Eucalyptus cloeziana is a large tree of Eucalyptus genus in the family of Myrtaceae, also known as Kunshilan’an. The tree height can reach 55 m, the natural distribution area is located in the central and northern areas of Queensland, Australia (44°44' ~ 152°52' E, 15°45' ~ 26°41' S), and the altitude ranges 25~950 m (Xiang et al., 2008; Wang et al., 2016). Relevant research showed that Eucalyptus cloeziana, with straight shape, high wood hardness, high strength, insect resistance, beautiful wood patterns and relatively uniform texture structure, is an excellent material for making high-grade furniture and solid wood flooring. The wood utilization rate of Eucalyptus cloeziana is more than 70%, and its economic value is much higher than that of short-rotation “Sufeng’an”. It is one of the medium and large diameter timber species with great cultivation value. It has been included in the list of national precious timber species and has very broad development prospects (Huang et al., 2018; Li et al., 2012a). Since 1972, Eucalyptus cloeziana has been introduced into China. In the 1980s, systematic studies such as introduction test, provenance selection, family test, correlation degree of wood properties and growth properties have been carried out in Guangxi, Guangdong, Hainan, Fujian, Hunan and Sichuan (Huang et al., 2018). In general, Eucalyptus cloeziana has strong growth potential and material characteristics with significant volume growth, for example, the average basic density of 18-year-old Eucalyptus cloeziana is 0.706 g/cm, and the variation range between provenances is 0.617~0.753 g/cm (Li et al., 2012b; Xiang et al., 2008); The heritability (H²) of different provenances of 25-year-old Eucalyptus cloeziana is 0.634~0.895, and the heritability (h²) of single plant is 0.136~0.342 (Wang et al., 2016).

Transcriptome is the sum of all mRNA transcribed by a specific cell or tissue at a certain developmental stage or functional state. With the development of a new generation of high-throughput sequencing technology, the generation of a large number of transcriptome sequencing data can provide rich sequence resources for animal and plant gene expression and function analysis, molecular marker development and related metabolic pathway research (Vaattovaara et al., 2019). The development of molecular biology of Eucalyptus cloeziana is slow, and the lack of genomic and transcriptome information limits the research on genetic improvement of Eucalyptus cloeziana. At present, only Zhu et al. (2018) annotated and analyzed the gene function of the transcriptome sequence of Eucalyptus cloeziana root. The existing SSR markers of Eucalyptus cloeziana all come from the reported near source Eucalyptus. For example, Lü et al. (2020) used 12 SSR loci to study the genetic variation level of free pollinated progeny forest of different provenances of Eucalyptus cloeziana; Deng et al. (2019) analyzed the genetic variation of Eucalyptus cloeziana population by using the SSR loci developed in near source Eucalyptus, but there is no report on the development of SSR markers for Eucalyptus cloeziana at present. The existing phylogenetic analysis of Eucalyptus shows that (Steane et al., 2011), Eucalyptus cloeziana is far from other eucalyptus trees in the same genus and belongs to an independent subgenus. Therefore, the research on the genome and transcriptome of this species will help to further explore its species characteristics.

In this study, the 2-year-old Eucalyptus cloeziana terminal buds were used as the material, the transcriptome sequence data were obtained by Illumina HiSeq X Ten high-throughput sequencing technology, and the gene function annotation and SSR locus retrieval and analysis were carried out for all Unigenes, so as to provide a theoretical basis for the functional gene mining, genetic map construction and SSR marker development of important traits such as growth and wood properties of Eucalyptus cloeziana.

