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. 2022 Dec 24;8(12):e12557. doi: 10.1016/j.heliyon.2022.e12557

Comparative phylogenetic analysis of oolong tea (Phoenix Dancong tea) using complete chloroplast genome sequences

Yaqun Liu 1,1, Liyun Lin 1,1, Dongjuan Yang 1, Xianghui Zou 1, Zhenxia Zhang 1, Mouquan Liu 1, Min Lin 1,∗, Yuzhong Zheng 1,∗∗
PMCID: PMC9834756  PMID: 36643327

Abstract

Phoenix Dancong tea, a variety of oolong tea, is produced in Chaozhou, Guangdong Province, China, and is characterized by numerous hybridizations and polyploidization. To assess the genetic diversity and phylogenetic relationships among Phoenix Dancong tea and other oolong teas, an integrated circular chloroplast genome was constructed for thirty species of Phoenix Dancong tea from Chaozhou. The genome of Phoenix dancong tea is a circular molecule of 157,041–157,137 bp, with a pair of inverted repeats (26,072-26,610 bp each) separated by a large single copy (86,615-86,658 bp) and small single copy (18,264-18,284 bp). A total of 135 unique genes were encoded, including 90 protein coding genes, 37 tRNAs and 8 rRNAs. A comparative analysis with the other seven species in the oolong tea family that have been sequenced to date revealed similarities in structural organization, gene content and arrangement. Repeated sequence analysis identified 17–23 tandem repeats, 20–24 forward repeats and 25–27 palindromic repeats. Additionally, a total of 65–70 simple sequence repeats were detected, with mononucleotide repeats being the most common. Phylogenetic analyses showed that Phoenix Dancong tea and Fujian oolong tea were clustered with other cultivated Camellia sinensis in the genus Camellia of the family Theaceae, while the two oolong tea species were relatively independently cross-embedded in the genus, Camellia. Close genetic relationships were observed between Phoenix Dancong tea and other oolong teas, and the overall chloroplast genomes of oolong tea showed patterns with low variations and conserved evolution. The availability of Phoenix Dancong tea chloroplast genomes not only elucidated the relationship among oolong teas from different origins in Guangdong and Fujian but also provided valuable genetic resources to assist further molecular studies on the taxonomic and phylogenomic resolution of the genus Camellia.

Keywords: Phoenix dancong tea, Oolong tea, Chloroplast genome, Genomic structure, Repeat analysis, Phylogeny

Phoenix Dancong tea; Oolong tea; Chloroplast genome; Genomic structure; Repeat analysis; Phylogeny.

1. Introduction

Oolong tea, a semioxidized tea famous for its elegant fruity and floral aroma, is one of the most important traditional economic beverages and is distributed widely in southern China. It is well known for its health benefits, which can regulate intestinal microbiota and increase digestion, fat transformation and also has other benefits [1]. Traditional oolong tea originated from Beiyuan tribute tea in Fujian Province during the Song Dynasty and has a history of more than 1000 years and is mainly distributed in Fujian, Guangdong and Taiwan [2]. Phoenix dancong (P. Dancong) tea is an important oolong tea variety with a long history and reputation lasting more than 900 years in Chaozhou, Guangdong Province. It is one of the most tasty and fragrant tea varieties in China. P. Dancong tea is an excellent single plant selected from local Phoenix Narcissus tea plant varieties through long-term artificial cultivation, each with its own distinctive form and taste [3]. Strikingly, relatively little is known about the similarities and differences of the cultivars from different geographical origins. Most oolong tea processing procedures are performed in the same way, resulting in similar appearance and flavor characteristics that are difficult to distinguish from each other. In addition, the phylogenetic relationships and interspecific boundaries are difficult to determine due to similar species origins, frequent hybridization and polyploidy during the cultivation process [4]. Traditional morphological and biological classification methods based on limited feature selection, such as novel artificial sensing tools [5] and headspace solid-phase microextraction/gas chromatography‒mass spectrometry (HS-SPME/GC‒MS) [6], are inherently dynamic and unreliable owing to genetic inheritance and other biological factors. Currently, there is a lack of research on the genetic background and resources of oolong tea. The lack of appropriate sequences and polymorphic genetic markers for phylogenetic analysis has long hindered understanding its phylogeny and increased the controversy over the genetic classification.

