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Article

Full-Length Transcriptome Characterization and Comparative Analysis of Chosenia arbutifolia

by
Xudong He
1,2,*,
Yu Wang
1,3,
Jiwei Zheng
1,2,
Jie Zhou
1,2,
Zhongyi Jiao
1,2,
Baosong Wang
1,2 and
Qiang Zhuge
3
1
Willow Engineering Technology Research Center of National Forestry and Grassland Administration, Jiangsu Academy of Forestry, Nanjing 211153, China
2
Willow Nursery of the Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Jiangsu Academy of Forestry, Nanjing 211153, China
3
College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Forests 2022, 13(4), 543; https://doi.org/10.3390/f13040543
Submission received: 11 March 2022 / Revised: 30 March 2022 / Accepted: 30 March 2022 / Published: 31 March 2022
(This article belongs to the Special Issue Tree Genetics: Molecular and Functional Characterization of Genes)
Browse Figures
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Abstract

:
As a unique tree species in the Salicaceae family, Chosenia arbutifolia is used primarily for construction materials and landscape planting in China. Compared with other Salicaceae species members, the genomic resources of C. arbutifolia are extremely scarce. Thus, in the present study, the full-length transcriptome of C. arbutifolia was sequenced by single-molecular real-time sequencing (SMRT) technology based on the PacBio platform. Then, it was compared against those of other Salicaceae species. We generated 17,397,064 subreads and 95,940 polished reads with an average length of 1812 bp, which were acquired through calibration, clustering, and polishing. In total, 50,073 genes were reconstructed, of which 48,174 open reading frames, 4281 long non-coding RNAs, and 3121 transcription factors were discovered. Functional annotation revealed that 47,717 genes had a hit in at least one of five reference databases. Moreover, a set of 12,332 putative SSR markers were screened among the reconstructed genes. Single-copy and special orthogroups, and divergent and conserved genes, were identified and analyzed to find divergence among C. arbutifolia and the five Salicaceae species. To reveal genes involved in a specific function and pathway, enrichment analyses for GO and KEGG were also performed. In conclusion, the present study empirically confirmed that SMRT sequencing realistically depicted the C. arbutifolia transcriptome and provided a comprehensive reference for functional genomic research on Salicaceae species.

1. Introduction

The fast-growing tree Chosenia arbutifolia (Pall.) A. Skv. has a wide distribution range over northeastern China, northeastern North Korea, the Honshu and Hokkaido Islands of Japan, and the Russian Far East. It has been extensively utilized as a source of construction materials and landscape planting [1]. It has unique morphological features that differentiate it from poplar and willow, such as missing glands, unique flower structures, and special leaves. Hence, C. arbutifolia was segregated into the genus Chosenia by certain taxonomists [2,3]. However, there is molecular evidence that Chosenia should be categorized into Salix [4,5]. C. arbutifolia was defined as synonymic to S. arbutifolia in the Plant List (http://www.theplantlist.org/tpl/record/tro-28300001, accessed on 10 January 2022). Unlike other Populus and Salix species, C. arbutifolia is not readily propagated through cuttings. Furthermore, C. arbutifolia seed germination requires severe conditions, such as water flow and sediment accumulation [6]. Extensive deforestation and poor reproductive capacity have diminished indigenous C. arbutifolia populations and this tree species is now classified as endangered in China.
Whole-genome sequencing (WGS) is used to explore gene structure and variation. However, the universal application of this technology is constrained by high costs and long run times, especially for species with complex genetic backgrounds. Recently, RNA sequencing based on high-throughput sequencing platforms has provided vital advances and can be effectively implemented in non-model species lacking reference genomes. RNA sequencing realistically reflects gene copy number, type, expression level, and structure, and reveals new genes, potential metabolic pathways, and genetic mechanisms [7,8]. Despite some challenges such as length bias, read accuracy, and high cost, the third-generation single-molecular real-time sequencing (SMRT) technology has the momentous advantages of super-long reads and high throughput, no GC bias and the tendentiousness of PCR amplification, and directly detects DNA base modifications compared with short-read sequencing technology [9]. Thus, full-length transcriptome sequencing (Iso-seq) obtains entire transcript sequences without assembly and identifies multiple forms of alternative splicing [10,11]. It also discovers new genes and analyzes fusion, homologous, superfamily, and allelomorphic genes [12,13], and has been massively utilized in several woody species, including Cinnamomum porrectum [14], Torreya grandis [15], Olea europaea [16], Vitis vinifera [17], Rhododendron lapponicum [18], and Cephalotaxus oliveri [19].
Despite the importance and endangered status of C. arbutifolia, few studies have been conducted on it. Moreover, they focused primarily on the ecological and biological characteristics [1,6], propagation methods [20], population protection [21], molecular markers development [22], gene family analysis [23,24,25], whole-chloroplast genome sequencing [5], and population structure evaluation [26,27] of C. arbutifolia. Only one published report evaluated the C. arbutifolia transcriptome based on the Illumina HiSeq [28]. Several Populus species such as P. deltoides × P. euramericana cv. ‘Nanlin895′ [29], P. wulianensis [30], and P. alba var. pyramidalis [31] have already been subjected to full-length transcriptome sequencing. In contrast, there are few available transcriptome resources for Salix species and especially C. arbutifolia. In this study, we sequenced the full-length transcriptome of C. arbutifolia through SMRT technology and compared it against five other Salicaceae species to elucidate the gene functions and phylogenetic relationships in the Salicaceae. The results of this work could provide substantial resources for functional genomic research and may help clarify the complex evolutionary architecture of the Salicaceae.