1 Results and Analysis

1.1 Transcriptome sequencing analysis of Eucalyptus cloeziana

56 157 538 Raw reads were obtained by sequencing the transcriptome of Eucalyptus cloeziana terminal buds, and the number of bases was 8 423 630 700 bp (8.4 Gb). After removing the low-quality original sequence, 55 203 802 clean reads were obtained, accounting for 98.3% of the original sequence. The amount of clean bases was 7 473 648 753 bp (7.5 Gb), the percentage of bases with quality of Q30 (the correct recognition rate of bases up to 99.9%) was more than 93.99%, and the content of GC was 49.97%. The above data showed that the transcriptional data of Eucalyptus cloeziana obtained by sequencing on Illumina hiseq platform was of good quality. Using Trinity software for assembly and splicing, a total of 26 587 Unigenes were obtained, with a total length of 34 023 022 bp, N50 of 1 851 bp, the longest and shortest sequences of 15 778 bp and 301 bp respectively, and the average length of 1 279.69 bp. There were 13 527 Unigenes between 301 and 1 000 bp, accounting for half of the total (50.87%); There were 7 836 Unigenes between 1001 and 2000 bp, accounting for 29.47% of the total; The length >2000 bp accounted for 19.65% (5 224 Unigenes) (Figure 1). In general, the transcriptome sequencing of Eucalyptus cloeziana terminal buds was of good quality, the sequence assembly was long, and the length range was relatively uniform.

1.2 Basic function annotations of transcriptome in Eucalyptus cloeziana terminal buds

All UniGenes (26 587) of the transcriptome in Eucalyptus cloeziana terminal buds obtained by sequencing were compared with seven related databases (NR, COG/KOG, GO, Swiss-Prot, eggNOG, KEGG and Pfam) by BlastX for annotation, gene function and metabolic pathway analysis (Figure 2). Finally, 22 099 Unigenes (83.12%) with annotation information were obtained, of which 4 777 (17.96%) were successfully annotated in all databases. The number of Unigenes annotated in NR database was the largest, with a total of 21 969 (82.63%); The number of Unigenes annotated in KEGG database was the least, with only 7 117 (26.77%). The number of Unigenes annotated in eggNOG, Swiss-Prot, Pfam, GO and KOG databases was 19 671 (73.99%), 15 469 (58.86%), 15 208 (57.20%), 14 105 (53.05%) and 11 507 (43.38%) respectively. The Unigenes of transcriptome in Eucalyptus cloeziana terminal buds obtained higher annotation rate.

1.2.1 NR function class

21 969 Unigenes of Eucalyptus cloeziana transcriptome were aligned with similar sequences in NR database. From the species distribution of homologous sequence (Figure 3), the matching similarity between Eucalyptus cloeziana and Eucalyptus grandis was high, and the number of Unigenes annotated to Eucalyptus grandis was as high as 20 767 (94.55% of the total Unigenes annotated in NR); A total of 1% of UniGenes was annotated to Punica granatum (86), Vitis vinifera (26), Arabidopsis thaliana (24), Theobroma cacao (22 articles), Quercus suber (22s) and Cephalotus follicularis (21); The remaining 2.73% (599) annotated to other species. The results reflected that Eucalyptus grandis had rich transcriptome data in NR database. Eucalyptus grandis and Eucalyptus cloeziana were of the same genus (Eucalyptus), so the number of Unigenes annotated to Eucalyptus grandis was the largest.

1.2.2 KOG function class

A total of 11 507 Unigenes were annotated into 25 KOG function classes, with a total number of 12 920. There were differences in the Unigene number in different function classes (Figure 4). The number of Unigenes annotated by general function prediction only (R) was the largest, including 2 399 Unigenes, accounting for 18.56% of the total number of annotations; Followed by post-translational modification, protein turnover, chaperones (O) and signal transduction mechanisms (T), the number of annotations were 1 382 (10.69%) and 1 239 (9.59%) respectively; Transcription (K), carbohydrate transport and metabolism (G), translation, ribosomal structure and biogenesis (J) and intracellular trafficking, secretion, and vesicular transport (U) were also more, with 717 (5.55%), 690 (5.34%), 595 (4.61%) and 585 (4.53%) genes respectively. Among all functional classes, nuclear structure (Y) (41), extracellular structures (W) (38) and cell motility (N) (6) was the least. The transcriptome of Eucalyptus cloeziana terminal buds was consistent with the annotation results obtained in most plants, and the most genes were enriched with general function and protein post-translational modification function.