Chloroplast genomes are extensively used in plant phylogeny, molecular evolution and population genetics studies [7]. Chloroplasts are one of the most important semiautonomous organelles in green plants. In addition to serving as sites of photosynthesis, chloroplasts also perform carbohydrate biosynthesis and other functions [8]. The chloroplast genome is relatively independent from the nuclear genome and is more conserved in its structure and organization [9]. In general, the chloroplast genome is a circular DNA molecule consisting of 120–160 kb, which encodes 120–130 protein coding genes (CDS), ribosomal RNA (rRNA) and transfer RNA (tRNA) associated with photosynthesis or other gene expressions [10]. It has a typical quadripartite structure comprising four major segments: a large single copy region (LSC, 80–90 kb), a small single copy region (SSC, 16–27 kb) and two inverted repeat regions (IRs, IRa and IRb, 20–28 kb) [11]. The chloroplast genomes of several oolong tea species have recently been published in GenBank (http://www.ncbi.nlm.nih.gov/genbank/), including Baijiguan (Accession number: MT773373), Bantianyao (Accession number: MW046255), Dahongpao (Accession number: MT773374), Rougui (Accession number: MT773375), Shuijingui (Accession number: MT773376), Tieluohan (Accession number: MT773377), and Wuyi Narcissus (Accession number: MT612435), all of which belong to Fujian oolong tea. However, most of these studies have focused on the genome compositions and phylogenetic relationships of other tea species. As a globally famous beverage crop, oolong tea has been studied in great depth in terms of its components and efficacy, but its genetic and evolutionary history deserves further exploration [2]. Unfortunately, to date, studies on the chloroplast genome of oolong tea have only been reported for those varieties cultivated in Fujian Province. Although they demonstrated the power of complete chloroplast genomes, their related species were not included. The genetic relationships among oolong teas from different origins have not been fully elucidated.

To investigate the relationship between the structural characteristics and phylogeny of the chloroplast genome in oolong teas, another important representative of oolong tea cultivated in Guangdong Province (also known as P. Dancong tea) was selected for this study. Here, we selected thirty excellent, representative species of P. Dancong tea and performed sequencing, assembly and characterization of the chloroplast genomes. Their features and structures were compared with those of Fujian oolong tea to infer the phylogenetic relationships and evolutionary mechanisms arising from the regional and varietal differences in oolong teas. Furthermore, these data will provide sufficient genomic resources for further research and application of tea and contribute to its complex evolutionary history and uncertain phylogeny.

2. Materials and methods

2.1. Materials and DNA extraction

Fresh young leaves of 30 P. Dancong tea varieties, namely, Youhuaxiang, Dongfanghong, Laoxianweng, Zongsuoxie, Zhilanxiang, Songzhong, Huangjinye, Jiangmuxiang, Baxian, Potou, Jiangxiang, Zhuye, Rouguixiang, Xiongdizai, Jilongkan, Juduozai, Dawuye, Leikouchai, Nanjiangxiang, Chengmen, Tuofuhou, Wuye, Lanrenxiang, Guihuaxiang, Milanxiang, Yashixiang, Yehuadancong, Shuixian, Hongyin and Baiyin, were used in this study and were collected from Chaozhou City, Guangdong Province, China. All P. Dancong teas used in the present study were screened from the Phoenix narcissus variety by generations of tea farmers. The research samples were identified as different varieties of P. Dancong tea by Dr. Shoujun Guo and Dongjuan Yang from the School of Life Science and Food Engineering, Hanshan Normal University. The other samples outside the experiment were stored in the herbarium of Hanshan Normal University. Total genomic DNA was extracted from young leaves using the cetyltrimethylammonium bromide (CTAB) method [12]. The extracted DNA quality was assessed by 1.0% (wt/v) agarose gel electrophoresis and a NanoDrop spectrophotometer (Thermo Scientific, USA).

2.2. Illumina sequencing, assembly, and annotation

Purified DNA was used to generate an insert size of 300 bp using a TruSeq DNA Sample Prep Kit (Vanzyme, China), and next-generation sequencing was conducted on an Illumina HiSeq 2500 sequencer (Illumina, USA) according to the standard Illumina library preparation procedure. Approximately 8,205,826 (Wuye) to 22,120,726 (Juduozai) raw reads were obtained with paired-end (PE) 150-bp length reads. To guarantee better results for the following mapping and assembly, the raw reads were evaluated using FastQC (version 0.11.8, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed using Fastp (version 0.19.5, https://github.com/OpenGene/fastp) to filter low-quality reads and remove adapters with a qualified quality phred cutoff of 20, unqualified percent limit of 20 and required length of 60 [13]. The clean high-quality reads were assembled using MetaSPAdes (version v3.13.0) with default parameters [14]. Here, the published chloroplast genome of Rougui (Accession number: MT773375) was selected as the reference genome. The P. Dancong tea chloroplast genome was annotated using CpGAVAS2 (http://47.96.249.172:16019/analyzer/home) [15]. The annotated circular chloroplast genome of P. Dancong tea was mapped and visualized using Chloroplot online software (https://irscope.shinyapps.io/chloroplot/) [16]. The codon usage, relative synonymous codon usage (RSCU) values and GC contents were calculated by MEGA7 (http://www.megasoftware.net) [17].

2.3. Genome comparison

The chloroplast genomes of Fujian oolong tea varieties (namely, Baijiguan, Bantianyao, Dahongpao, Rougui, Shuijingui, Tieluohan and Wuyi Narcissus) were used for comparisons with the P. Dancong tea genomes obtained in this study, which were downloaded from the GenBank database. Multiple alignments of these 37 oolong tea varieties were carried out by MAFFT Version 7 (https://mafft.cbrc.jp/alignment/software/) using the auto mode option and were adjusted manually where necessary [18]. Sequential comparisons of chloroplast genomes with sequence annotation information were visualized using the mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) program in Shuffle-LAGAN mode [19]. Boundaries between the IR and SC regions and gene order rearrangements of the oolong tea species were compared using the online program, IRscope (https://irscope.shinyapps.io/irapp/) [20]. Nucleotide diversity (Pi) was calculated by sliding window analysis in DnaSP v5.0 with a step size of 200 bp and sliding window of 600 bp [21].