2. Materials and Methods

2.1. Plant Materials and RNA Extraction

Twigs and flower branches were sampled from a single superior C. arbutifolia individual in Fusong, Jilin Province, China (41°47′10.55″ N, 127°55′56.13″ E) and brought to the laboratory. The plant material was hydroponically cultivated in a greenhouse. Young leaves, buds, male flowers, roots, and stem bark were sampled and their total RNAs were isolated with an Omega Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA). Extracted RNA purity and concentration were evaluated through 0.8% agarose gels electrophoresis and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

2.2. cDNA Library Construction and Iso-Seq

Equivalent RNAs from diverse tissues were mixed as a single sample. Oligo (dT) was utilized for enrichment of mRNAs containing poly-A. The SMARTer PCR cDNA Synthesis Kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) was used for cDNA reverse transcription. Partial cDNAs were screened with BluePippin for fragments > 4 kb and amplified by PCR. The full-length cDNAs were joined with a dumbbell-shaped SMRT adapter after damage and end repair. Sequences without adapter ligation were removed by exonuclease digestion. The complete SMRT bell library was constructed via primer ligation and DNA polymerase binding, evaluated by QC, and subjected to Iso-seq on a PacBio Sequel platform [9].

2.3. Data Processing

The PacBio official software packages, including ccs, lima, and isoseq3, were employed to process the raw offline data. The subread sequences were generated by filtration and removal of adapters and sequences < 50 bp. They were then calibrated in ccs to obtain circle consensus sequences (CCS). Based on the existence of 3′-primers, 5′-primers, or poly-A tails, the CCS were then separated with lima into full-length and non-full-length sequences. After removing poly-A with isoseq3, high-quality sequences were acquired by clustering and polishing the consensus sequences.

2.4. Sequences Analysis and Functional Annotation

The software programs PLEK [32], CNCI [33], CPC [34], and the Pfam database [35] were used to identify long non-coding RNAs (LncRNAs). The software program iTAK [36] was employed to predict transcription factors (TFs). The open reading frames (ORFs) were captured using software ANGEL [37]. For functional annotation, the reconstructed genes were compared against various protein databases, including non-redundant (NR), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), clusters of orthologous groups (COG), and SWSS with the software program BlastX at E ≤ 1.0 × 10−5. MISA (http://pgrc.ipkgatersleben.de/misa/misa.html, accessed on 5 February 2021) program in Perl script was run to screen SSRs with di-, tri-, tetra-, penta-, and hexa-nucleotide motifs at minimum repeats of 6, 5, 5, 5, and 5, respectively.