1.2.3 GO function class

The GO functional annotation classification of Eucalyptus cloeziana terminal buds was carried out (Figure 5). A total of 14 105 Unigenes were annotated into the GO database, accounting for 53.05% of the total Unigenes, which were respectively matched to 50 GO function categories of biological pathways (processes), cellular components and molecular functions. Further analysis found that the total number of genes performing biological processes in go database was 49 872, which was divided into 22 terms. Among them, the number involved in cellular process (9400) and metabolic process (7565) was the largest, followed by response to stimulus (4623) and biological regulation (4457), and the expression of biological adhesion and cell killing was the lowest, only 11 and 10; The total number of gene annotations of cell components was 51 804, which was divided into 13 terms. Among them, the expression of cell (11 853) and cell part (11 835) was the highest, followed by organelle (9 220), and the proportion of nucleoid (45) was the lowest; The total number of annotated genes performing molecular functions is 17188, which is divided into 15 functional regions. The most representative genes are those related to binding (8 238) and catalytic activity (6 721), followed by transporter activity (1 085). The proportion of receptor regulator activity was the lowest, only 9 sequences. The number of genes involved in cellular and metabolic processes was the largest, which was consistent with expectations, reflecting the rapid growth of young Eucalyptus cloeziana, vigorous cell division and active metabolism in the meristem region at the top of the stem tip.

1.2.4 KEGG metabolic pathway

After KEGG functional annotation of Eucalyptus cloeziana transcriptome UniGenes, a total of 7 117 Unigenes were found to be involved in 127 metabolic pathways (Figure 6). According to their functions, these successfully annotated Unigenes were divided into five categories: metabolism, genetic information processing, cellular processes, environment information processing and organismal systems, and the number was 2 775, 1 578, 315, 278 and 195 respectively. The five metabolic pathways were further divided into 18 subclasses, and the metabolic pathways related to metabolism was the most abundant, with 10 in total. Among them, the genes involved in carbohydrate metabolism (660, 16.03%) accounted for the largest proportion, followed by amino acid metabolism (409, 9.93%) and energy metabolism (353, 8.57%); There are 4 metabolic pathways related to genetic information processing, among which the genes involved in translation (642, 15.59%) accounted for the largest proportion, followed by folding, sorting and degradation (526, 12.77%); The other pathways mainly involved transport and catabolism (315, 7.65%), signal transduction (253, 6.14%) and environmental adaptation (195, 4.74%). The proportion of genes involved in metabolic pathways related to carbohydrate metabolism in Eucalyptus cloeziana terminal buds transcriptome was the largest, which was consistent with the gene annotation results of more metabolic processes obtained by GO function, which can provide bioinformatics basis for functional gene mining and metabolomics of important traits such as fast-growing and wood properties of Eucalyptus cloeziana.

1.2.5 Gene transcription factors (TFs)

Transcription factors (TFs) are proteins that bind to specific DNA sequences, which play a key role in the regulation of gene expression by controlling the transcription of genetic information from DNA to RNA. Among 22 099 Unigenes with annotation information, 1 021 were annotated to the transcription factor database, distributed in 65 families (Figure 7). In the family, bHLH (68), MYB (65), MYB-related (62), NAC (59) and C2H2 (53) accounted for the largest proportion of 6.66%, 6.37%, 6.07%, 5.78% and 5.19% respectively (Figure 7). The transcription factor family such as AP2/ERF-RAV, HRT, S1Fa-like, STAT and ULT had only one Unigene. TFs plays an important role in cell activities, especially bHLH (Yu et al., 2019), MYB (Chen et al., 2019) and other transcription factors play an important role in cell signal transduction and plant stress resistance.

