE. To evaluate the differences of molecular response among all samples, the expression level of the unigenes was calculated by FPKM. The top 20 expressed genes from EC, PE, CE and MCE libraries were shown in Table 3. Some genes including glutathione S-transferase(GST), lipid-transfer protein༈LTP༉, peroxidase༈POD༉indole-3-acetic acid-amido synthetase GH3.1, ADP-ribosylation factor, catalase isozyme, and polyubiquitin were highly expressed in four satges.

In order to reveal the potential key factors and deep understand the regulatory network of SE, the unigenes of each libarary of H. brasiliensis SE were analyzed comparatively under the condition of − 1.0 ≥ Log₂ [FC] ≥ 1.0 and FDR < 0.01. A total of 9415 DEGs were obtained in EC vs. PE, PE had 5260 up-regulated and 4155 down-regulated gene. In PE vs. CE, CE had 1483 genes up-regulated and 2366 down-regulated. In CE vs. MCE, 6449 DEGs were obtained, of which 4016 DEGs were up-regulated, whereas 2433 DEGs were down-regulated. The 2820 DEGs were found in PE vs. AE with 1300 up-regulated and 1520 down-regulated DEGs. In AE vs. WAE, 5590 DEGs were obtained, of which 3318 DEGs were up-regulated, whereas 2272 DEGs were down-regulated. In AE vs. CE, 1536 DEGs were found with 556 up-regulated and 980 down-regulated DEGs. The 3307 DEGs were found between WAE vs. MCE with 1938 up-regulated and 1369 down-regulated DEGs (Fig. 4).

GO analysis of DEGs between cotyledonary embryo and abnormal embryo

To further demonstrate the unigenes functions, gene ontology (GO) assignments were carried out using the Blast2GO program. In AE vs. CE, 843 DEGs were classified to three major categories: biological processes (BP), cellular components (CC) and molecular function (MF). A total of 41 GO subcategories were enriched over three major functional categories. The main subcategories were shown in Fig. 5a. The six major subcategories of biological process were metabolic process, cellular process, single-organism process, biological regulation, localization and response to stimulus. The five major subcategories of cellular component were membrane, cell, cell part, organelle and membrane part. The four major subcategories of molecular function were binding, catalytic activity, transporter activity and nucleic acid binding transcription factor activity.

In WAE vs. MCE, 1927 DEGs were classified to three major categories: biological processes (BP), cellular components (CC) and molecular function (MF). The 41 GO subcategories were enriched over three major functional categories (Fig. 5b). The major subcategories of three categories were consistent with the result in AE vs. CE.

KEGG Pathways of DEGs between cotyledonary embryo and abnormal embryo

There were 376 DEGs in AE vs. CE, which were assigned to 46 KEGG pathways (Fig. 6a). The most representative pathways were phenylpropanoid biosynthesis (25 unigene, Fig. S4A), plant hormone signal transduction (21 unigenes, Fig. S4B), starch and sucrose metabolism (20 unigenes, Fig. S4C), phenylalanine metabolism (19 unigenes), carbon metabolism (15 unigenes), biosynthesis of amino acid (14 unigenes) and glutathione metabolism (14 unigenes).

In WAE vs. MCE, the 771 DEGs were assigned to 57 KEGG pathways (Fig. 6b). The 7 most represented pathways were phenylpropanoid biosynthesis (63 unigenes, Fig. S5A), starch and sucrose metabolism (49 unigenes, Fig. S5B), plant hormone signal transduction (46 unigenes, Fig. S5C), carbon metabolism (31 unigenes), photosynthesis (30 unigenes), phenylalanine metabolism (29 unigenes) and cyanoamino acid metabolism (29 unigenes). The results indicated that phenylpropanoid biosynthesis, phytohormones signaling pathway and sucrose and starch metabolism played importance roles during H. brasiliensis late SE.

Differential expression of hormone signal transduction related genes between cotyledonary embryo and abnormal embryo

Various phytohormones induced SE and regeneration in several plants have already been reported. To further understand hormone regulation, FPKMs of hormonal signal transduction related genes were analyzed (Fig. 7a and Table S1). Among all auxin signal transduction related genes, AUX-like5, IAA9-like, IAA28-like and GH3.1 were up-regulated in CE. SAUR71-like were higher expression in AE than CE. AUX22D-like, AUX28-like, AUX-like1, AUX-like2, SAUR32-like, IAA14-like and IAA27-like were highly expressed in MCE. ARF5-like was low expression in CE but highly expressed in MCE. These genes participated in auxin signaling pathway, which was important for cell enlargement and plant growth (Fig. 7b).