2.4. Analysis of long repeats and single sequence repeats

REPuter software (https://bibiserv.cebitec.uni-bielefeld.de/reputer) [22] was used to identify the sizes and locations of forward and palindrome repeats with a Hamming distance of 3, minimal repeat size of 30 bp and maximum computed repeats of 90 bp. Tandem Repeats Finder software (https://tandem.bu.edu/trf/trf.html) [23] was used to identify tandem repeats with default parameters. Simple sequence repeats (SSRs) were detected using MIcroSAtellite software (MISA, https://webblast.ipk-gatersleben.de/misa/) [24] with thresholds of ten repeat units for mononucleotide SSRs, five repeat units for dinucleotide SSRs, four repeat units for trinucleotide SSRs, and three repeat units for tetra-, penta-, and hexanucleotide SSRs. The long repetitive sequences and single sequence repetitive sequences in oolong tea were clustered and analyzed. Samples containing the gene regions were labeled "Gene", and those without were labeled "No Gene", and these datasets were analyzed by hierarchical clustering using correlation as a similarity measure. A clustering graph was generated using the OmicStudio tools (https://www.omicstudio.cn/) [25].

2.5. Phylogenetic analysis

The chloroplast genome sequences of 37 oolong tea varieties, together with those of 69 Theaceae species, were aligned using the MAFFT program version 7. Chloroplast genome sequences were obtained from the GenBank database (Supplementary Table S1). Gaps in alignments were stripped. Phylogenetic trees were constructed using maximum likelihood (ML) methods implemented using RAxML version 8 under the general time reversible (GTR) model of nucleotide substitution and the gamma (Γ) distribution of rate heterogeneity [26, 27]. Coffea arabica (Accession number: NC_008535) and Arabidopsis thaliana (Accession number: NC_000932) were used as outgroups [28, 29, 30]. The phylogenetic trees were visualized with the Interactive Tree of Life v6 (iTOL, https://itol.embl.de/) [31].

3. Results

3.1. Chloroplast genome features of P. Dancong tea

The complete chloroplast genomes of the thirty P. Dancong teas ranged from 157,041 bp (Potou) to 157,137 bp (Zhuye), with differences less than 96 bp. All of these exhibited typical quadripartite structures, including large single copy regions of 86,615-86,658 bp and small single copy regions of 18,264-18,284 bp, which were separated by two equal length inverted repeat regions of 26,072-26,610 bp (Figure 1). The average overall guanine-cytosine (GC) content of these chloroplast genomes was 37.30%, while the GC contents of each region were different. The GC content of the IRs was 43.00%, which was higher than that of LSCs (35.30%) and SSCs (30.50%) (Supplementary Table S2). In the statistical analysis, the comparative differences among the sequences of the 30 species were not obvious, and both the lengths and gene contents of each region were similar.

Figure 1.

Figure 1

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Complete chloroplast genome map of P. Dancong tea. The genes shown in the outer circle are transcribed clockwise, and those in the inner circle are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray colors in the inner circle indicate the GC content of the chloroplast genome. The red box indicates the missing gene, orf42.

Each chloroplast genome was found to harbor a total of 135 genes, of which 109 were unique in LSC and SSC, and 26 were duplicated in the IR regions, including 90 protein coding genes, 37 transfer RNA genes and 8 ribosomal RNA genes. These genes belong to several categories with different functions and can be divided into self-replication (tRNA, rRNA, ribosome subunits and DNA-dependent RNA polymerase); photosynthesis (NADH oxidoreductase, photosystem subunits, rubisco, cytochrome and ATP synthase); biosynthesis (maturase, envelope membrane protein, acetyl-CoA subunits, translational initiation factor and protease); and conserved open reading frames. We identified 63 protein coding genes and 22 tRNA genes located in LSCs, 16 protein coding genes, 14 tRNA genes and 8 rRNA genes located in IRs, and 11 protein coding genes and one tRNA gene located in SSCs. Among them, three pairs of adjacent genes, psbD/psbC, atpE/atpB and rps3/rpl22, were observed in LSCs, with overlapping regions of 52 bp, 3 bp and 15 bp, respectively. It should be noted that the two open reading frames ORF42 in IRs were absent in P. Dancong tea (Figure 1).

A total of 21 genes (13 protein coding genes and 8 tRNAs) contained introns, all of which were single intron genes except for ycf3 and clpP (with two introns). Although intron-containing genes are very conserved, it can be clearly seen that the differences in total gene lengths depend mainly on the differences in intron lengths, which hardly vary across genera. The intron lengths and related gene lengths in IRs were consistent with those in LSC and SSC regions (Figure 2). In addition, the 5′-end and 3′-end exons of the rps12 gene are located in LSC and IR regions, respectively. matK, the gene encoding maturase, is located within the largest intron of trnK-UUU.