1.2.6 Plant resistance gene (PRG)

Plant resistance gene (PRG) (http://prgdb.crg.eu /wiki/Main_Page) database contains more than 112 disease resistance genes and 104 335 candidate disease resistance genes. This time, a total of 3 274 UniGenes annotated to the PRG database, distributed in 13 classes. The results of PRG comparison showed that the largest number of matched genes were RLP (785) and TNL (712), accounting for 23.98% and 21.74% of the total annotations, respectively; Followed by CNL (539) and NL (527), accounting for 16.46% and 16.10% of the total annotations, respectively (Figure 8). In the normal growth environment and state, RLP and TNL resistance gene families were specifically amplified in Eucalyptus cloeziana terminal buds, which may indicate that Eucalyptus cloeziana had potential strong adaptability and stress resistance, which provided a reference for the subsequent research on the stress resistance of Eucalyptus cloeziana to environmental and biological stresses, and also provided enlightenment for the resistance breeding of Eucalyptus cloeziana in the future.

1.3 Distribution characteristics of SSR loci in Eucalyptus cloeziana terminal buds

A total of 12 366 SSR loci were found from 26 587 Unigenes, which were distributed in 8 218 UniGene sequences, and the frequency was 46.51%, with an average of one SSR locus per 2.75 kb. 116 repeat motifs were found in the transcriptome of Eucalyptus cloeziana terminal buds, of which dinucleotide and trinucleotide were the main repeat motifs, accounting for 46.6% and 34.20% of the total SSRs respectively; The dominant repeat motifs were AG/CT and CCG/CGG, accounting for 44.94% and 12.53% respectively; SSR repeat number was mainly low repeat number (5~10 times) (71.28%); The average length of SSR motifs was 19.7 bp, and 63.75% of the motif length was concentrated between 12 and 20 bp. On the whole, Eucalyptus cloeziana transcriptome had high frequency of SSR, rich types of repeat motifs, and high repeat number (10 or more) accounted for a large proportion (38.8%), which had great development potential (Table 1).

2 Discussion

As a valuable timber tree species, Eucalyptus cloeziana has broad development prospects. Abundant terminal bud transcriptome information obtained by high-throughput sequencing can provide rich resources for molecular assisted breeding of Eucalyptus cloeziana. In this study, through high-throughput sequencing and analysis of cDNA library of Eucalyptus cloeziana terminal buds, a total of 56 157 538 Raw reads were obtained, and a total of 8.4 Gb sequencing data were obtained. The Q30 was more than 93.99%, the GC content was 49.97%, and the base number distribution was stable. After splicing and assembly, a total of 26 587 high-quality Unigenes were obtained, with a total length of 34 023 kb, an average length of 1 279.69 bp, the length range from 301 to 1000 bp (13 527, accounting for 50.87% of the total), and N50 of 1851 bp. Based on the same sequencing technology, 53 433 Unigenes were obtained from the transcriptome sequences of Eucalyptus cloeziana roots. The length of N50 was 1 587 bp, the average length was 890 bp, the content of G+C was 46.86%, and 50.73% of Unigenes were concentrated between 200 and 500 bp (Zhu et al., 2018); The transcriptome of the near source genus Syzygium samarangense (Wei et al., 2018) was assembled to obtain 87 538 Unigenes, with an average length of 858.56 bp, and 75.84% of the Unigenes distributed between 300 and 1 000 bp; Based on the same platform, the average length of transcriptome sequences of Pseudolarix amabilis leaves was 699 bp and the length of N50 was 1 300 bp (Zhang, 2019); The average length of transcriptome of Corylus avellana axillary buds was 1 189 bp, and the length of N50 was 1 916 bp (Kavas et al., 2019); The average length of transcriptome Unigene of Camellia sinensis mature seed was 854 bp and the length of N50 was 1 480 bp (Jin et al., 2018). In comparison, the sequence of Eucalyptus cloeziana terminal buds was longer, the length distribution was more uniform, and the integrity of sequence assembly was better.