2.5. Comparative Transcriptome Analysis

Five whole-genome sequences for the Salicaceae species S. suchowensis (PRJNA668632), S. brachista (PRJNA472210), P. alba (PRJNA491245), P. deltoides (PRJNA598948), and P. trichocarpa (PRJNA10772) were accessed from the NCBI database to perform comparative analyses. The amino acid sequences were aligned using OrthoFinder v. 2.3.3 [38] under the default parameters to obtain homologous genes. Single-copy ortholog sequences in all six species were selected and subjected to multiple sequence alignment using software MAFFT [39]. The output was utilized to construct a phylogenetic with FastTree software [40]. Goatools [41] and KOBAS [42] based on Fisher’s exact test (p < 0.05) were employed for the GO and KEGG enrichment analyses, respectively.

3. Results

3.1. Full-Length Transcriptome Sequencing

We filtered the raw offline sequencing data and generated 25.78 Gb of subreads by full-length transcriptome sequencing. There were 17,397,064 subreads and their lengths were in the range of 51–189,250 bp. The average length was 1482 bp (Table 1). Of these, 272,952 circle consensus sequences (CCS; mean, minimum, and maximum lengths 1759 bp, 62 bp, and 16,567 bp, respectively) were obtained after calibration (Table 1). The detection and elimination of 3′-primers, 5′-primers, and poly-A tails among the CCS yielded a net 232,288 full-length non-chimeric reads (FLNC), namely full-length transcripts. A total of 95,940 polished reads ranging from 51 bp to 13,247 bp were obtained for the following analysis after FLNC clustering and polishing. The average length was 1812 bp (Table 1). Overall, the N50 were 1616 bp, 1939 bp, and 2086 bp for the subreads, CCS, and polished reads, respectively (Table 1).
As there was alternative splicing, the genome coding area should be reconstructed with high-quality polished reads to form a complete coding area. The numbers of reconstructed genes and related transcripts were 50,073 (95,646,387 bp) and 95,940 (173,883,048 bp), respectively, and their average lengths were 1910 bp and 1812 bp, respectively (Table 1). A total of 48,174 ORFs and 4281 LncRNAs were predicted, and their average lengths were 986 bp and 1120 bp, respectively. In total, 3121 transcription factors (TFs) were identified, of which 2288 were true TFs and the remaining 833 were transcriptional regulators. Out of the 3121 TFs, 2942 were classified into 90 families, while the remaining 179 were sorted into other taxa. Table 2 shows the top 10 TF families.

3.2. Functional Classification and Annotation

The putative functions of all 50,073 reconstructed genes were compared against the NR, GO, COG, KEGG, and SWSS databases (Figure 1A). We annotated 47,678 (95.22%) and 44,765 (89.40%) genes in the NR and GO databases, and 38,606 (77.10%), 33,587 (67.08%), and 21,499 (42.94%) in the SWSS, COG, and KEGG databases, respectively. Moreover, 18,096 (37.76%) genes were significantly correlated with sequences in all databases and 47,717 (95.29%) genes had hits in at least one database (Table 3).
For the GO term classification, all 44,765 genes assigned to three primary categories were divided into 59 subgroups (Figure 1B). ‘Biological process’ had 27 subgroups, and ‘cellular process’ was the most abundant (28,978), followed by ‘metabolic process’ (23,279) and ‘biological regulation’ (17,382). The ‘cellular component’ category had 19 subcategories and consisted mainly of ‘cell’ (38,644), ‘cell part’ (38,644), and ‘organelle’ (29,774). The ‘molecular function’ category contained 13 subcategories enriched with ‘catalytic activity’ (19,462), ‘binding’ (16,182), and ‘transcription regulator activity’ (3328). Compared with the KEGG database, the metabolic pathway analysis demonstrated that 21,499 genes were involved in 400 pathways. The most enriched pathways were ‘global and overview maps’ (5677), followed by ‘signal transduction’ (5984), and ‘carbohydrate metabolism’ (3817) (Figure 1C). Furthermore, 33,587 genes were separated into 25 categories for COG classification (Figure 1D). The ‘function unknown’ part explained the largest proportion (3937; 11.72%), followed by ‘posttranslational modification, protein turnover, chaperones’ (3818; 11.37%) and ‘signal transduction mechanisms’ (3254; 9.69%). Moreover, ‘cell motility’, ‘general function prediction only’, ‘extracellular structure’, and ‘nuclear structure’ were each <1% (Figure 1D).