Abscisic acid (ABA) signal transduction related genes, PYL2-like was down-regulated in CE. PYL4-like was down-regulated in AE. Jasmonic acid (JA) signal transduction related genes, JAZ7 was higher expression in CE than AE. JAZ5 were up-regulated in AE. Ethylene (ET) signal transduction related genes, RAP2-3 were up-regulated in CE and AE. RAP2-12-like and WRI1-like were highly expressed in CE. ERF4-like were up-regulated in MCE. ERF018-like were only up-regulated in AE. In brief, these genes involved in the hormones signaling transduction pathways, including auxin, ABA, JA, ET, implying that these hormones were indispensable role in their complicated crosstalk process during H. brasiliensis late SE.

Differential expression of TFs and SE-related genes between cotyledonary embryo and abnormal embryo

In the embryogenic callus formation, transcription factors (TFs) possessed important functions. 219 TFs were identified in CE vs. AE of H. brasiliensis late SE. The following TFs families were overrepresented: WRKY, MYB, MADS-box, AP2/ERF, bHLH. The expression profiles of 19 TFs in CE, AE, MCE and WAE are shown in Fig. 8a and Table S2. WRKY40 and WRKY70 were up-regulated in CE and down-regulated in AE. WRKY23 were higher expression in AE than CE. MYB26-like and MYB98-like were up-regulated in AE. MYBS3-like and MYB1R1-like were up-regulated in MCE. AGL11 and AGL15 were up-regulated in AE. BBM2 was highly expressed in AE. AIL6 was higher expression in CE than AE. bHLH93-like was highly expressed in CE. The expression of bHLH94-like was obviously up-regulated in AE. The results implied these TFs may be play a key role in H. brasiliensis late SE.

Some SE-related genes, such as CAM [37], SERK [38, 39], LEA [40, 41], have been identified to play vital role during plant embryogenesis. CML13 and CML36 were up-regulated in CE but down-regulated in AE. CAM-5-like and CAM (LOC110641724) were up-regulated in AE but had not changed in CE. CAM-7 were up-regulated in CE but down-regulated in AE. SERK1 were up-regulated in CE. LEAD-34-like and SERK2-like showed higher expression in AE than CE. LEAD-29-like were up-regulated in MCE. The dynamic variation of the FPKM of these somatic embryogenesis-related genes suggested that they were critical for H. brasiliensis late SE.

Differential expression of histone modifications related genes between cotyledonary embryo and abnormal embryo

The plant growth regulators and abiotic stress can contribute to induce SE. In the meantime these factors may contribute to induce epigenetic modifications [42]. Histone modification is one of the most important epigenetic modifications and play a key role in the regulation of gene expression [43]. Therefore, the expression levels of histone modifiers were analyzed and shown in Fig. 8b and Table S3. The most genes related to histone methylation showed higher expression in CE than AE. The histone H3 lysine 9 methyltransferase genes (SUVH1-like, SUVH3-like, SUVH4-like and SUVH9), SUVR3-like, EZA1-like and ASHH3-like were expressed at a higher level in CE. In addition, histone demethylation related genes, LSD1-homolog 1-like were highly expressed in CE. LSD1-homolog 2 were up-regulated in MCE. The increased expression of genes in CE or MCE suggested that it is likely to have a function during late SE.

Histone acetyltransferases (HATs) may mediate hyperacetylation and activate transcription. 10 of the 11 genes related to histone acetylation showed significant differential expression in CE vs. AE. HAG6, HAC12-like, MCC1 and GCN5-like were up-regulated in CE. HAG11, HAG16, HAG18 and HATB-like were obvious high accumulation in AE. 7 of the 13 genes related to histone deacetylation showed obvious difference in expression in CE vs. AE. HDAC15-like and HDAC19 were highly expressed in CE. HDAC6-like, HDAC9 and SAP18-like were obvious high accumulation in AE. The histone phosphorylation related genes were only highly expressed in AE. These genes highly expressed in late SE can be used as candidate genes for in-depth study in vitro embryogenesis.

qPCR verification of selected DEGs

Twenty genes related to SE were selected to carry out expression level analysis using qRT-PCR across 6 different tissues of H. brasiliensis: EC, PE, CE, AE, MCE and WAE (Fig. 9). The results validated that the expression levels of 19 genes were highly consistent with transcriptome data.

