Complete Chloroplast Genome Sequence of Camellia sinensis: Genome
Structure, Adaptive Evolution, and Phylogenetic Relationships

doi:10.21203/rs.3.rs-2749264/v1

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Research Article

Complete Chloroplast Genome Sequence of Camellia sinensis: Genome Structure, Adaptive Evolution, and Phylogenetic Relationships

https://doi.org/10.21203/rs.3.rs-2749264/v1

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published 28 Jun, 2023

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The chloroplast (cp) genome holds immense potential for a variety of applications including species identification, phylogenetic analysis, and evolutionary studies. In this study, we utilized Illumina NovaSeq 6000 to sequence the DNA of Camellia sinensis L. cultivar ‘Zhuyeqi’ , followed by the assembly of its chloroplast genome using SPAdes v3.10.1, with subsequent analysis of its features and phylogenetic placement. The results showed that the cp genome of Zhuyeqi was 157,072 bp, with a large single copy- region (LSC, 86,628 bp), a small single copy- region (SSC, 18,282 bp), and two inverted repeat regions (IR, 26,081 bp). The total AT and GC contents of the cp genome of Zhuyeqi were observed to be 62.21% and 37.29%, respectively. Thecp genome encoded 135 unique genes, including 90 protein-coding genes (CDS), 37 tRNA genes, and 8 rRNA genes. Moreover, 31 codons and 247 simple sequence repeats (SSRs) were identified. The cp genomes of Zhuyeqi was found to be relatively conserved, with particular conservation observed in the IR region, which showed no evidence of inversions or rearrangements.The five regions with the largest variations were identified, with four regions (rps12, rps19, rps16, and rpl33) located in the LSC region and one divergent region (trnI-GAU) in the IR region. Phylogenetic analysis revealed that Camellia sinensis(KJ996106.1) was closely related to Zhuyeqi, indicating a close phylogenetic relationship between these two species. These findings could provide important genetic information for further research into breeding of tea tree, phylogeny and evolution of Camellia sinensis.

Zhuyeqi

chloroplast genome

assembly

SSR loci

phylogeny

Chloroplast(cp) is an important organelle widely present in higher plants, which provides energy required for plant growth and development through photosynthesis. As a semi-autonomous organelle, chloroplast has a complete genetic information expression system. The cp genome of higher plants is highly conserved, with a typical quadripartite structure consisting of a large single copy- region (LSC), a small single- copy region (SSC), and two inverted repeat regions a (IRa) and b (IRb) [1]. Compared with the mitochondrialand nuclear genomes, the cp genome exhibits maternal inheritance and is more conserved in terms of gene type and structure than mitochondrial and nuclear genomes[2]. Therefore, the cp genome has become one of the important means for studying the phylogenetic status of plants [3–5]. With the rapid development of bioinformatics analysis and high-throughput sequencing technology, there are increasing studies using cp genome to investigate the evolution of plant species. Relevant studies on the characterization of chloroplast genomes of various plants, such as apple (Malus domestica) [6], mango (Mangifera indica) [7], strawberry (Fragaria × ananassa Duch) [8], blueberry (Vaccinium Spp) [9], grape (Vitis vinifera) [10], pepper (Capsicum annuum) [11], Cypripedium corrugatum Franch [12], Argyranthemum frutescens[13], and Viola philippica [14], provide important theoretical support for the study of genetic improvement, genomics, and phylogenetic status of higher plants.

The tea plant [Camellia sinensis (L.) O. Kuntze] is an important woody economic crop in China, and is highly heterozygous in its genetic composition as a perennial cross-pollinating crop, with a long conventional breeding cycle and low efficiency. Zhuyeqi is one of the asexually propagated varieties of tea, bred using single plant breeding methods from An-hua populations by the Tea Research Institute of Hunan Academy of Agricultural Sciences from 1957 to 1974. The leaves of Zhuyeqi are elliptical in shape, yellow-green in color with a glossy surface, relatively flat, tapering towards the tip, and with soft texture. The bud and leaves are yellow-green in color. Its leaves contain nutrients such as catechins, theaflavins, amino acids, caffeine, flavonoids, and other substances, with high nutritional and health value. While the chloroplast genome of crops such as tobacco(Nicotiana tabacum L.)[15], potato(Solanum tuberosum)[16], rice(Oryza sativa L.)[17], and wheat(Triticum aestivum L.)[18] have already been sequenced, research on the tea plant chloroplast genome began relatively late. Therefore, in this study, the chloroplast genome of Zhuyeqi was sequenced, assembled and annotated using Illumina NovaSeq 6000, and the chloroplast genome sequence was uploaded to NCBI database. Bioinformatics software was used to analyze the structural features, simple sequence repeat (SSR) sites, codon usage bias, IR boundaries, and phylogenetic relationships of the chloroplast genome of Zhuyeqi. This provides molecular basis and reference for tea variety identification, genetic breeding and phylogenetic research.