3.3. SSR Mining

MISA screened 12,332 putative SSRs in 50,073 reconstructed genes. Of these, 2110 (4.2%) sequences contained at least two SSRs. The occurrence frequencies were one SSR per 4.06 genes and 7.76 kb in sequence length in C. arbutifolia. Table 4 shows that the di-nucleotide repeat motif (6640) accounted for 53.8% and was the most abundant, followed by tri- (4996; 40.5%), tetra- (442; 3.5%), and hexa-nucleotide (141; 1.1%), whereas penta-nucleotide (113; 0.9%) was the least. Twelve different motifs were observed for the di-nucleotide, and AG/CT was the most frequent (53.4%). Of the 60 types of tri-nucleotides and 94 types of tetra-nucleotides, AAG/CTT and AAAT/ATTTA were the most abundant, with frequencies of 20.0% and 8.6%, respectively. The penta- and hexa-nucleotides had 64 and 102 types of motifs, respectively. AGGGG/CCCCT and AAGGAG/CCTTCT were the most frequent, taking 11.5% and 9.9%, respectively.

3.4. Comparative Analysis

The whole-genome sequences for three Populus and two Salix species were downloaded from NCBI, and included P. alba, P. deltoides, P. trichocarpa, S. suchowensis, and S. brachista. The amino acid sequences of all six species were clustered by similarity with Orthofinder. The homologous genes in each species are shown in Figure 2. A total of 2732 single-copy orthogroups were detected for all six species. The 19,704 most special orthogroups were screened in C. arbutifolia, followed by P. deltoides (6479), S. suchowensis (5181), S. brachista (3697), and P. trichocarpa (3063), whereas P. alba (1030) had the fewest special orthogroups (Figure 2A). The single-copy ortholog sequences of all species were selected for multiple sequence alignment using MAFFT. Based on the alignment results, a whole genome level phylogenetic tree was established using FastTree (Figure 2B). C. arbutifolia was assigned to one clade, while the other five species were relegated to another clade. The main clades were divided in the branches Populus and Salix. P. alba, P. deltoides, and P. trichocarpa were clustered in the former, while S. brachista and S. suchowensis were clustered in the Salix latter.
All 19,704 special orthogroups in C. arbutifolia were analyzed for GO and KEGG annotations. As shown in Figure 3, a total of 17,511 special orthogroups had hits and were assigned to 58 subcategories for GO term classification. ‘Cellular process’ (11,546; 66.04%), ‘cell and cell part’ (15,285; 87.29%), and ‘catalytic activity’ (8026; 45.83%) were the most plentiful in the ‘biological process’, ‘cellular component’, and ‘molecular function’ categories, respectively. The KEGG pathway analysis demonstrated that 312 pathways were enriched in 9112 (46.24%) of the detected special orthogroups. The pathway involving the greatest number of special orthogroups was ‘metabolic pathways’ (3059), followed by ‘biosynthesis of secondary metabolites’ (1669) and ‘biosynthesis of antibiotics’ (854). Moreover, the results of the functional enrichment are displayed in Figure 4. For the GO enrichment, 13,301 genes involved in ‘molecular function’ were identified, followed by 11,145 genes related to ‘cytoplasm’, whereas the fewest 188 genes were detected in ‘fungal-type vacuole’ (Figure 4A). For the KEGG enrichment, ‘microbial metabolism in diverse environments’ was the most enriched pathway (759), followed by ‘glycolysis/gluconeogenesis’ (290) and ‘starch and sucrose metabolism’ (221) (Figure 4B).
The divergent orthologous genes under positive selection (Ka/Ks > 1) and conserved orthologous genes under selection constraints (Ka/Ks < 0.1) were evaluated. Of the 2732 single-copy orthogroups in C. arbutifolia, 153 divergent and 41 conserved genes were identified. Of these, 122 (79.7%) divergent and 39 (95.1%) conserved genes had specific functions. The GO enrichment analysis demonstrated that for the divergent genes, eight genes were involved in ‘nuclease activity’ and ‘endonuclease activity’, seven were associated with ‘RNA modification’, and six were related to ‘multicellular organism reproduction’ (Figure 5A). For the conserved genes, 16 genes were associated with ‘cellular component organization’ and ‘cellular component organization or biogenesis’, 14 were related to ‘protein-containing complex’, and 5 were associated with ‘vesicle-mediated transport’ (Figure 5C). The KEGG enrichment results disclosed that two genes were related to the ‘thermogenesis’, ‘retrograde endocannabinoid signaling’, and ‘nucleotide excision repair’ pathways, while the other 24 pathways only had one gene for the divergent genes (Figure 5B). For the conserved genes, four genes were involved in the ‘endocytosis’ pathway and three genes were associated with ‘necroptosis’ (Figure 5D).