Somatic embryogenesis is a promising and rapid vegetative propagation technique for plant regeneration. We have established an efficient rubber tree SE system using immature anthers. The transcriptome analysis of plant SE revealed a large number of potential key factors of embryogenesis [25, 26, 44–46]. In this study, we obtained the transcriptome analysis of rubber tree SE derived from EC, PE, CE, AE, MCE and WAE. The de novo assembly generated 36937 unigenes. We found the regenerate competence of cotyledonary embryo and abnormal embryo had obvious differences during late SE. Therefore, this study mainly focused on DEGs in CE vs. AE and MCE vs. WAE. In CE vs. AE, 376 DEGs were provided and assigned to 50 KEGG pathways. The 771 DEGs were also assigned to 50 KEGG pathways in MCE vs. WAE. The most representative pathways were phytohormones signaling pathway, biosynthesis of phenylpropanoid, and sucrose and starch metabolism in CE vs. AE and MCE vs. WAE. The significant role of phenylpropanoid biosynthesis in plant SE development has been studied. The phenylpropanoid biosynthesis related genes were significantly enriched in papaya embryogenic callus [25]. The similar result was reported about enrichment of the phenylpropanoid pathway in strawberry embryogenic callus [19]. In addition, external stimuli and plant hormones related genes played a key role in the SE process [47, 48]. Sucrose were added to culture medium as exogenous carbon sources in conifers SE [49, 50]. The germination of Norway spruce (Picea abies) somatic embryos was affected by carbohydrates [51]. These suggested genes involved in the three pathways which can play important role in H. brasiliensis late SE.

Auxin are critical regulators in different developmental stages of SEs [52–54]. The addition of exogenous auxin can affect the expression level of endogenous IAA [48, 55–57]. Dynamic change of endogenous IAA has been proved to induce plant SE and improved SE competency [58]. Auxin/Indole-3-Acetic Acids (Aux/IAAs), Gretchen Hagen3s (GH3s), small auxin upregulated RNAs (SAURs) and auxin response factor (ARF) have been identified as auxin-responsive genes in auxin signaling and homeostasis [59–61], can regulate downstream genes precisely and rapidly, and further regulated plant growth and developmental processes. Aux/IAA family play a key role in inhibiting the expression levels of genes transcriptional activated by ARFs [62, 63]. In low auxin levels, Aux/IAA proteins interacted with ARFs and inhibited activation of auxin-responsive genes. In high auxin levels, these proteins can interact with TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) receptors to be ubiquitinated and subsequently resolved by the 26S proteasome [64–66]. The liberated ARFs regulated the expression of auxin-responsive genes (Fig. 7b). There were 29 Aux/IAA family members in Arabidopsis, but not all genes were induced by auxin [67]. Many Aux/IAA genes have also been identified in other plants, such as, Eucalyptus grandis [68], Solanum Lycopersicon [69], Cucumis sativus [70], Populus trichocarpa [71], Zea mays [72] and Oryza sativa [73–74]. SAUR genes were consisted of a large multigene family, played crucial roles in regulating plant growth and development [75, 76]. GH3 family participated in a series of hormone-dependent processes in plant, including root growth, flowering [77, 78]. In our study, Auxin signal transduction related genes, IAA9-like, IAA28-like and GH3.1 were high accumulation in CE. AUX-like1, SAUR32-like, IAA14-like, IAA27-like and ARF5-like were up-regulated in MCE. JA and ET have also been reported to play a role in SE induction [79]. JAZ7, RAP2-12-like and WRI1-like were highly expressed in CE. The phytohormones signaling pathway related genes displayed intricate regulation during H. brasiliensis late SE. The regulatory mechanisms of these genes in H. brasiliensis late SE will be confirmed in the future study.