2.1 DNA Extraction and Genome Sequencing

Fresh young leaves of Camellia sinensis (L.) O. Kuntze variety‘Zhuyeqi’ were collected from Meng Er Chong Agricultural Technology Co., Ltd. in Hunan province, China(27°47′29.01″N ,110°51′50.86″E). The total genomic DNA of ‘Zhuyeqi’ was extracted using the TaKaRa DNA extraction kit (TIANGEN, D9194) according to the manufacturer's protocols. The purified DNA was then fragmented by mechanical disruption (sonication). Then, the paired-end (PE) library was constructed using VAHTS Multiplex Oligos set 4 for Illumina (Vazyme, Nanjing, China) and VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Nanjing, China), according to the manufacturer’s protocols. Finally, the qualified libraries were sequenced on the Illumina NovaSeq 6000 platform, according to the paired-end PE150 sequencing strategy. Approximately 6.4 Gb of raw data were obtained. All of the above works were carried out by Genepioneer Biotechnologies Co. Ltd. (Nanjing, China).

2.2 Genome structure analysis

The gene types and numbers of the annotated genes in the cp genome were exported to a CSV file using Geneious software. The LSC, SSC, and IR regions of each cp genome sequence were extracted using Geneious, and the full genome and each section were exported as .fas files. The AT and GC contents of the cp genome were calculated using BioEdit software [19].

2.3 Codon preference analysis

The CDS coding sequences of the Zhuyeqi cp genome were extracted using Geneious. The number of codons and the relative usage of synonymous codons were analyzed using codon W 1.4.4[20]. Relative synonymous codon usage (RSCU) refers to the relative probability of usage of a synonymous codon when coding for a specific amino acid. RSCU > 1 indicates that the codon is frequently used, RSCU = 1 indicates no preference, and RSCU < 1 indicates low usage frequency.

2.4 Repeat sequence analysis

Simple sequence repeats (SSRs) were detected using the MIcroSAtellite identification tool (MISA)[21]. Mononucleotide repeats were set at ≥ 8, dinucleotide and trinucleotide repeats were set at ≥ 4, and tetranucleotide, pentanucleotide, and hexanucleotide repeats were set at ≥ 3 [22]. Dispersed repeat sequences were detected using the online software REPuter [23], which provides four types of repeats: palindromic, forward, reverse, and complementary. The parameters were set as follows: repeat size between 30 and 1000, Hamming distance of 3, and default edit distance.

2.5 IR/SC boundary comparison analysis

In this study, the tea chloroplast genome(NC_038198.1) was used as the reference sequence, and the IRscope online software (https://irscope.shinyapps.io/irapp/) was used to draw the IR region boundary map of the chloroplast genome[24].

2.6 Nucleotide substitution rate (Ka/Ks) analysis

Using the tea chloroplast genome sequence as a reference, a total of 135 protein-coding genes shared by the Zhuyeqi chloroplast genome were extracted in Geneious. After alignment with MAFFT, the .fas format was converted to .aln format using the online program ALTER (https://www.sing-group.org/ALTER/), and the .aln format file was then converted to .axt format using AXTConvertor in Linux system under KaKs.Calculator [25]. Finally, KaKs.Calculator was used to calculate the ratio of non-synonymous substitutions (Ka) to synonymous substitutions (Ks). The NG model was selected, and the genetic code type was set to 11 (bacterial and plant plastid codon). When Ka = 0 and Ks = 0, the value of Ka/Ks was represented by NA.

2.7 Phylogenetic analysis

Whole-genome was used for evolutionary tree analysis, and the circular sequence was set to the same starting point. The sequences between species were aligned using the MAFFT software (v7.427, --auto mode). The aligned data was then analyzed using the RAxML v8.2.10 software (https://cme.h-its.org/exelixis/software.html) with the GTRGAMMA model and rapid Bootstrap analysis with a bootstrap value of 1000 to construct the maximum likelihood evolutionary tree.