Figure 2.

Figure 2

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Statistical analysis of intron-containing genes of P. Dancong tea. (A) Lengths and numbers of introns. (B) Lengths and numbers of total intron-containing genes.

There are 26796 codons (including stop codons) in the chloroplast genome of P. Dancong tea, and 20 amino acids are translated. The most prevalent amino acid is leucine (L, 10.50%), followed by isoleucine (I, 8.70%) and serine (S, 7.90%), while the rarest amino acid is cysteine (C, 1.10%) among the proteins encoded by chloroplast genes. Subsequently, slight differences were found in the RSCU values in the P. Dancong tea chloroplast genome. It is well known that codon usage preferences are related to RSCU values [32]. RSCU values greater than 1 were found for 29 codons, of which 28 were A/T-ending codons (except TTG), suggesting that they are biased codons. Thirty codons had RSCU values less than 1, of which 28 were C/G-ending codons (except CTA and ATA). Usage of the start codons, AUG and UGG, with the latter encoding tryptophan, has no bias (RSCU = 1). CGC had the lowest RSCU value (0.335), AGA had the highest RSCU value (1.886), and both encode arginine (Figure 3). To conclude, CGC was negatively biased, and AGA was positively biased.

Figure 3.

Figure 3

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Heatmap of RSCU values and corresponding amino acid analysis of P. Dancong tea.

3.2. Comparison of chloroplast genome sequence variations in Fujian oolong tea

3.2.1. Structural variations in the chloroplast genome of oolong tea

To facilitate the subsequent phylogenetic analyses and smooth plant identification, 7 published Fujian oolong teas were selected for comparison with the 30 P. Dancong teas to estimate the sequence similarities of teas from different regions. The overall sequence identities of the 37 oolong tea chloroplast genomes were relatively conserved and similar, including gene order and number. They were more conserved in the IR and SSC regions than in the LSC region and varied more in the noncoding regions than in the coding regions (Figure 4). Furthermore, the nucleotide diversity, Pi, was calculated to determine the sequence divergence levels among the oolong tea chloroplast genomes. All aligned sequences demonstrated surprisingly low divergences, with all regions displaying low variations (Pi < 0.006). The Pi values ranged from 0 to 0.003 with an average of 0.000132, and these small differences were not statistically significant (data not shown). Both results indicate high sequence similarity across the chloroplast genomes, suggesting that the chloroplast genome of oolong tea was highly conserved in gene order and structure.

Figure 4.

Figure 4

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Visualization alignment of oolong tea chloroplast genome sequences. The mVISTA-based identity plots show the sequence identities with Wuyi Narcissus as a reference. Only the variant alignments are shown because of the large number of samples. The x-axis represents the coordinates in the chloroplast genome. The y-axis indicates the average percentage identity of sequence similarity in the aligned regions, ranging between 50% and 100%. Genome regions are color-coded as protein coding, rRNA coding, tRNA coding or conserved noncoding sequences (CNS).

3.2.2. Contraction and expansion of inverted repeats

The IR region (size from 24,399 bp to 26,110 bp) of 37 oolong tea chloroplast genomes was highly conserved, but structural variations remained in the IR/SC boundary regions. To further observe potential contraction and expansion of the IR regions, we compared the gene variants in the IR/SSC and IR/LSC boundary regions. rps19-rpl2-trnH and ycf1-ndhF were located in the JLB and JLA regions and JSB and JSA regions, respectively. The rps19 gene crossed the LSC/IRb region with 46 bp located at the IRb region. Two copies of the ycf1 gene crossed JSB and JSA with nearly 1 bp in the SSC region and 4553/1069 bp in the IRa region. The trnH gene in the LSC region was contracted by 1 bp from the junctional region of the IRa-LSC boundary (Fig. S1). Overall, the contraction/expansion events of the IR region detected in these chloroplast genomes were extremely stable.

3.2.3. Long repeat structure analysis

Three categories consisting of long repeats, tandem repeats (TR), forward repeats (FR) and palindromic repeats (PR), were detected in oolong tea chloroplast genomes. The numbers of the three repeated types were consistent as follows: TRs ranged from 17 (Nanjiangxiang, Chengmen, and Guihuaxiang) to 23 (Wuyi Narcissus); FRs ranged from 20 (Youhuaxiang and Dongfanghong) to 24 (Shuixian and Tieluohan); and PRs ranged from 25 (Zongsuoxie, Shuixian, and Tieluohan) to 27 (Zhilanxiang, Jiangmuxiang, and Baxian). The TR lengths were 14–25 bp, and the lengths of the other two categories were 18–56 bp (Figure 5ABC). These repeat loci were distributed in the TR sites, accounting for 17.06% of the total repeats (one site was counted), 38.26% in the FR and 44.78% in the PR sites (two sites were counted) (Figure 5D). The IR regions had the greatest number repeat sites (53.20%), followed by LSC (34.04%) and SSC (12.76%). The maximum number of repeat sites was located in intergenic spacer (IGS) regions (42.73%), and a minority were located in introns (35.88%), which were petD and ndhA. Only a few types of genes (e.g., petG, rpoB, clpP, rps19, rpl2, ycf1, and ycf2) possessed repetitive elements, with the ycf2 gene containing the highest number of repeat sites (27.71%) (Figure 5EFG).