Transcription factors are key factors in plant embryogenesis and development. Many researches on SE development showed that complicated transcription regulation networks existed in cell maintaining embryogenic competency, embryogenic callus formation [52, 80]. The WRKY family has been reported to be involved in biotic or abiotic stresses responses, and were up-regulated in plant SE [81]. The WRKY genes showed higher expression and be related to embryogenic callus formation [82]. The up-regulated WRKY genes were high ratio in embryogenic callus formation of bread wheat [44]. In addition, MYB family was also involved in plant development and growth [83–86], hormone signal transduction [87, 88]. In our study, WRKY40, WRKY70, MYBS3-like and MYB1R1-like were highly expressed in CE, they can be used as marker genes for H. brasiliensis late SE.

AtEMK, a member of the AP2/ERF family, was ectopical expressed and promote the initiation of somatic embryos in Arabidopsis and H. brasiliensis [14, 89]. BABY BOOM (BBM) had been reported as a marker in Brassica napus SE [90]. The over-expression of BBM can enhance SE and regeneration ability in tobacco, sweet pepper, cacao [91–93]. The bHLH family are involved in developmental, growth and abiotic stress responses [94], axillary meristem formation [52]. They also participate in abscisic acid and brassinosteroid signaling in Arabidopsis and rice [95]. A member of bHLH protein BIM1 regulated Arabidopsis SE and be involved in auxin and BR signaling pathways [96]. In our study, AIL6 and bHLH93-like were highly expressed in CE, they might play a key role in H. brasiliensis late SE.

SERK has been proved as a key factor in plant SE. AtSERK1 was higher expression during Arabidopsis embryogenic formation [97]. SERK was abundant in embryogenic tissues in Dactylis glomerate [98]. However, SERKs were also tested in non-embryogenic tissues in maize, rice and wheat [38, 99, 100]. Ca²⁺ has been identified to play a mediating role during plant SE [37, 101]. LEA5, a late embryogenesis abundant proteins gene, was highly expressed in late embryogenesis [102]. In our study, SERK1, CML13, CML36 and CAM-7 were up-regulated in CE. LEAD-29-like were up-regulated in MCE. These genes can have various regulatory functions in H. brasiliensis late SE.

The histone modifications played important roles in gene expression, DNA replication and transcription, chromatin compaction [103, 104]. KRYTONITE (KYP), a histone H3 lysine 9 methyltransferase, also showed higher expression level in Arabidopsis somatic embryos [16]. In our study, the histone methylation related genes, SUVH1-like, SUVH3-like, SUVH4-like, SUVH9, SUVR3-like, EZA1-like and ASHH3-like were expressed at a higher level in CE. In addition, histone demethylation related genes, LSD1-homolog 1-like were highly expressed in CE. LSD1-homolog 2 were only up-regulated in MCE. These genes can play important role in H. brasiliensis late SE.

Some HATs including HAG1, HAF2, HAC1, HAC2, HAC4, HAC5 and HAC12 have been identified in Arabidopsis [16, 105, 106]. HAC2, HAG2 and HAG3 showed more accumulation in somatic embryos as compared to leaf tissues [16]. Similarly, in our study, histone acetylation related genes (HAG6, HAC12-like, MCC1 and GCN5-like) and histone deacetylation related genes (HDAC15-like, HDAC19) showed obvious high accumulation in CE. The potential function histone acetylation/ deacetylation related genes in H. brasiliensis late SE will be further studied.

In this study, the transcriptome data for rubber tree SE was generated. A comparative analysis of gene expression profiles during rubber tree late SE provided a series of DEGs that regulated late SE in rubber tree. We revealed the expression level of some genes related to phytohormones signaling pathway such as auxin, JA and ET signaling pathway, implying their possible roles in rubber tree late SE. The transcript factors such as WRKY, MYB, AP2 and bHLH, as well as CAM, SERK and LEA were related to rubber tree late SE, might play a key role in SE. Histone modification might have crucial roles during late SE. This study provides novel insights into the molecular regulation mechanisms during rubber tree late SE.

Plant material and induction of somatic embryogenesis

H. brasiliensis clones of reyan7-33-97 were planted in National Rubber Tree Varieties Resource Garden of the Chinese Academy of Tropical Agriculture Sciences, Danzhou, Hainan, China.