3.1 Genome Structure of Zhuyeqi Chloroplast Genome

Using the tea (Camellia sinensis) chloroplast genome (NC_038198.1) as a reference, we assembled and annotated the complete cp genome of Zhuyeqi using Illumina NovaSeq 6000 sequencing platform. The cp genome of Zhuyeqi has a typical quadripartite structure, consisting of one large single-copy (LSC) region, one small single-copy (SSC) region, and a pair of inverted repeats (IRa and IRb), with lengths of 86,628 bp, 18,282 bp, and 26,081 bp, respectively (Fig. 1, Table 1). The overall nucleotide composition were A of 31.12%, T of 32.11%, C of 18.78% and G of 17.99%, resulting a total AT content of the cp genome of 63.23% and GC of 36.77%. A total of 135 genes were annotated in the Zhuyeqi cp genome, including 90 protein-coding genes(CDS), 37 transfer RNA genes (tRNAs), and 8 ribosomal RNA genes (rRNAs). The AT content of the Zhuyeqi chloroplast genome is 62.71%, which is significantly higher than the GC content (37.29%). The GC content of the LSC, SSC, and IR regions is 35.31%, 30.54%, and 42.95%, respectively, with the GC content of the IR regions being higher than that of the LSC and SSC regions.

Table 1

Characteristics of Zhuyeqi cp genome
Category	Item	Describe
Chloroplast genome structure	Cp gene/bp	157072
	LSC/bp	86628
	SSC/bp	18282
	IRA/IRB/bp	26081
Gene composition	Cp gene	135
	CDS	90
	tRNA	37
	rRNA	8
GC Content (%)	Cp gene	37.29
	LSC	35.31
	SSC	30.54
	IRA/IRB/	42.95

According to functional classification, the genes in the Zhuyeqi chloroplast genome can be divided into four categories: photosynthesis genes (44), self-replication genes (74), other genes (6), and genes of unknown function (10) (Table 2). There are a total of 20 duplicated genes in the Zhuyeqi chloroplast genome, including one NADH dehydrogenase subunit gene (ndhB), four self-replication genes (rpl2, rpl23, rps12, rps7), four rRNA genes (rrn16, rrn23, rrn4.5, rrn5), six tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC), and four unknown function protein genes (orf42, ycf1, ycf15, ycf2). Most of the genes in the Zhuyeqi chloroplast genome are non-coding, with only a few genes containing one or two introns. Among them, ndhA, ndhB, petB, petD, atpF, rpl16, rpl2, rps12, rps16, rpoC1, trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, trnT-CGU, trnV-UAC contain one intron, while clpP and ycf3 contain two introns. It is worth noting that further research using advanced molecular techniques is needed to determine the functions of the 10 unknown function genes identified in the Zhuyeqi chloroplast genome.

Table 2

Genes present in chloroplast genome of Zhuyeqi
Category of genes	Group of genes	Names 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
	Subunits photochlorophyllide reductase	-
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-UCC, trnH-GUG, trnI-CAU(2), trnI-GAU(2), trnK-UUU, trnL-CAA(2), trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU(2), trnP-UGG, trnQ-UUG, trnR-ACG(2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-CGU, trnT-GGU, trnT-UGU, trnV-GAC(2), trnV-UAC, trnW-CCA, trnY-GUA, trnfM-CAU
Other genes	Maturase	matK
	Protease	clpP**
	Envelope membrane protein	cemA
	Acetyl-CoA carboxylase	accD
	c-type cytochrome synthesis gene	ccsA
	Translation initiation factor	infA
	other	-
Genes of unknown function	Conserved hypothetical chloroplast ORF	orf42(2), ycf1(2), ycf15(2), ycf2(2), ycf3**, ycf4
Note: * indicate one intron in the gene; ** indicate two introns in the gene; Gene (2): multiple copy gene

3.2 Analysis of codon preference

There are significant differences in the pattern of codon preference among different species. Each amino acid is corresponded to at least one codon, and up to six codons. This unequal usage of synonymous codons is referred to as codon preference. Natural selection and base mutation are considered to be the main factors influencing codon preference. Codon W 1.4.4 software was used to analyze the relative synonymous codon usage (RSCU) of each amino acid in the Zhuyeqi chloroplast genome (Fig. 2 and Table 3). The results showed that there were 31 high-frequency codons with RSCU > 1, among which 13 and 16 high-frequency codons ended with A and U, respectively, accounting for 93.55% of the total. Only two high-frequency codons ended with G, and there were no high-frequency codons ending with C. It can be seen that the Zhuyeqi chloroplast genome has a preference for using A or U to end codons.

