Complete Chloroplast Genome Structural Characterization and Comparative Analysis of Viburnum japonicum (Adoxaceae)

Abstract

Viburnum japonicum (Thunb.) Sprengel is an endangered species endemic to coastal regions of eastern Asia (China, Japan, and South Korea). However, its systematic position has been controversial. In this study, we present the complete chloroplast (cp) genome of V. japonicum (GenBank OP644292) sequenced using the Illumina NovaSeq platform. The cp genome has a total length of 158,606 bp and a G+C contents of 38.08%. It consists of a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs) regions measuring 87,060 bp, 18,510 bp, and 26,516 bp, respectively. A total of 131 genes were identified, including 87 protein-coding genes, 36 transfer RNA genes (tRNAs), and 8 ribosomal RNA genes(rRNAs). Additionally, a total of 44 dispersed repeats were detected, including three types: forward, palindromic, and reverse. Among the 38 SSR loci that were discovered, the majority were mononucleotide loci composed of A/T. Furthermore, we found that 15 genes (accD, atpF, ndhA, ndhB, petB, petD, rpl16, rpl2, rpoC1, rps12, rps16, trnA-UGC, trnI, trnK-UUU, and trnL-UAA) contain one intron, while clpP and ycf3 have two introns. The relative synonymous codon usage (RSCU) analysis detected 31 high-frequency codons, where A/U bases accounted for 93.55% of the total, indicating an asymmetry in chloroplast gene and a presence for A/U bases. Comparative analysis of genome structure and sequences data of V. japonicum chloroplast genomes in comparison with other closely related species demonstrated a high level of conservation in their structure and organization. Furthermore, three mutation hotspots (psbH, rps19, and trnL) were identified, which could be valuable for future phylogenetic and population genetic research. Phylogenetic analysis revealed that the two accessions of V. japonicum are closely related to a group of V. setigerum, V. erosum, and V. fordiae within the Viburnum genus. In conclusion, this study provides important insights for accurately identifying and understanding the phylogeny of Viburnum species through the complete cp genome sequencing of V. japonicum.

1. Introduction

Viburnum japonicum (Thunb.) Sprengel 1825, also known as Japanese viburnum, belongs to the Adoxaceae family and is a valuable species for ornamental and economic purposes. For example, chavicol extracted from V. japonicum and its related effective compounds can significantly inhibit drosophila larvae and adults, making it a promising source of biopesticides [1]. It is easily distinguished from other recorded Viburnum species due to its rounded and evergreen shrubs, broad leaves, and glabrous texture. Previously, it was believed to be distributed only along the coast of Japan, mainly Honshu, Kyushu, and Ryukyu Islands. However, in 1994, Chinese plant taxonomists discovered the species on the east coast of Zhejiang Province. Since then, it has been recognized as a plant species with extremely small populations (PSESP) and included in Zhejiang’s list of the key protected wild plants. In 2003, V. japonicum was also observed in South Korea on Gageo-do Island [2], indicating its potential to spread beyond its native range. In recent years, research on V. japonicum has primarily focused on physiological resistance [3,4,5], breeding techniques [6,7], molecular marker development [8] and population genetics [9]. However, limited genomic exploration has been conducted, with only a few brief reports from South Korean scholars regarding chloroplast [10,11,12], and no further genomic information was accessible. The taxonomic status and phylogenetic position of V. japonicum remain controversial due to inadequate species sampling within the Viburnum genus.

The chloroplast genome of plants plays a crucial role in elucidating chloroplast functions, species identification, population genetics, phylogenetics, and conservation of plant resources [13,14,15,16]. In this study, we conducted a comprehensive analysis of the complete cp genome sequence of V. japonicum using second-generation sequencing technology. This analysis involved annotating functional genes, analyzing codon preference, examining simple sequence repeats (SSR), and comparing chloroplast genomes. The main objective was to deepen our understanding of the genomic information specific to this species. Consequently, the findings of this study will serve as a reliable reference genome for future investigations focused on molecular evidence and phylogenetic relationships among Asian Viburnum species.

2. Materials and Methods

2.1. Plant Material, DNA Extraction, and Sequencing

One individual of V. japonicum (Figure 1) was collected, which was deposited at the Arboretum of Zhejiang Academy of Forestry, Hangzhou, Zhejiang Province, China (30°12′ N, 120°01′ E) with a voucher number of HZ2022-05 (contact: Hong Zhu, Email: zh1107401987@126.com). The total genomic DNA was extracted from fresh leaves of the collected V. japonicum using a modified CTAB method [17]. Paired-end (PE) reads of 2 × 150 bp were conducted on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) at Personalbio Biotech (Shanghai, China).