In this experiment, all Unigenes (26 587) of Eucalyptus cloeziana terminal buds were compared with seven public databases, and 83.12% (22 099) sequences were successfully annotated. It was much higher than 64.94% of the transcriptome of Eucalyptus cloeziana roots (Zhu et al., 2018), which may be related to the short sequence fragment of the latter and the lack of annotation information of the root. Through the analysis of NR database, the matching similarity between Eucalyptus cloeziana and Eucalyptus grandis was very high (94.55%), which was consistent with the NR comparison results of roots, which may be related to the integrity of biological information data of Eucalyptus grandis; In the GO database, 14 105 Unigenes were annotated and matched to 50 functional gene regions in three classes: biological function, cell component and molecular function. The regions executing biological processes accounted for the largest proportion, with a total of 49 872 annotation information, of which the number involved in cellular process and metabolic process was the largest, indicating that cell division and differentiation and metabolic activities were vigorous in the process of growth and differentiation of Eucalyptus cloeziana terminal buds. A large number of studies showed that cellular process and metabolic process were the most significant subclasses of GO functional biological process, especially in buds (Bian et al., 2019; Chang et al., 2019; Kavas et al., 2019); 7 117 Unigenes were annotated to KEGG function and were involved in 127 metabolic pathways, of which the genes involved in carbohydrate metabolism (660, 16.03%) accounted for the largest proportion, indicating that the apical meristem region of Eucalyptus cloeziana carried out frequent mitosis and vigorous physiological and metabolic activities.

Transcription factors (TFs) are proteins that can bind to specific DNA sequences and play a key role in the regulation of gene expression. In this study, compared with TF database, a total of 1 021 Unigenes were annotated into the transcription factor database and distributed in 65 families, of which bHLH (68) accounted for the largest proportion, followed by MYB (65), MYB-related(62), NAC(59) and C2H2(53) families. bHLH is one of the largest transcription factor families in plants. Yu et al. (2019) reviewed in detail that bHLH transcription factors play an important role in biological processes such as plant signal transduction, growth and stress resistance. MYB protein is the largest and most diverse transcription factor family in plants, which is mainly involved in cell cycle regulation, tissue and organ morphogenesis, secondary metabolite synthesis and stress resistant biological processes (Chen et al., 2019); Araújo (2018) revealed that the expression of MYB and NAC family members in the stems of Eucalyptus grandis and Eucalyptus globulus was significantly higher than that of the control under low temperature stress. This study also found that a total of 3 274 Unigenes were annotated into 13 resistance gene categories in PRG database, of which RLP was the largest, accounting for 23.98% of the total annotation. Activation of RLP protein (RuBisCO-like protein) usually leads to rapid accumulation of active oxygen species (AOS), changes in cell ion flux, activation of protein kinase cascade, changes in gene expression, and possible targeted protein degradation (Tabita et al., 2008). A large amount of information obtained through transcriptome analysis will provide a strong basis for the study on growth and stress resistance of Eucalyptus cloeziana.

SSR molecular marker is an ideal marker to study the polymorphism of plant genetic resources, molecular marker assisted breeding and genetic mapping. In this study, 12 366 SSR loci were obtained from 26 587 Unigenes in the transcriptome of Eucalyptus cloeziana terminal buds, with a frequency of 46.51% and a distribution density of 1/2.75 kb. Dinucleotide and trinucleotide were the main repeat motifs, accounting for 46.6% and 34.20% of the total SSR respectively. The distribution density of SSR loci was consistent with the analysis results of 71 115 EST sequences based on database Eucalyptus (E. grandis, E. globosus, E. saligna 和 E. urophylla) reported by Ceresini et al. (2005): 20 530 SSR loci with an average distribution distance of 1/2.7 kb; The distribution density of SSR loci was higher than that of other Eucalyptus transcriptome (1/3.7 kb) reported by He et al. (2015), and the frequency of SSR in E. globulus transcriptome was 23.15% reported by Acuña et al. (2012); The distribution density of SSR loci was lower than that of Camellia sinensis (1/2.61 kb) (Jin et al., 2006) and Hevea brasiliensis (1/2.25 kb, the frequency of SSR was 63.71%) (Feng et al., 2009). In general, SSR loci in transcriptome of Eucalyptus cloeziana terminal buds have high frequency, high distribution density and rich types of repeat motifs, which will have great application potential in genetic diversity research, molecular marker assisted breeding and genetic mapping of Eucalyptus cloeziana.