Immature male flowers were gathered from the rubber tree of reyan7-33-37. Immature male flowers were surface-sterilized with 75% (v/v) ethanol for 30 s, and followed to immerse in 0.2% (v/v) mercuric chloride solution for 10 min, and then washed four times with distilled water. The immature anthers were cultured in solid Murashige and Skoog (MS) medium containing 1 mg l^− 1 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg l^− 1 kinetin (KT) and 0.5 mg l^− 1 naphthaleneacetic acid (NAA). After an additional 5–6 weeks of growth, embryogenic calluses (EC) were obtained in the darkness and 26–28℃. These samples of primary embryo (PE), cotyledonary embryo (CE), abnormal embryo (AE), mature cotyledonary embryo (MCE) and withered abnormal embryo (WAE) were collected successively. All samples were rapidly frozen in liquid nitrogen, and stored at -80℃ until RNA extraction. Three biological replicates were prepared for each sample.

Construction of cDNA library and sequencing

Total RNA was extracted using RNAprep pure plant Kit (Polysaccharides and Poly phenolics-rich, QIAGEN). RNA degradation and contamination was monitored on 1% agarose gels. The quality of RNA was detected by using the NanoDrop 2000 spectrophotometer (IMPLEN, CA, USA). The mRNA was enriched from total RNA by magnetic beads containing Oligo (dT) and broken into small fragments. First strand cDNA was then synthesized by these cleaved mRNA fragments. The second-strand cDNA was synthesized using DNA polymerase I and RNaseH. The purified cDNAs were carried out end repair and an addition of an “A” base. Finally, PCR was performed to generate cDNA libraries. The library quality was examined using the Qsep100 Analyzer (BIOptic Inc, Taiwan, China). The cDNA libraries were deep sequenced on the Illumina novaseq6000 cDNA sequencing platform.

Transcriptome de novo assembly and annotation

High quality, clean reads were acquired by filtering the reads with adaptor sequences and ambiguous “N” base more than 1% and base quality less than Q15. All unigenes were obtained by comparing with the H. brasiliensis genome from NCBI. All unigenes were annotated by the blast search against a number of public databases of using BLASTx (E-value–5 ≤ 10). The public databases included the Clusters of Orthologous Groups of proteins database (COG) (http://www.ncbi.nlm.nih.gov/COG), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOG) (http:/ /eggnog5.embl.de/), the Gene Ontology database (GO) (http://www.geneontology. org/), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg), the Clusters of Protein homology database (KOG) (http://www.ncbi.nlm.nih.gov/KOG/ ).

Analysis of differentially expressed genes (DEGs)

The expression level of the unigenes was calculated by FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced). The genes with − 1.0 ≥ Log₂ [FC] ≥ 1.0 and the threshold of FDR < 0.01 were regarded as DEGs.

Expression profiles of genes in H. brasiliensis SE

FPKM was applied to analyze the gene expression level. The heat map was created using log₂ [FPKM] with the pheatmap package [107].

Quantitative PCR (qPCR)

Twenty genes were chosen for validation by qPCR. The samples of EC, PE, CE, AE, MCE and WAE were used for RNA extraction, and then reverse transcribed into cDNA as template. Each sample included three biological replicates. QPCR specific primers for the twenty genes were designed by using Primer Premier software 6.0 (Table S4). HbACT7 was amplified as a standard control. qPCR was performed on a Mx3005P Real-Time PCR system using a SYBR Premix EXTaq II™ Kit (TaKaRa, China). All reactions were performed at 95 °C for 30 s, 40 cycles at 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 25 s. The 2^−ΔΔCt method was used to calculate the relative expression levels of genes [108]. The statistical differences were analyzed by ANOVA (One-way analysis of variance) based on Fisher’s LSD test (P < 0.05 and P < 0.01) [109].

2,4-D

2,4-dichlorophenoxyacetic acid; ABA:Abscisic acid; AE:Abnormal embryo; ARF:Auxin response factor; BP:Biological process; CC:Cellular component; cDNA:Complementary DNA; CE:Cotyledonary embryo; COG:Clusters of orthologous groups of proteins database; DEGs:Differentially expressed genes; EC:Embryogenic calluses; eggNOG:Evolutionary genealogy of genes:non-supervised orthologous groups; ET:Ethylene; FPKM:Fragments per kilo base of transcript sequence per millions base pairs sequenced; GEO:Gene expression omnibus; GO:Gene ontology; JA:Jasmonic acid; KEGG:Kyoto encyclopedia of genes and genomes; KOG:Clusters of Protein homology database; KT:Kinetin; MF:Molecular function; LEA:Late embryogenesis abundant protein; NAA:Naphthaleneacetic acid; PE:Primary embryo; qPCR:Quantitative polymerase chain reaction; SE:SERK:Somatic embryogenesis receptor-like kinase; Somatic embryogenesis; SRJCs:self-rooted juvenile clones; TFs:transcription factors; MCE:Mature cotyledonary embryo; Non-supervised Orthologous Groups (eggNOG)WAE:Withered abnormal embryo.