Table 3

RSCU analysis of each amino acid in the Zhuyeqi chloroplast genome.
AminoAcid	Symbol	Codon	No.	RSCU
*	Ter	UAA	42	1.4001
*	Ter	UAG	26	0.8667
*	Ter	UGA	22	0.7332
A	Ala	GCA	399	1.1384
A	Ala	GCC	226	0.6448
A	Ala	GCG	137	0.3908
A	Ala	GCU	640	1.826
C	Cys	UGC	75	0.4966
C	Cys	UGU	227	1.5034
D	Asp	GAC	205	0.3674
D	Asp	GAU	911	1.6326
E	Glu	GAA	1060	1.5068
E	Glu	GAG	347	0.4932
F	Phe	UUC	552	0.7164
F	Phe	UUU	989	1.2836
G	Gly	GGA	746	1.6496
G	Gly	GGC	188	0.4156
G	Gly	GGG	303	0.67
G	Gly	GGU	572	1.2648
H	His	CAC	140	0.4274
H	His	CAU	515	1.5726
I	Ile	AUA	746	0.9597
I	Ile	AUC	465	0.5982
I	Ile	AUU	1121	1.4421
K	Lys	AAA	1084	1.4818
K	Lys	AAG	379	0.5182
L	Leu	CUA	380	0.8112
L	Leu	CUC	209	0.4464
L	Leu	CUG	185	0.3948
L	Leu	CUU	587	1.2528
L	Leu	UUA	887	1.893
L	Leu	UUG	563	1.2018
M	Met	AUC	2	0.009
M	Met	AUG	661	2.982
M	Met	GUG	2	0.009
N	Asn	AAC	304	0.4582
N	Asn	AAU	1023	1.5418
P	Pro	CCA	336	1.1884
P	Pro	CCC	198	0.7004
P	Pro	CCG	146	0.5164
P	Pro	CCU	451	1.5952
Q	Gln	CAA	719	1.5216
Q	Gln	CAG	226	0.4784
R	Arg	AGA	506	1.872
R	Arg	AGG	165	0.6102
R	Arg	CGA	389	1.4388
R	Arg	CGC	90	0.333
R	Arg	CGG	114	0.4218
R	Arg	CGU	358	1.3242
S	Ser	AGC	120	0.3408
S	Ser	AGU	431	1.2246
S	Ser	UCA	417	1.1844
S	Ser	UCC	331	0.9402
S	Ser	UCG	181	0.5142
S	Ser	UCU	632	1.7952
T	Thr	ACA	416	1.2236
T	Thr	ACC	253	0.744
T	Thr	ACG	141	0.4148
T	Thr	ACU	550	1.6176
V	Val	GUA	539	1.4952
V	Val	GUC	175	0.4856
V	Val	GUG	198	0.5492
V	Val	GUU	530	1.47
W	Trp	UGG	483	1
Y	Tyr	UAC	201	0.3956
Y	Tyr	UAU	815	1.6044

3.3. Detection of Repeat Sequences and SSRs in the cp genome of Zhuyeqi

In the genome of Zhuyeqi chloroplast, 40 repeat sequences were identified, among which palindrome repeat sequences were the most common, accounting for 50% of the total repeats (20 repeats), while forward repeat sequences accounted for the other 50% (20 repeats). No reverse or complementary repeat sequences were found. There were 14 repeats with a length ranging from 30 to 38 bp, and 25 repeats with a length ranging from 39 to 82 bp. One repeat sequence had a length of 26,081 bp(Fig. 3).

The SSR analysis of the Zhuyeqi chloroplast genome was performed using MISA v1.0 software (Fig. 4). A total of 247 SSR loci that met the criteria were identified in the Zhuyeqi chloroplast genome, with 143, 50, and 54 SSR loci located in the LSC, SSC, and IR regions, respectively, accounting for 57.9%, 21.9%, and 20.2% of the total. Among all SSRs, there were 157 mononucleotide repeats, 4 dinucleotide repeats, 72 trinucleotide repeats, 12 tetranucleotide repeats, and 2 pentanucleotide repeats. The most commonly repeated base element was A/T, followed by AT/TA, ATA, TAA, and TTC. These results indicated that A/T bases were dominant and had a base preference, which was consistent with the AT content of 62.71% in the Zhuyeqi chloroplast genome, suggesting that the ease of strand separation of AT bases compared to GC bases may be related.