3 Materials and Methods

3.1 Test materials

The test materials were the stem terminal buds of 2-year-old Eucalyptus cloeziana, which were collected from the Eucalyptus cloeziana Test Base of Dongmen Forest Farm, Fusui County, Chongzuo City, Guangxi Zhuang Autonomous Region (22°17'22.30"N, 107°14'108.00"E). After the sample was collected, it was quickly put into liquid nitrogen quick freezing for preservation. In October 2019, total RNA was extracted and cDNA library was constructed by the OEbiotech, and the original reads were obtained through Illumina HiSeq X Ten high-throughput sequencing platform.

3.2 Assembly and splicing of transcriptome sequence

The raw data obtained in the sequencing process were tested, filtered and quality controlled to obtain high-quality clean reads, and then De-novo splicing and assembly was carried out based on Trinity software (Grabherr et al., 2011), that is, under the condition of not relying on the reference genome, the reads with overlap were connected into a longer sequence and spliced into Contigs after continuous extension, according to the sequence similarity and length, the longest one was selected as Unigene, and then bioinformatics statistical analysis was carried out.

3.3 Function annotation of transcriptome

By using diamond (Buchfink et al., 2015)software, all Unigene sequences of Eucalyptus cloeziana were annotated and compared with NR (nonredundant protein database, https://www.ncbi.nlm.nih.gov/), KOG (karyotic orthologous groups, http://www.ncbi.nlm.nih.gov/COG), GO (gene ontology, http://www.geneontology.org), Swiss-Prot (Swiss Prot protein database, http://web.expasy.org/docs/swiss-prot_guideline.html), eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups, http://eggnog.embl.de/version_3.0/), KEGG (Kyoto encyclopedia, http://www.genome.jp/kegg/) biological information database; The predicted amino acid sequence of Unigene was aligned to Pfam database (http://pfam.xfam.org/) by using HMMER (E-value ≤ 1e-10) software (Mistry et al., 2013) to obtain the function annotation information of Unigene; By Using iTAK (Zheng et al., 2016), Unigene was aligned to its own database to obtain the annotation information of transcription factors (TFs); The Unigene sequence was aligned to PRG (plant resistance gene) database, and the best one with E value less than 1e-5 was screened as the annotation information of the Unigene.

3.4 SSR analysis of transcriptome

SSR search and statistical analysis were performed on all Unigenes by using MISA (MIcroSAtellite identification tool) (http://pgrc.ipk-gatersleben.de/misa/). The SSR search conditions were set as follows: the repeat motif was 1~6 bp, meaning mono-, di-, tri-, tetra-, penta- and hexa-nucleotide respectively. The minimum repeat number was 10, 6, 5, 5, 5 and 5 times respectively. The recognition condition of composite SSR was that the distance between two SSR sites was ≤100 bp.

Authors’ Contributions

LJ and JWX jointly completed the experimental design, data analysis and the writing of the first draft of the manuscript; ZL and LXY participated in sampling and partial data analysis; BTD was the conceiver and person in charge of the project, guiding the experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by Key Research and Development Project of Guangxi (2018AB44025) and the Scientific Research Project of Guangxi University “Research on Integrated Technology of Eucalyptus cloeziana Plantation” (20180493).

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