Acknowledgements

We thank the GENE DENOVO Technology Corporation (Guangzhou, China) for RNA-seq sequencing.

Authors’ contributions

The study wasconceived and directed bySQP.YW and SQPconducted the analysis and wrote the manuscript.YW,YKZ, HLL,DG and HJZ performed experiments and carried out the data analysis. All authors read and approvedthe final manuscript.

Funding

This study was supported by the National Key R&D Program of China (Grant No. 2018YFD1000502) and the National Natural Science Foundation of China (Grant No. 31670611).

Availability of data and materials

The generated RNA-seq data have been depositedin NCBI-SRA database under the accession of PRJNA646309.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table 1 Pre-processing statistics and quality control statistics
Sample	Raw Reads	Clean Reads	Raw Bases(Gb)	Clean Bases(Gb)	Effective Rate(%)	Q30 content(%)
EC-1	5.2E+07	50059934	7.86	7.56	96.21	94.81
EC-2	5.1E+07	49524648	7.73	7.48	96.73	94.81
EC-3	5.1E+07	49118950	7.68	7.42	96.61	94.78
PE-1	5E+07	48319634	7.53	7.25	96.29	97.01
PE-2	5.1E+07	49061282	7.64	7.36	96.33	96.86
PE-3	5.1E+07	48891852	7.6	7.33	96.46	96.9
CE-1	5.1E+07	49805096	7.73	7.52	97.32	94.74
CE-2	5.2E+07	50906314	7.88	7.69	97.56	94.91
CE-3	5.1E+07	50054842	7.76	7.56	97.4	94.84
MCE-1	5.1E+07	49771578	7.7	7.47	96.96	95.89
MCE-2	5E+07	48654566	7.54	7.3	96.85	94.92
MCE-3	5E+07	48974062	7.52	7.35	97.72	95.62
AE-1	5E+07	48881230	7.56	7.33	97.05	96.81
AE-2	5.1E+07	48970492	7.6	7.35	96.7	96.75
AE-3	5.1E+07	48844568	7.59	7.33	96.52	96.88
WAE-1	5.1E+07	49843978	7.71	7.53	97.67	94.71
WAE-2	5E+07	49076246	7.6	7.41	97.49	94.65
WAE-3	5E+07	49093144	7.59	7.41	97.71	94.72

Table 2 The number and distribution of unigenes annotated in the databases
Database	Annotated Number	300<=length<1000	length>=1000
COG	13421	4142	9153
GO	19619	5980	13639
KOG	20954	7547	13097
eggNOG	36362	14578	21038
All	36937	14983	21176