2.2. Genome Assembly and Annotation

The raw data obtained underwent a filtering process where low-quality reads were removed, and high-quality reads were evaluated using the FastQC v0.120 software [18] (http://www.bioinformatics.babraham.ac.uk/projects/fastqc accessed on 2 August 2023). The filtering criteria were as follows: (1) Removing the sequencing linker and primer sequence from the reads; (2) Discarding reads with an average quality value lower than Q5; (3) Filtering out any reads with a length of 50 bp or less. Based on these cleaning reads, the cp genome of V. japonicum was assembled via Fast-Plast v1.2.9 software [19] (https://github.com/mrmckain/Fast-Plast accessed on 2 August 2023). The genome was annotated using GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html accessed on 2 August 2023). The annotated V. erosum (MN218778) chloroplast genome was used as a reference. The OGDRAW program (https://chloro-box.mpimpgolm.mpg.de/index.html accessed on 2 August 2023) was used to draw a detailed physical map of the V. japonicum complete cp genome. The CPGView tool [20] (http://www.1kmpg.cn/cpgview accessed on 2 August 2023) was utilized to visualize the structures of splicing genes within the chloroplast genome (Figures S1 and S2). The final annotated genome sequence of V. japonicum was submitted to the GenBank database and assigned the accession number OP644292.

2.3. Repeat Sequence Analysis

The MISA software [21] (https://webblast.ipk-gatersleben.de/misa/ accessed on 2 August 2023) was utilized to identify SSR sites in the chloroplast genome of V. japonicum. Dispersed repeats of four types (forward repeats (F), reverse repeats (R), complement repeats (C), and palindromic repeats (P)) were detected in the chloroplast whole genome using the REPute v2.5 software (https://bibiserv.cebitec.uni-bielefeld.de/reputer accessed on 2 August 2023). The parameters were set as follows: minimum length of 30 bp and hamming distance of 3.

2.4. Relative Condon Usage Analysis

The analysis of relative synonymous codon usage (RSCU) in the chloroplast genome coding genes was conducted using the CodonW v1.4.2 software [22] (https://www.softpedia.com/get/Science-CAD/CodonW.shtml accessed on 2 August 2023), with default parameter settings.

2.5. Genome Comparison Analysis

The differences in boundary sequences were visualized using the IRscope v2.01 software (https://irscope.shinyapps.io/irapp/ accessed on 2 August 2023). Homologous gene sequences of different species were compared globally using MAFFT software v 7.520 [23], and nucleotide variability (π) values were calculated for each gene using DnaSP v5 software [24], and scatter fold plots were plotted.

2.6. Phylogenetic Analysis

To clarify the phylogenetic position of V. japonicum within the genus Viburnum, 17 additional complete cp genome sequences (Dipsacus japonicus MZ934745, Sambucus chinensis MW455170, and S. williamsii KX510276 as outgroups) were aligned using the program MAFFT v7.520. The software RAxML v8.2.12 [25] was used for phylogenetic tree construction based on the maximum likelihood (ML) algorithm under the parameter model of GTRGAMMA and 1000 bootstrap replicates.

3. Results and Discussion

3.1. Genome Sequencing Assembly and Characterization

A total of 23,763,912 raw read and 3,588,350,712 raw bases were gained. The proportion of Q20 and Q30 was 97.45% and 92.92%, respectively. As a result, we obtained a scaffold with its high quality of assembled 155,827 bp in length. Finally, the complete cp genome of V. japonicum was successfully assembled with an average read coverage depth of 650X (Figure S3) and a typical circular structure of 158,606 bp in length, consisting of four subregions: a large single-copy (LSC) of 87,060 bp, a small single-copy (SSC) region of 18,510 bp, and a pair of inverted repeat regions (IRs), each of 26,516 bp (Figure 2). The overall nucleotide composition of the cp genome sequence was: A (30.62%), T (31.29%), C (19.38%), and G (18.71%), and the total G + C contents of the cp genome were 38.08%; LSC (36.42%), SSC (31.97%), and IRs (42.97%) (

Table 1. Base composition of the chloroplast genome of V. japonicum (Thunb.) Sprengel.