4. Discussion

In the recent decade, high-throughput sequencing technology and bioinformatics have progressed and have been extensively applied in genetic and genomic research. Full-length transcriptome sequencing with SMRT technology is efficient and reliable in transcriptome reconstruction, especially for non-model organisms lacking de novo assembly [12,13]. Unlike short-read sequencing, Iso-seq provides much longer reads and can overcome certain limitations, such as assembly errors and artifacts [17]. Here, the full-length transcriptome of C. arbutifolia was sequenced on the third-generation PacBio Sequel platform. We obtained 17,397,064 subreads with a mean length of 1482 bp. In contrast, previous reports on second-generation sequencing for Salix spp. yielded only 398 bp for S. suchowensis [43], 432 bp for S. babylonica [43], 703 bp for S. integra [44], 713 bp for S. psammophila [45], 918 bp for S. arbutifolia [28], and 944 bp for S. matsudana [46]. For the clustered and polished reads, the sequence quality index N50 was 2086 bp (Table 1). In contrast, N50 was only 1191 bp for S. integra [44], 1274 bp for S. psammophila [45], 1315 bp for S. babylonica [43], 1445 bp for S. suchowensis [43], and 1468 bp for S. matsudana [46]. Nevertheless, the foregoing values were comparable to the N50 for S. arbutifolia (1952 bp) [28]. A comparison against other Populus species subjected to the same SMRT technology disclosed that the average subread length for C. arbutifolia was shorter than those of P. wulianensis (2177 bp) [30] and P. × canadensis (2057 bp) [47], possibly because of the diverse genetic backgrounds among these species.
LncRNAs are regulatory factors with important functions in several biological processes, including development, growth, secondary metabolism, and abiotic stress responses. They regulate gene expression by interacting with miRNA networks [48]. Here, a total of 4281 LncRNAs were identified, which is considerably greater than those of P. alba var. pyramidalis (3410) [31], P. deltoides × P. euramericana cv. ‘Nanlin895′ (1187) [29], and P. × canadensis (753) [47]. A few studies explored the functions of LncRNAs [47,49]. However, relatively little is known about those of C. arbutifolia. TFs play vital roles in regulating gene expression to biotic and abiotic stress responses. Of the 3121 TFs screened in the present study, 2942 were classified into 90 families. The latter number is comparable to that which was reported for C. oliveri [19]. The top 10 TF families, including bHLH, AP2-ERF, MYB, C3H, Bzip, C2H2, MYB-related, GRAS, NAC, and WRKY (Table 2), were similar to those reported for C. oliveri [19]. The properties of the foregoing TF families associated with plant immunity, regulation, and signaling pathways have been investigated [50].
In genetic and genomic studies, SSRs are the preferred ideal molecular markers. The development of SSR markers through high-throughput sequencing technology is highly efficient, and may be applied to those plants without available genomic resources. In our work, a total of 12,332 candidate SSRs were identified in 50,073 reconstructed genes. The coverage (24.6%) was extremely higher for the SSR markers derived from EST resources by short-read sequencing, such as S. suchowensis and S. babylonica (4.3%) [51], and S. psammophila (7.86%) [45]. The high frequency of occurrence for C. arbutifolia may be explained by the use of multiple tissues and advanced sequencing technology. However, the same mining criterion and SMRT technology returned a frequency of occurrence of 26.0% for P. wulianensis [30]. Substantial bias among repeat types can be observed for various plants. Hence, di- and tri-nucleotides are common but the other nucleotide types are rare. Tri-nucleotide repeats should be the most abundant because of genetic code selection [51]. Nevertheless, di-nucleotide repeats accounted for 53.8% of the total in C. arbutifolia (Table 4). These results are consistent with those for S. psammophila [45] but differ from those for P. wulianensis [30], S. suchowensis, and S. babylonica [40]. In the latter cases, tri-nucleotides were the most abundant. Motifs that differed in terms of their repeats were analyzed to disclose any bias in C. arbutifolia. For the di- and tri-nucleotides, AG/CT and AAG/CTT were the most frequent, respectively, as previously reported [30,51]. In contrast, GAA/TTC was the dominant motif in S. psammophila [45]. Although numerous EST-SSR markers have been developed for Salix [43], the SSRs in C. arbutifolia still have great potential to provide useful resources in genetic breeding research.
The taxonomically difficult genus Salix comprises over 500 species and the phylogenetic position of C. arbutifolia is still highly controversial. In the present study, we constructed a phylogenetic tree containing three Populus species and two Salix species. The construction was based on the alignment results for single-copy ortholog sequences (Figure 5B). C. arbutifolia occupied one branch, while another was occupied by Populus and Salix species. In the early stage, Chosenia was reckoned a transitional type during the genetic differentiation from Populus to Salix, based on certain morphological divergence indices. Chosenia was proposed as a separate genus in Salicaceae phylogeny [1,2,3], and this recommendation was consistent with our study. In contrast, whole cp genome sequences [4,52,53], ribosomal DNA [54,55], and rbcL or matK gene [55,56] sequences revealed that C. arbutifolia is closely related to Salix species and should be assigned to the genus Salix. As the resources in the database are limited, only five whole-genome sequences for Salicaceae species were downloaded and applied in the phylogeny analysis. Hence, the information returned was restricted and additional species should be used in future studies.