Figure 5.

Figure 5

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Analysis of long repeated sequences in oolong tea chloroplast genomes. (A) Numbers and lengths of TRs. (B) Numbers and lengths of FRs. (C) Numbers and lengths of PRs. (D) Total lengths of the three repeat types. (E) Identification, distribution and clustering of TR loci. (F) Identification, distribution and clustering of FR loci. (G) Identification, distribution and clustering of PR loci. (H) Summary of unique and shared TR loci. (I) Summary of unique and shared FR loci. (J) Summary of unique and shared PR loci. The plot on the left shows the total number of related genes, and the plot on the right shows the number of unique repeat loci, followed by the intersections/overlaps (connected dots) between branching pairs.

Oolong tea chloroplast genomes have a total of 32 identical long repeat sequences (9 TR, 10 FR and 13 PR). Based on the quadripartite structure and gene regions, the results also showed that the divergent regions of chloroplast genomes were associated with various repeat sequences. The distribution of TR was rather conserved, except for Wuyi Narcissus, Shuijingui and Zhilanxiang, which were clustered into three classes due to rrn23/trnA-UGC and ycf15/rps7. While both FR and PR clustered into 16 classes, only trnQ-UUG/psbK, trnS-GCU/trnG-GCC, trnG-GCC, atpH/atpI, psbM/trnD-GUC, trnG-UCC, trnP-UGG, ycf2, ycf1 and ndhA (itron) were identical in FR, and the other repeat sites were different. The diversity of PR sites is mainly caused by atpA/atpF, trnA-UGC/rrn23 and ndhG/ndhI (Figure 5). The specific data are shown in Supplementary Table S3.

3.2.4. Simple sequence repeat analysis

Simple sequence repeats (SSRs) are tandem iterations of single nucleotides or short oligonucleotides with sizes of 1–6 bp [33]. We found that 65–70 SSRs were distributed in the oolong tea chloroplast genome, with sizes ranging from 10 to 20 bp. The incidence of SSRs was nonuniform to the region size, accounting for 66.45%, 14.94% and 18.61% in LSC, SSC, and IRs, respectively. The SSRs were located in CDSs with an average of 18, IGSs with an average of 49 and introns with an average of 9 (Figure 6A).

Figure 6.

Figure 6

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Comparison of SSR distributions in oolong tea chloroplast genomes. (A) Numbers and lengths of different SSR types. (B) Identification, distribution and clustering of SSR loci. (C) Numbers of mono-, di-, tri-, tetra- and hexa-nucleotides. (D) Summary of unique and shared SSR loci. The plot on the left shows the total numbers of related genes, and the plot on the right shows the numbers of unique repeat loci, followed by intersections/overlaps (connected dots) between branching pairs.

Five kinds of SSRs were found in the chloroplast genome of oolong tea, with the number of mononucleotide repeats ranging from 0 to 20 (79.63% in LSC, 7.41% in SSC and 12.96% in IRs), followed by dinucleotide repeats and trinucleotide repeats at 4 and 1, respectively, tetranucleotide repeats ranging in number from 8 to 10 and 2 hexanucleotide repeats. However, no pentanucleotide repeats were found. The longest repeat was an 18 bp stretch of the (AAAAAG/CTTTTT)3 hexanucleotide. It is noteworthy that all of the mononucleotide and dinucleotide repeats consisted of A/T. Among the trinucleotide and tetranucleotide repeats, AT and GAA occurred most frequently (Figure 6C). The distributions and clustering of SSRs of Fujian oolong tea and Chaozhou P. Dancong tea were more significant. There were a total of 40 identical loci for SSRs, and their distribution was rather conserved in the oolong tea chloroplast genome (Figure 6D). These unique repeats were mainly found in ndhK/ndhC, psaI/ycf4, trnP-UGG/psaJ (IGS), clpP, rps8 (CDS) and ndhA (itron), which are located in the LSC and SSC regions (Figure 6B). The specific data are shown in Supplementary Table S4.