Table 3 The top 20 expressed genes in EC, PE, CE and MCE library

No	Gene-ID	Database-ID	FPKM-EC	Description
1	gene10318	XM_021818345.1	3266.37	metallothionein-like protein type 2
2	gene23077	XM_021779607.1	2803.41	probable indole-3-acetic acid-amido synthetase GH3.1
3	gene24550	XM_021781891.1	2587.98	peptidyl-prolyl cis-trans isomerase-like
4	gene37167	XM_021801099.1	5591.156667	pathogenesis-related protein PR-4-like
5	gene41379	XM_021807510.1	3354.676667	metallothionein-like protein type 3
6	gene41538	XM_021807736.1	1336.78	peroxidase 12-like
7	gene42156	XM_021808475.1	3150.052519	L-ascorbate peroxidase, cytosolic-like
8	gene548	XM_021811448.1	1719.636667	thioredoxin H-type-like
9	gene11066	XM_021819455.1	1216.968149	catalase isozyme 2-like
10	gene1185	XM_021821602.1	6138.533333	metallothionein-like protein type 2
11	gene15002	XM_021825368.1	2578.13	glucan endo-1,3-beta-glucosidase, basic isoform-like
12	gene18326	XM_021830411.1	2459.693333	endochitinase EP3-like
13	gene19193	XM_021831939.1	1835.053335	glutathione S-transferase F9-like
14	gene33311	XM_021795239.1	1328.469977	pathogenesis-related protein PR-4-like
15	gene3644	XM_021801975.1	1588.716667	thaumatin-like protein 1b
16	gene41464	XM_021807622.1	2882.38	endochitinase EP3-like
17	gene5134	XM_021810359.1	2157.947846	catalase isozyme 2
18	gene12558	XM_021821637.1	1745.217667	cysteine synthase
19	gene21974	XM_021836019.1	1238.357898	40S ribosomal protein S25-3-like
20	gene24408	XM_021781690.1	1518.806667	polyubiquitin
No	Gene-ID	Database-ID	FPKM-PE	Description
1	gene17338	XM_021828886.1	448.8675164	ADP-ribosylation factor
2	gene24550	XM_021781891.1	1090.893333	peptidyl-prolyl cis-trans isomerase-like
3	gene25944	XM_021784022.1	517.586	polyubiquitin
4	gene37168	XM_021801110.1	1051.049333	pathogenesis-related protein PR-4-like
5	gene37235	XM_021801218.1	424.1643333	probable glutathione S-transferase
6	gene5278	XM_021810573.1	700.6816667	probable aquaporin TIP3-2
7	gene548	XM_021811448.1	651.8526667	thioredoxin H-type-like
8	gene1185	XM_021821602.1	691.8516667	metallothionein-like protein type 2
9	gene17500	XM_021829184.1	651.8516667	uncharacterized
10	gene19193	XM_021831939.1	444.690335	glutathione S-transferase F9-like
11	gene19425	XM_021832135.1	4129.713333	non-specific lipid-transfer protein 1-like
12	gene22222	XM_021836400.1	475.7673333	histone H2B
13	gene23940	XM_021780963.1	563.119	osmotin-like protein
14	gene37576	XM_021801775.1	574.5693333	thaumatin-like protein
15	gene12558	XM_021821637.1	419.428	cysteine synthase
16	gene35575	XM_021798790.1	464.6649333	copper transport protein ATX1-like
17	gene30702	XM_021791318.1	1738.72	peroxidase 42-like
18	gene23545	XM_021780391.1	2407.276667	peroxidase 42-like
19	gene33942	XM_021796208.1	577.7063333	peptidyl-prolyl cis-trans isomerase 1
20	gene24408	XM_021781690.1	472.838	polyubiquitin
No	Gene-ID	Database-ID	FPKM-CE	Description
1	gene17338	XM_021828886.1	1145.143911	ADP-ribosylation factor
2	gene18178	XM_021830179.1	943.5483996	protein translation factor SUI1 homolog 2-like
3	gene25944	XM_021784022.1	1762.396667	polyubiquitin
4	gene37168	XM_021801110.1	9026.456667	pathogenesis-related protein PR-4-like
5	gene37235	XM_021801218.1	2538.033333	probable glutathione S-transferase
6	gene5278	XM_021810573.1	1959.873667	probable aquaporin TIP3-2
7	gene5809	XM_021811329.1	1199.044333	metallothionein-like protein type 2
8	gene7973	XM_021814772.1	1108.26	glutaredoxin
9	gene9140	XM_021816591.1	4259.97	metallothionein-like protein type 2
10	gene17500	XM_021829184.1	995.952	uncharacterized
11	gene19425	XM_021832135.1	3340.51	non-specific lipid-transfer protein 1-like
12	gene20309	XM_021833577.1	1361.93341	ubiquitin-conjugating enzyme E2 28
13	gene12558	XM_021821637.1	1265.565333	cysteine synthase
14	gene25797	XM_021783808.1	1133.149667	L-ascorbate peroxidase, cytosolic
15	gene30702	XM_021791318.1	2923.166667	peroxidase 42-like
16	gene23545	XM_021780391.1	3234.236667	peroxidase 42-like
17	gene24345	XM_021781508.1	1190.793333	translationally-controlled tumor protein homolog
18	gene36607	XM_021800241.1	1155.013333	aquaporin TIP1-1-like
19	gene41316	XM_021807427.1	1017.603343	aquaporin PIP1-3-like
20	gene31451	XM_021792523.1	1867.496667	probable aquaporin PIP1-2
No	Gene-ID	Database-ID	FPKM-MCE	Description
1	gene17338	XM_021828886.1	1126.976	ADP-ribosylation factor
2	gene18178	XM_021830179.1	1004.106	protein translation factor SUI1 homolog 2-like
3	gene25944	XM_021784022.1	2101.800	polyubiquitin
4	gene33318	XM_021795235.1	1202.313	pro-hevein
5	gene37168	XM_021801110.1	18664.897	pathogenesis-related protein PR-4-like
6	gene39161	XM_021804156.1	918.860	2-methylbutanal oxime monooxygenase
7	gene41379	XM_021807510.1	864.198	metallothionein-like protein type 3
8	gene41597	XM_021807803.1	791.620	elicitor-responsive protein 3-like
9	gene42156	XM_021808475.1	2095.956	L-ascorbate peroxidase, cytosolic-like
10	gene548	XM_021811448.1	831.757	thioredoxin H-type-like
11	gene9140	XM_021816591.1	5217.597	metallothionein-like protein type 2
12	gene11066	XM_021819455.1	1380.437	catalase isozyme 2-like
13	gene1185	XM_021821602.1	1598.003	metallothionein-like protein type 2
14	gene19425	XM_021832135.1	2686.840	non-specific lipid-transfer protein 1-like
15	gene20309	XM_021833577.1	1215.717	ubiquitin-conjugating enzyme E2 28
16	gene5134	XM_021810359.1	1842.001	catalase isozyme 2
17	gene19423	XM_021832115.1	1306.773	non-specific lipid-transfer protein 1-like
18	gene23545	XM_021780391.1	1473.353	peroxidase 42-like
19	gene24345	XM_021781508.1	1151.487	translationally-controlled tumor protein homolog
20	gene31451	XM_021792523.1	821.413	probable aquaporin PIP1-2