5. Conclusions

The present study is the first full-length transcriptome analysis of C. arbutifolia, which, to our knowledge, is classified as an endangered tree species in China. The C. arbutifolia transcriptome was sequenced and characterized through SMRT technology. The single-copy and special orthogroups, as well as divergent and conserved genes in C. arbutifolia and other five Salicaceae species were also compared and analyzed. Given the limited available information in databases, more species should be added for further analysis. Overall, the full-length transcriptome sequences generated for C. arbutifolia will be beneficial for understanding the genomic architecture and providing useful genetic resources for future research on Salicaceae species.

Author Contributions

X.H. and Q.Z. conceived and designed the experiments; X.H. wrote the paper; Y.W. performed the experiments; J.Z. (Jiwei Zheng) and J.Z. (Jie Zhou) analyzed the data; Z.J. and B.W. collected the samples. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 31670662) and the Independent Scientific Research Project of the Jiangsu Academy of Forestry (Grant No. ZZKY202101).

Data Availability Statement

All SMRT sequencing reads obtained for C. arbutifolia were deposited to the sequence read archive (SRA) database at NCBI under the accession number PRJNA788330.

Acknowledgments

The authors are grateful to Shanghai BIOZERON Biotechnology Co., Ltd. for their assistance with the bioinformatics analysis.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Gene annotation and classification for Chosenia arbutifolia. (A) Venn diagrams of reconstructed genes annotated in different databases. (B) Gene ontology term classifications for the reconstructed genes. (C) General pathway assignment based on KEGG database. (D) Histograms depicting clusters of orthologous groups.
Figure 1. Gene annotation and classification for Chosenia arbutifolia. (A) Venn diagrams of reconstructed genes annotated in different databases. (B) Gene ontology term classifications for the reconstructed genes. (C) General pathway assignment based on KEGG database. (D) Histograms depicting clusters of orthologous groups.
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Figure 2. Analysis of orthologous genes in Chosenia arbutifolia and five other Salicaceae species. (A) Venn diagrams of orthologous genes. Numbers in middle circle represent total single-copy ortholog sequences for all six species. Numbers at interior edges of oval represent special orthogroups. Numbers outside oval represent total orthogroups. (B) Phylogenetic tree of Chosenia arbutifolia and five other Salicaceae species.
Figure 2. Analysis of orthologous genes in Chosenia arbutifolia and five other Salicaceae species. (A) Venn diagrams of orthologous genes. Numbers in middle circle represent total single-copy ortholog sequences for all six species. Numbers at interior edges of oval represent special orthogroups. Numbers outside oval represent total orthogroups. (B) Phylogenetic tree of Chosenia arbutifolia and five other Salicaceae species.
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Figure 3. Gene ontology (GO) term classifications of special orthogroups in Chosenia arbutifolia.