3.3. Phylogenomic analysis

Chloroplast genome sequences are valuable genomic resources for elucidating evolutionary histories and have been widely applied in phylogenetic studies [25]. To determine the phylogenetic position of oolong tea, 76 complete chloroplast genome sequences of Theaceae species were obtained from the GenBank database (Supplementary Table S1). A ML phylogenetic tree was constructed based on these chloroplast genome data using Coffea arabica and Arabidopsis thaliana as outgroups. The phylogenetic analysis indicated that P. Dancong tea and Fujian oolong tea clustered together with other cultivated Camellia sinensis in the genus Camellia of the family Theaceae. Apparently, the two oolong tea species clustered in the same clade and were embedded in the family, Camellia, which corroborated the close relationship between these two families (Figure 7A). We found that five Fujian oolong tea species formed a monophyletic clade along with P. Dancong tea. Wuyi Narcissus and Shuijingui are closely related and relatively independent from the others. Both Baijiguan-Bantianyao and Dahongpao-Rougui reside on a separate, highly supported branch within the Tieluohan clade. The results also revealed that the P. Dancong teas, Hongyin and Baiyin, are sisters to Fujian oolong teas, Bantianyao and Rougui, respectively. In addition, we note that Leikouchai-Baxian, Zhuye-Potou, Dongfanghong-Youhuaxiang, Dawuye-Tuofuhou, Lanrenxiang-Jilongkan, Nanjiangxiang-Guihuaxiang, Juduozai-Wuye, Huangjinye-Milanxiang and Rouguixiang-Laoxianweng are highly conserved (Figure 7B).

Figure 7.

Figure 7

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Phylogenetic tree constructed based on the complete chloroplast genomes of P. Dancong teas, oolong teas and other Theaceae species. (A) Phylogenetic tree of oolong tea in the family Theaceae. Circles from outside to inside: outermost circle-dark blue: Theaceae family; second circle-light red: Camellia family; third circle-light green: Camellia sinensis; innermost circle-purple: oolong tea, -green: other Camellia, -yellow: other Theaceae, -red: outgroup. (B) Phylogenetic tree based on the complete chloroplast genomes of P. Dancong teas and Fujian oolong teas. Circles from outside to inside: outermost circle-light green: P. Dancong tea that is on the same branch as Tieluohan; second circle-purple: P. Dancong tea that is on the same branch as Baijiguan-Bantianyao and Dahongpao-Rougui; third circle-dark green: P. Dancong tea that is on the same branch as Bantianyao and Rougui; innermost circle-pink: P. Dancong tea Leikouchai-Baxian, Zhuye-Potou, Dongfanghong-Youhuaxiang, Dawuye-Tuofuhou, Lanrenxiang-Jilongkan, Nanjiangxiang-Guihuaxiang, Juduozai-Wuye, Huangjinye-Milanxiang and Rouguixiang-Laoxianweng are on the same branch.

4. Discussion

Oolong tea, a unique kind of tea in southern China, has long been classified and identified according to its phenotypic characteristics [2]. In recent years, with the development of high-throughput sequencing technology and efficient large-scale data analysis, an increasing number of studies on plant evolutionary relationships have been carried out. Chloroplasts are specialized photosynthetic organelles in plants that are highly conserved and maintain relatively small copy numbers, making them easy to sequence for further evolutionary studies [7].

In this study, we sequenced the complete chloroplast genome of P. Dancong tea by using Illumina high-throughput sequencing technology. The genomes ranged from 157,041 to 157,137 bp, which is consistent with the known sizes of other Camellia species, such as C. japonica (157,001 bp, Accession number: MW602996.1) [34] and C. rostrata (156,547 bp, Accession number: MW755303.1) [35]. Its quadripartite organization consists of two copies of IRs separating LSC and SSC, which are almost identical to previously reported angiosperm chloroplast genomes [11]. The sizes of the LSC, SSC and IR regions of P. Dancong tea were also well within the size range of other sequenced Camellia species [26]. The structures and sizes of intraspecific and interspecific genomes are extremely conserved, which may be related to the slow molecular evolution of plant chloroplasts [36]. ORF42 was absent in the IRs of P. Dancong tea, which could be associated with sequencing/assembly errors or the occurrence of gene loss. However, ORF42 was absent in IRa and IRb in all 30 P. Dancong teas. Loss of ORF42 has been reported in another other cultivated tea, Camellia sinensis var. assamica cv. Yunkang 10 [29]. It is speculated that the gene was lost in P. Dancong tea with a high probability.

The overall GC contents of the chloroplast genomes ranged from 37.29% to 37.31%, with the IR region being the highest (43.00%), followed by LSC (35.30%) and SSC (30.50%), which may be due to the presence of rRNAs (e.g., rrna4.5, rrna5, rrna23 and rrna16), which are GC-rich in the IR region. It should be noted that although the chloroplast genomes of most angiosperms generally encode 74 CDSs [37], P. Dancong tea, like other teas, harbors 90 CDSs (including accD, ycf1, ycf2, rpl23 and infA), and other plants also have the same numbers, such as Rosa filipes [38] and Cerasus humilis [39]. Relative synonymous codon usage analysis showed that the starting codons of rps19 and ndhD had been replaced by GTG and ended with TAA. A similar situation occurs for the chloroplast genomes of other plants, such as Neotropical Bulbophyllum [40] and Nelumbo nucifera [41]. Leucine and isoleucine are the most common essential amino acids of plants and are used with the highest frequency in P. Dancong tea [42]. Furthermore, almost all of the A/T-ending codons showed RSCU values greater than 1, whereas the C/G-ending codons showed RSCU values less than 1, indicating that A/T ending codons are used more frequently.