Region	Percentage of Bases
	AT (%)	GC (%)	A (%)	T (%)	G (%)	C (%)
Total	61.91	38.08	30.62	31.29	18.71	19.38
IRa	57.03	42.97	28.61	28.42	22.25	20.72
IRb	57.03	42.97	28.42	28.61	20.72	22.25
SSC	69.03	31.97	33.97	34.06	15.22	16.75
LSC	63.58	36.42	31.18	32.40	17.76	18.66

).

A total of 131 genes were annotated, including 87 protein-coding genes (PCGs), 8 ribosomal RNA genes (rRNAs), and 36 transfer RNA genes (tRNAs). Of these genes, 15 (accD, atpF, ndhA, ndhB, petB, petD, rpl16, rpl2, rpoC1, rps12, rps16, trnA-UGC, trnI, trnK-UUU, and trnL-UAA) contained one intron, while another two genes (clpP and ycf3) had two introns (Figure S1). The functions of the major genes were broadly divided into three categories: photosynthesis-related genes, self-replication-related genes, and other genes. Photosynthesis- and self-replication-related genes constituted most of the chloroplast genome (

Table 2. Annotation table of functional genes in the chloroplast genome of V. japonicum.

Categories	Group of Genes	Name of Genes
Photosynthesis	Subunits of photosystem I	psaA, psaB, psaC, psaI, psaJ
	Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Subunits of NADH dehydrogenase	ndhA *, ndhB * (2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	Subunits of cytochrome b/f complex	petA, petB *, petD *, petG, petL, petN
	Subunits of ATP synthase	atpA, atpB, atpE, atpF *, atpH, atpI
	Large subunit of rubisco	rbcL
Self-replication	Proteins of large ribosomal subunit	rpl14, rpl16 *, rpl2 * (2), rpl20, rpl22, rpl23 (2), rpl32, rpl33, rpl36
	Proteins of small ribosomal subunit	rps11, rps12 ** (2), rps14, rps15, rps16 *, rps18, rps19, rps2, rps3, rps4, rps7 (2), rps8
	Subunits of RNA polymerase	rpoA, rpoB, rpoC1 *, rpoC2
	Ribosomal RNAs	rrn16 (2), rrn23 (2), rrn4.5 (2), rrn5 (2)
	Transfer RNAs	trnA-UGC * (2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnH-GUG, trnI * (2), trnK-UUU *, trnL-CAA (2), trnL-UAA *, trnL-UAG, trnM-CAU (4), trnN-GUU (2), trnP-UGG, trnQ-UUG, trnR-ACG (2), trnR-UCU, trnS-CGA *, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (2), trnW-CCA, trnY-GUA
Other genes	Maturase	matK
	Protease	clpP **
	Envelope membrane protein	cemA
	Acetyl-CoA carboxylase	accD *
	c-type cytochrome synthesis gene	ccsA
	Translation initiation factor	infA
Genes of unknown function	Conserved hypothetical chloroplast ORF	ycf1, ycf15 (2), ycf2 (2), ycf3 **, ycf4

* Genes containing one intron, ** genes containing two introns, (2): gene with two copies.

).

3.2. SSR Analysis and Repeat Sequence Statistics

According to MISA analysis, a total of 38 SSRs loci were detected in the V. japonicum chloroplast genome. Among them, there were 36 mononucleotide loci and two dinucleotide loci, and no other types of SSR were found. Among the mononucleotide loci, the most frequent simple repeat sequence was A/T, accounting for 97.22%, while only one locus was composed of C/G. As for the dinucleotide loci, they were all composed of AT/AT, indicating a preference for base composition in SSR genes (Figure 3).

In the V. japonicum chloroplast genome, a total of 44 dispersed repeats were detected, involving 3 types, including 18 of F type, 23 of P type, and 3 of R type. However, the C type was not found (Figure 4). The length of these dispersed repeats ranged from 30 to 64 base pairs, with the majority of longer repeats found in the ycf gene and the intergenic spacer (IGS).