3.4. IR Expansion and Contraction

Taking the reported chloroplast genomes of 7 species or varieties in Camellia family and Camellia sinensis L. var Zhuyeqi together, the IR/LSC and IR/SSC boundaries were compared (Fig. 5). The total length of the chloroplast genome of Zhuyeqi (157,072 bp) was the shorter than Camellia var. ‘pubilimba’ and Camellia sinensis var. ‘Dahongpao’, while showed longer than the other six Camellia varieties. The main marker genes of rps19, ndhF, ycf1, and trnH in chloroplast were detected to be at the LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC boundaries, respectively. Among these genes, rps19 mainly located at LSC region or crossed LSC/IRb boundary, showing − 284–52 bp within the IRb region. The rpl2 gene in the IR regions was 106 and 112 bp from the LSC in the 8 species (106 bp for Camellia sinensis var ‘Zhuyeqi’, Camellia taliensis, Camellia sinensis,Camellia clevfera, Camellia gymnogyna, Camellia sinensis var ‘assamica’ and Camellia japonica; 112 bp for Camellia sinensis var ‘pubilimba’). It is noteworthy that in all the compared 8 Camellia species or varieties, ycf1 crossed the IRb/SSC boundary with 899–1,071 bp within the IRb region, with 2–26 bp within SSC region. In addition, ycf1 gene located also at SSC/IRa boundary with 4,517–4,717 bp within the SSC region, with 967-1,105 bp within IRa region. The ndhF gene was located within the SSC region, and the distance to the IRb/SSC junction was 5, 56, 61, 64, 143 and 154 bp.The trnN gene was located entirely within IRa and was contracted by 971–1,381 bp. The trnH gene in the LSC region was contracted by 1 and 2 bp from the connection region of IRa/LSC (1bp for Camellia sinensis var ‘Zhuyeqi’, Camellia taliensis, Camellia sinensis var ‘pubilimba’, Camellia sinensis,Camellia clevfera, and Camellia gymnogyna; 2 bp for Camellia sinensis var ‘assamica’).

3.6 Ka/Ks analysis in the cp of Zhuyeqi

The diversity of nucleic acids can reveal the variation in nucleic acid sequences among different species, and regions with high variation can provide potential molecular markers for population genetics. The KaKs.Calculator was used to calculate the nucleotide nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and their ratio (Ka/Ks) for the protein-coding genes in the Zhuyeqi chloroplast genome(Fig. 6). The Pi value range of the entire Zhuyeqi chloroplast genome was 0-0.01408, with a mean value of 0.00126. The average Pi value was highest in the SSC region (0.00143), followed by the LSC region (0.00128), and lowest in the IR region (0.00106). Additionally, five highly divergent regions were detected, including trnI-GAU (0.01408), rps12 (0.01323), rps19 (0.00952), rps16 (0.00852), and rpl33 (0.00616). The LSC region contained four different regions (rps12, rps19, rps16, and rpl33), and the IR region contained one divergent region (trnI-GAU).

3.7 Phylogenetic analysis

In order to determine the evolutionary position of Zhuyeqi in the family Theaceae, the obtained Zhuyeqi chloroplast genome sequence was aligned with the complete chloroplast genomes of 21 species Theaceae that have been published on the NCBI website using multiple sequence alignment, and an evolutionary tree was constructed using RAxML v8.2.10 software. As shown in Fig. 7, the species in different genera within the same family are relatively distant from Zhuyeqi, indicating that these species are more distantly related to Zhuyeqi. Five different Theaceae species and Zhuyeqi clustered together, with Camellia sinensis (KJ996106.1) being the closest to Zhuyeqi, indicating that Zhuyeqi is most closely related to this tea variety.

In this study, Illumina NovaSeq 6000 was used to sequence the DNA of Zhuyeqi, and SPAdes v3.10.1 was used to assemble the chloroplast genome. The chloroplast genome features and phylogenetic relationships were analyzed. Overall, the chloroplast genome of Zhuyeqi showed high similarity with those of other reported tea plant chloroplast genomes in terms of genome length, structure, and composition. No rearrangement was observed, and a good collinearity relationship was found. Chloroplast have independent and highly conserved genomes, and the complete chloroplast genome length of Zhuyeqi and seven other Camellia species (KF156839.1, KF562708.1, KF156838.1, KF156835.1, KF156833.1, KF156834.1, KF156837.1) ranged from 156,576 to 157,103 bp, with a difference in genome length of no more than 600 bp. Moreover, the number, type, and order of genes were also basically the same, which once again illustrates the highly conserved characteristics of chloroplast genomes.