Fig.S5.pdf
KEGG annotation pathway of phenylpropanoid biosynthesis(A), plant hormone signal transduction (B), starch and sucrose metabolism (C) of DEGs in MCE vs. WAE. The enzymes labeled in the red box were associated with up-regulated genes, and the enzymes labeled in the green box were associated with down-regulated genes. The enzymes labeled in the blue box were related to both up-regulated and down-regulated genes. The number in the box represented the EC number of the enzyme.
Fig.S4.pdf
KEGG annotation pathway of phenylpropanoid biosynthesis(A), plant hormone signal transduction (B), starch and sucrose metabolism (C) of DEGs in CE vs. AE. The enzymes labeled in the red box were associated with up-regulated genes, and the enzymes labeled in the green box were associated with down-regulated genes. The enzymes labeled in the blue box were related to both up-regulated and down-regulated genes. The number in the box represented the EC number of the enzyme.
FigureS3.tif
egg NO G function of classification of consensus
FigureS2.tif
KOG function of classification of consensus
FigureS1.tif
GO function of classification of consensus
TableS4.docx
qRT-PCR Primer
TableS3.xlsx
FPKM and annotation of histone modifications related genes
TableS2.xlsx
FPKM and annotation of TFs and SE-related genes
TableS1.xlsx
FPKM and annotation of hormonal signal transduction related genes

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published 12 Mar, 2021

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Editorial decision: Major revision
10 Nov, 2020
Review #1 received at journal
07 Oct, 2020
Review #2 received at journal
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Reviewer #2 agreed at journal
16 Sep, 2020
Reviewer #1 agreed at journal
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Reviewers invited by journal
22 Aug, 2020
Editor assigned by journal
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Transcriptomes Analysis Reveals Novel Insight Into the Molecular Mechanisms of Somatic Embryogenesis in Hevea Brasiliensis

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Induction of somatic embryogenesis

Transcriptome analysis of rubber tree SE

Global analysis of gene expression during rubber tree

GO analysis of DEGs between cotyledonary embryo and abnormal embryo

KEGG Pathways of DEGs between cotyledonary embryo and abnormal embryo

Differential expression of hormone signal transduction related genes between cotyledonary embryo and abnormal embryo

Differential expression of TFs and SE-related genes between cotyledonary embryo and abnormal embryo

Differential expression of histone modifications related genes between cotyledonary embryo and abnormal embryo

qPCR verification of selected DEGs

Discussion

Conclusions

Materials And Methods

Plant material and induction of somatic embryogenesis

Construction of cDNA library and sequencing

Transcriptome de novo assembly and annotation

Analysis of differentially expressed genes (DEGs)

Quantitative PCR (qPCR)

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1