The gene content and order of arrangement of the chloroplast genome of P. Dancong tea are basically the same as those of Fujian oolong tea and other unorganized Camellia [26]. Therefore, the exact IR boundary locations and adjacent genes of the oolong tea chloroplast genomes from two different origins were compared in this study. Expansion and contraction of the IR region are critical for the size variations of the chloroplast genome and play an important role in its stability and evolution [43, 44]. The IR region of oolong tea was not only highly conserved in terms of length, but the locations and sizes of the boundary genes, rps19, ycf1 and trnH were also relatively stable. Although the positions of these boundary genes are slightly different from those of other plants, the gene types are almost the same, which also exists in Stemona sessilifolia [45], Myristicaceae [46], and Brassicaceae [47]. Accordingly, we infer that there should be no simple phylogenetic signals at the IR boundary.

It is obvious that the chloroplast genome of oolong tea contains repeats other than IRs, which can be of great help in further research on genome rearrangements and phylogenetic analyses. There were three long repeats (TR, FR and PR) in the chloroplast genome of oolong tea, of which the number in PR were the largest and were mainly distributed in the IGS. These corresponding regions are potential hotspots of gene reconstruction and can provide information sources for genetic markers. The genes, ycf1 and ycf2, appeared simultaneously in three long repeats in all samples, suggesting that these long repeats may be shared among oolong teas. Although a large number of loci with long repeated sequences have been discovered, as in other plants, the origin mechanism and function remain unclear [26].

Five types of SSRs (e.g., mononucleotides, dinucleotides, trinucleotides, tetranucleotides and hexanucleotides) were detected, ranging in number from 65 to 70 and in size from 10 bp to 20 bp. They are mostly located in noncoding regions, especially in intergenic spacer regions, which usually consist of A/T. The SSR loci identified in this study may be useful in population and evolutionary research. Many of these loci, such as rps16/trnQ-UUG, psbK/psbI, atpH/atpI, rps2/rpoC2, and atpB/rbcL, have been applied in research on the interspecific phylogenetic relationships and intraspecific genetic variations of Oryza [48], Bamboo [49], Brassica [50], and Impatiens [51]. Introns of trnK-UUU, rps16 and rpl16 were also the focus of chloroplast gene sequence variation analyses [52]. In addition, there is a rare SSR locus, rps8/rpl14, in the chloroplast genome of oolong tea, which may contribute to its biochemical genetic identification and population genetic studies. Little research has been reported on the development and application of SSR molecular markers for the chloroplast genome associated with oolong tea. Based on the available data, we can develop SSR molecular markers to obtain a more comprehensive understanding of the evolutionary history and genetic polymorphisms of oolong tea, which is the direction of our future research.

The Theaceae family is taxonomically and phylogenetically challenging due to the similarity of its morphological characteristics. A comparative analysis of the chloroplast genome of P. Dancong tea with the previously sequenced family, Theaceae, revealed the characteristics of chloroplasts in terms of gene contents and structural organizations. The complete chloroplast genomes of P. Dancong tea and other tea species all displayed conservative quadripartite circular structures and stable structural arrangements. The chloroplast genomes are accompanied by sequence repeats and indels among different tea species, which are mainly reflected in the variations in lengths and orientations of the IR region. This result coincided with the previously proposed view that sequence variations were the most important evolutionary dynamic contributing to the diversification of the tea chloroplast genome [53]. Molecular phylogenetic analysis showed that P. Dancong tea and Fujian oolong tea were derived from Camellia sinensis, a genus Camellia of the family Theaceae, and formed a closely intersecting branch. This suggested that the chloroplast genome of P. Dancong tea is most similar to that of Fujian oolong tea, which may be caused by closely homologous relatives sharing their genome contents. From the perspective of oolong tea, Fujian oolong teas, Wuyi Narcissus, Shuijingui and Tieluohan, are ancient members of an early differentiation, among which Tieluohan forms a sister relationship with P. Dancong tea. Baijiguan, Bantianyao and Rougui are closer to Shuixian, Hongyin and Baiyin, respectively. On this basis, it is considered that a single chloroplast phylogenetic lineage has developed over a long period of evolution. This phenomenon also explains the fact that the chloroplast genome is more conserved and evolves much more slowly than the nuclear genome, which is more susceptible to environmental influences. Therefore, molecular data from the nuclear genome and population-level genetic analyses are necessary to thoroughly explore the evolutionary mechanisms. Our results suggested the phylogenetic relationships of some P. Dancong tea species according to the available chloroplast genomes, but more comprehensive genome sequences are required to resolve the overall phylogeny of this species. In future research, it is necessary to collect more species and use more molecular data to verify the interspecific relationships and interspecific differentiation of P. Dancong teas.