3.3. Relative Codon Preference Analysis

In the chloroplast genome of V. japonicum, there are a total of 26,248 codons, encoding 21 amino acids. In terms of the number of codons used, the codons encoding leucine (Leu), arginine (Arg), and serine (Ser) are the most diverse, with six codon types each. On the other hand, the codons encoding tryptophan (Trp) are the least diverse, with only one codon (UGG). Leucine (Leu) has the highest frequency of usage (10.5%, 2746 codons), while the codons UAG and UGA, which serve as termination codons, are the least used. Among the 65 codons used to encode the 21 amino acids, 31 codons exhibit preference (RSCU > 1), and only one codon for Trp does not show codon preference (UGG). Additionally, 33 codons encoding amino acids are used with lower frequency (RSCU < 1) (Figure 5 and

Table 3. Relative synonymous codon usage (RSCU) in V. japonicum chloroplast genome.

Amino Acid	Codon	Number	RSCU	Amino Acid	Codon	Number	RSCU
Termination codon	UAA	40	1.40	Met	AUG	619	2.00
	UAG	23	0.80		GUG	1	0.00
	UGA	23	0.80	Asn	AAC	284	0.45
Ala	GCA	390	1.10		AAU	968	1.55
	GCC	223	0.63	Pro	CCA	327	1.19
	GCG	161	0.46		CCC	208	0.76
	GCU	639	1.81		CCG	153	0.56
Cys	UGC	76	0.52		CCU	411	1.50
	UGU	215	1.48	Gln	CAA	705	1.50
Asp	GAC	209	0.38		CAG	232	0.50
	GAU	894	1.62	Arg	AGA	484	1.81
Glu	GAA	1027	1.49		AGG	185	0.69
	GAG	354	0.51		CGA	364	1.36
Phe	UUC	543	0.74		CGC	107	0.40
	UUU	916	1.26		CGG	122	0.46
Gly	GGA	704	1.57		CGU	341	1.28
	GGC	196	0.44	Ser	AGC	129	0.38
	GGG	328	0.73		AGU	400	1.18
	GGU	565	1.26		UCA	416	1.22
His	CAC	138	0.43		UCC	326	0.96
	CAU	503	1.57		UCG	184	0.54
Ile	AUA	685	0.93		UCU	584	1.72
	AUC	477	0.65	Thr	ACA	402	1.20
	AUU	1053	1.43		ACC	253	0.76
Lys	AAA	1028	1.48		ACG	152	0.45
	AAG	357	0.52		ACU	531	1.59
Leu	CUA	380	0.83	Val	GUA	523	1.48
	CUC	195	0.43		GUC	178	0.50
	CUG	175	0.38		GUG	214	0.60
	CUU	597	1.30		GUU	500	1.41
	UUA	811	1.77	Trp	UGG	468	1.00
	UUG	588	1.28	Tyr	UAC	203	0.42
					UAU	761	1.58

).

3.4. Comparative Genomic Analysis

To analyze whether there is a contraction or expansion phenomenon in the boundaries of the chloroplast genome of V. japonicum, we compared the distribution of boundary genes in seven species of the Viburnum genus, including V. japonicum. The overall gene differences among the six species of the Viburnum genus are small, around 158,000. The JLB boundary region is relatively conserved among the six Adoxaceae species, falling within the rps19 gene. The JSB boundary region of V. opulus, V. fordiae, and V. japonicum is located between the trnH and ndhF genes, while the other two species of Viburnum do not have the ndhF gene in the IRb and SSC regions. The JSA boundary falls within the ycf1 gene, with the ycf1 gene of V. japonicum spanning 5036 bp in the IRa region, while the ycf1 genes of the other five species span over 5600 bp in the IRa region. The bases of the ycf1 gene in the IRa region are relatively longer in length. The JLA boundary region falls between the rpl2 and trnH genes. Except for V. japonicum, the JLA boundary of the other six Viburnum species is relatively close to the trnH gene, with distances of 6, 0, 2, 0, 3, and 0 bp, respectively. However, the JLA boundary of V. fordiae is 88 bp away from the trnH gene (Figure 6).

DnaSP software v5.0 was used to analyze nucleotide polymorphism in the chloroplast genomes of V. japonicum and its closely related species. The results showed that their nucleotide variability (π) ranged from 0 to 0.016, with an average value of 0.003. Among them, the psbH, rps19, and trnL regions exhibited relatively high variation, with all π values exceeding 0.01. The highest polymorphism was observed in the trnL region located in the IR region (π = 0.016), while the other two regions originated from the LSC region (Figure 7).

3.5. Phylogenetic Analysis of Viburnum Species

The ML tree showed that all the Viburnum species clustered into two clades (Clade A and Clade B) by high bootstrap support. The two accessions of V. japonicum were a sister group to a small clade of three Viburnum species including V. setigerum, V. erosum, and V. fordiae, and were placed in a monophyletic group with two other Viburnum species (V. luzonicum and V. melanocarpum) in Clade A, while the remaining six Viburnum species were placed in a sister Clade B (Figure 8).