Figure 3. Gene ontology (GO) term classifications of special orthogroups in Chosenia arbutifolia.
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Figure 4. Bubble charts of enrichment for special orthogroups. (A) GO enrichment. (B) KEGG enrichment.
Figure 4. Bubble charts of enrichment for special orthogroups. (A) GO enrichment. (B) KEGG enrichment.
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Figure 5. Bubble charts of enrichment for two types of orthologous genes. (A) GO enrichment of divergent genes. (B) KEGG enrichment of divergent genes. (C) GO enrichment of conserved genes. (D) KEGG enrichment of conserved genes.
Figure 5. Bubble charts of enrichment for two types of orthologous genes. (A) GO enrichment of divergent genes. (B) KEGG enrichment of divergent genes. (C) GO enrichment of conserved genes. (D) KEGG enrichment of conserved genes.
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Table
Table 1. Characteristics of Chosenia arbutifolia transcriptome sequences.
Table 1. Characteristics of Chosenia arbutifolia transcriptome sequences.
ItemNumberAverage Length (bp)N50Min_LengthMax_Length
Subreads17,397,0641482161051189,250
CCS272,952175919396216,567
Polished reads95,940181220865113,247
Cogent genes50,0731910NA5113,835
ORF48,174986NA1479693
LncRNA42811120NA1326754
Table
Table 2. Top 10 TF families in Chosenia arbutifolia transcriptome sequences.
Table 2. Top 10 TF families in Chosenia arbutifolia transcriptome sequences.
FamilyNumberPercent (%)
bHLH1424.83
AP2-ERF1344.55
MYB1274.32
C3H1214.11
bZIP1173.98
C2H21153.91
MYB-related1033.50
GRAS1023.47
NAC923.13
WRKY923.13
Table
Table 3. Functional annotation of reconstructed genes.
Table 3. Functional annotation of reconstructed genes.
DatabaseAnnotated GenesPercent (%)
NR47,67895.22
GO44,76589.40
COG33,58767.08
KEGG21,49942.94
SWSS38,60677.10
In_all_DB18,09637.76
At_least_one_DB47,71795.29
Total_genes50,073100
Table
Table 4. SSR occurrence and composition in Chosenia arbutifolia.
Table 4. SSR occurrence and composition in Chosenia arbutifolia.
Repeat MotifNumberNo. TypesDominant MotifNo. Dominant Motifs
Di-nucleotide6640 (53.8%)12AG/CT3546 (53.4%)
Tri-nucleotide4996 (40.5%)60AAG/CTT999 (20.0%)
Tetra-nucleotide442 (3.5%)94AAAT/ATTT38 (8.6%)
Penta-nucleotide113 (0.9%)64AGGGG/CCCCT13 (11.5%)
Hexa-nucleotide141 (1.1%)102AAGGAG/CCTTCT14 (9.9%)
Total12,332 (100%)336
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He, X.; Wang, Y.; Zheng, J.; Zhou, J.; Jiao, Z.; Wang, B.; Zhuge, Q. Full-Length Transcriptome Characterization and Comparative Analysis of Chosenia arbutifolia. Forests 2022, 13, 543. https://doi.org/10.3390/f13040543

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He X, Wang Y, Zheng J, Zhou J, Jiao Z, Wang B, Zhuge Q. Full-Length Transcriptome Characterization and Comparative Analysis of Chosenia arbutifolia. Forests. 2022; 13(4):543. https://doi.org/10.3390/f13040543

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He, Xudong, Yu Wang, Jiwei Zheng, Jie Zhou, Zhongyi Jiao, Baosong Wang, and Qiang Zhuge. 2022. "Full-Length Transcriptome Characterization and Comparative Analysis of Chosenia arbutifolia" Forests 13, no. 4: 543. https://doi.org/10.3390/f13040543

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