5. Conclusions

In this work, we present for the first time the complete chloroplast genome of P. Dancong tea using Illumina sequencing technology. Its size and structure were fully characterized and compared with the chloroplast genomes of related species. A comparative analysis indicated that the chloroplast genomes of P. Dancong teas and Fujian oolong teas exhibited similar gene contents and organizations. By analyzing the chloroplast genome characteristics, alignments, codon usage biases, repeated sequences and phylogenies, the hypothesis that the chloroplast genome of oolong tea has a low variation rate and conservative evolutionary history was proposed, which means that the known data can be used as a reference for annotating the chloroplast genomes of other oolong tea species. Furthermore, P. Dancong tea and Fujian oolong tea clustered together in a monophyletic group, suggesting a clear family relationship. These genomic information and analysis results will provide a theoretical basis for subsequent research on the phylogeny, taxonomy and biogeography of oolong tea.

Declarations

Author contribution statement

Yaqun Liu: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Liyun Lin; Dongjuan Yang; Xianghui Zou: Performed the experiments; Contributed reagents, materials, analysis tools or data.

Zhenxia Zhang; Mouquan Liu: Contributed reagents, materials, analysis tools or data.

Min Lin; Yuzhong Zheng: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

Yuzhong Zheng was supported by Guangdong Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products [No. 2021B1212040015].

PhD Yaqun Liu was supported by Doctor Initiating Project of the Hanshan Normal University [No. QD202125].

Min Lin was supported by Doctor Initiating Project of the Hanshan Normal University [No. QD20190527], Guangxi Provincial Natural Science Foundation [No. 2019JJD140052 & No. 2020JJA140656], Education Department Project of Guangdong Province [No. 2019-GDXK-0032 & No. 2020KZDZX1146], Eastern Guangdong Technological Engineering Research Center [No. P19004].

Data availability statement

Data associated with this study has been deposited at GenBank (Accession number: MZ379786 for Youhuaxiang; OL690346-OL690374 for Dongfanghong, Laoxianweng, Zongsuoxie, Zhilanxiang, Songzhong, Huangjinye, Jiangmuxiang, Baxian, Potou, Jiangxiang, Zhuye, Rouguixiang, Xiongdizai, Jilongkan, Juduozai, Dawuye, Leikouchai, Nanjiangxiang, Chengmen, Tuofuhou, Wuye, Lanrenxiang, Guihuaxiang, Milanxiang, Yashixiang, Yehuadancong, Shuixian, Hongyin and Baiyin).

Declaration of interest's statement

The authors declare no competing interests.

Additional information

No additional information is available for this paper.

DNA sequences and GenBank accession numbers

All data generated or analyzed during this study are included in this published article. The chloroplast genome sequences of P. Dancong tea were submitted to GenBank (Accession number: MZ379786 for Youhuaxiang; OL690346-OL690374 for Dongfanghong, Laoxianweng, Zongsuoxie, Zhilanxiang, Songzhong, Huangjinye, Jiangmuxiang, Baxian, Potou, Jiangxiang, Zhuye, Rouguixiang, Xiongdizai, Jilongkan, Juduozai, Dawuye, Leikouchai, Nanjiangxiang, Chengmen, Tuofuhou, Wuye, Lanrenxiang, Guihuaxiang, Milanxiang, Yashixiang, Yehuadancong, Shuixian, Hongyin and Baiyin). The accession numbers corresponding to the additional datasets used and analyzed in this study can be found in the Supplementary Table 1.

Acknowledgements

We thank technician Youcai Li and other laboratory members at Genesky Biotechnology Inc. (Shanghai, China) for their technical help and collaboration. We also thank the Weiye Tea Plantation for providing the raw materials used in this research and for helpful discussions. We are especially grateful to our team members for their important ideas and active participation in this study.

Contributor Information

Min Lin, Email: konfutea@hotmail.com.

Yuzhong Zheng, Email: zhengyuzhong@gmail.com.

Appendix A. Supplementary data

The following are the supplementary data related to this article:

mmc1
mmc1.docx (825.1KB, docx)
mmc2
mmc2.docx (34.4KB, docx)
mmc3
mmc3.docx (25.4KB, docx)
mmc4
mmc4.docx (235KB, docx)
mmc5
mmc5.docx (165KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1
mmc1.docx (825.1KB, docx)
mmc2
mmc2.docx (34.4KB, docx)
mmc3
mmc3.docx (25.4KB, docx)
mmc4
mmc4.docx (235KB, docx)
mmc5
mmc5.docx (165KB, docx)

Data Availability Statement

Data associated with this study has been deposited at GenBank (Accession number: MZ379786 for Youhuaxiang; OL690346-OL690374 for Dongfanghong, Laoxianweng, Zongsuoxie, Zhilanxiang, Songzhong, Huangjinye, Jiangmuxiang, Baxian, Potou, Jiangxiang, Zhuye, Rouguixiang, Xiongdizai, Jilongkan, Juduozai, Dawuye, Leikouchai, Nanjiangxiang, Chengmen, Tuofuhou, Wuye, Lanrenxiang, Guihuaxiang, Milanxiang, Yashixiang, Yehuadancong, Shuixian, Hongyin and Baiyin).

Articles from Heliyon are provided here courtesy of Elsevier

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