4. Discussion

Using Illumina high-throughput sequencing technology, we obtained the complete chloroplast sequence of V. japonicum. The results showed that the complete sequence length of the chloroplast genome of V. japonicum (158,606 bp) closely resembles recently reported data for other Viburnum members, such as V. erosum (158,624 bp) [12], and exhibits near-identity with an individual from South Korea (158,614 bp) [10]. The complete chloroplast genome displayed a distinct quadripartite structure with a conserved arrangement within the chloroplast genome of V. japonicum, showing adherence to the typical length range (120–160 kb) observed in angiosperms [26] as well as the total GC content found in Adoxaceae [27]. These findings provide compelling evidence for the highly conserved and slow evolution of the chloroplast genome in this genus. This inference is further validated by thorough analyses of contraction and expansion at the inverted repeat region boundaries (Figure 6) as well as comprehensive whole-genome alignments. When comparing with the article published by South Korean scholars Cho et al. [10], this study supplements and improves the analysis conducted in their previous article. Our results had a more comprehensive analysis in terms of repeat sequence, SSR analysis, RCSU analysis, IR boundaries, and sliding window analysis. As a result, a more comprehensive understanding of their chloroplast genome has been achieved. Compared to the Internal Transcribed Spacer (ITS), chloroplast genes, and intergenic spacer regions, the chloroplast genome provides a greater wealth of informative data.

The number and types of SSRs are species-dependent and can be utilized for species identification, phylogenetic analysis, and molecular-assisted breeding [28]. In this study, 38 SSR loci were identified from the chloroplast genes of V. japonicum. These loci primarily consisted of the A/T form, which were comparatively fewer in number than in other Viburnum species, such as V. henryi and V. nervoum [27]. In the arrangement of the entire SSR, A and T bases occurred significantly more frequently than G and C, aligning with the higher proportion of A/T in the complete genome. In the genome, AT base pairs are less stable than GC base pairs [29], thus SSRs with more A/T repeats could lead to more mutation sites. Consequently, these SSR loci can be further developed as molecular markers for genetic variation and species identification purposes within the Viburnum genus and even the Caprifoliaceae family. In addition, V. japonicum exhibits three types of SSR repeats and lacks the C-type, which is similar to species from the same genus. The biased usage of codons is a result of the long-term adaptive evolution of species to the environment and is influenced by multiple factors. The RSCU value reflects the codon usage pattern of different genes, with higher values indicating higher codon usage frequency. Among the 31 high-frequency codons, those ending with A/U accounted for 93.55%, consistent with the codon usage bias in other plant chloroplast genomes, suggesting the presence of asymmetry in chloroplast gene codons and a preference for A/U bases.

Comparative analysis of the complete chloroplast (cp) genome sequences has proven to be a reliable tool for supporting the study of plant phylogeny and taxonomy [30]. Korean botanists have proposed a close relationship between V. japonicum and V. utile [10], while others have suggested a sister relationship with V. erosum [11,12,31]. However, the inconsistencies and limitations in these research conclusions can be attributed to insufficient sampling of species within the Viburnum genus. To clarify the phylogenetic relationships of V. japonicum with other Viburnum species, this study constructed a phylogenetic tree using published chloroplast genome data of 15 Viburnum species, with three species serving as outgroups. Based on the support values of the phylogenetic tree, our study divided the Viburnum genus into two clades. V. japonicum was placed in Clade A and exhibited a relatively close evolutionary relationship with V. setigerum, V. erosum, and V. fordiae. The results above indicate that despite a complete cp genome, species in young evolutionary lineages may still be indistinguishable. Therefore, the conclusions of the phylogenetic analysis should take into account specific characteristics of the nuclear genome.

5. Conclusions

In this study, we sequenced, characterized, and analyzed the second complete chloroplast genome sequence of V. japonicum. In conjunction with the published chloroplast genome sequences of other Viburnum species, we conducted comparative genomic analysis and constructed a phylogenetic tree to elucidate the systematic relationships between V. japonicum and other species within the genus. This study aimed to provide more comprehensive and in-depth information. It establishes a foundation for species identification in the genus Viburnum and may also contribute to the development of new conservation and management strategies to support efforts in species conservation.