The study of codon preference is not only helpful for the evolutionary process of species, but also for optimizing the expression of exogenous genes in chloroplasts, predicting gene function, and gene expression levels[26]. In plant chloroplast genomes, codons are often biased towards the use of A or U bases[27]. In Zhuyeqi, 31 high-frequency codons were identified, of which 29 end with A or U. This result may be caused by natural selection and mutation[28]. Li et al. (2021) found 37 high-frequency codons in the chloroplast genome of the "Wuyi narcissus" tea plant, and the third base of the codon also preferred to use A or U[29].

In the chloroplast genome of higher plants, a large number of repeats have been identified. Due to unconventional recombination and slippage, repeat sequences play an important role in genome rearrangement and variation [30–31]. Generally speaking, due to the process of species evolution, most of the repeat sequences in the genome are distributed in non-coding regions. Organisms try to retain the least amount of genetic information possible to increase their genetic efficiency. In the chloroplast genome of Zhuyeqi, 40 repeat sequences were found, among which palindrome repeat sequences were the most common, with 20 accounting for 50%, and 20 were forward repeat sequences, also accounting for 50%. No reverse or complementary repeat sequences were found. There were 14 repeat sequences with a length between 30–38 bp and 25 repeat sequences between 39–82 bp. These results indicated that the chloroplast genome of Zhuyeqi retained rich genetic information. Chloroplast Simple Sequence Repeats (cpSSR) not only have the advantages of high polymorphism, codominance, and wide distribution as microsatellite markers, but also have the characteristics of conserved chloroplast genome sequences, simple structure, and maternal inheritance. They are widely used in species evolution and genetic diversity analysis[32–33]. A total of 247 SSR sites that met the conditions were identified in the chloroplast genome of Zhuyeqi, with 143, 50, and 54 sites distributed in the LSC, SSC, and IR regions, respectively, accounting for 57.9%, 21.9%, and 20.2%. Among all repeat types, the majority were A/T repeats, which is consistent with the high AT content in the chloroplast genome of Zhuyeqi. Studies have shown that SSRs are usually composed of polyadenine (poly A) or polypyrimidine (poly T) repeats, and G/C repeat types are less common[35–37]. The results of this study support the conclusion that SSRs in the chloroplast genome are usually composed of poly A or poly T repeats.

Nucleotide synonymous substitution rates and non-synonymous substitution rates are important markers in genetic evolution research[38–39]. In most genes, except for those that evolve rapidly, the frequency of non-synonymous substitutions is lower than that of synonymous substitutions due to the purifying selection. When Ka/Ks < 1 (especially when it is smaller than 0.5), the gene is subject to purifying selection; when Ka/Ks > 1, the gene is subject to positive selection; if the value of Ka/Ks is close to 1, it indicates neutral evolution[40–41] This study found that the Ka/Ks value range of the Zhuyeqi chloroplast genome was 0-0.01408, with an average value of 0.00126, indicating that protein-coding genes of the Zhuyeqi chloroplast genome underwent strong purifying selection during the evolution process.

In molecular systematics research, the effective informative sites provided by single genes or gene combinations are limited, and differences in the evolution of different gene sequences result in low resolution of the constructed phylogenetic tree, making it difficult to clarify the evolutionary relationships among family Theaceae of the Zhuyeqi[42–43]. Compared with traditional molecular systematics research, the chloroplast genome provides a large number of effective informative sites for phylogenetic research, greatly improving the resolution of the system evolutionary tree of some low-level taxa or phylogroups with slow molecular evolution rates and low molecular-level variation, and can effectively solve the system evolutionary relationships of different taxonomic levels of angiosperms [44–45]. In this study, the chloroplast genomes of Zhuyeqi and 20 other species of the Theaceae family were aligned and analyzed for phylogenetic relationships. The results showed that Zhuyeqi is on the same branch as Camellia sinensis (KJ996106.1), and their relationship is the closest, consistent with the classification recorded in the "Flora of China[46]". This study clarified the phylogenetic position of Zhuyeqi and its phylogenetic relationships with other genera in the Theaceae family, laying a foundation for further tea variety identification and domestication analysis; this study also revealed the structural characteristics, SSR sites, and phylogenetic analysis of the chloroplast genome of Zhuyeqi, providing useful genetic information for future genetic evolutionary analysis of tea varieties.

This article reported for the first time the complete genome sequence of the chloroplast of Zhuyeqi and conducted a systematic phylogenetic analysis with other Camellia genus of the Theaceae family, providing a useful reference for the classification and phylogenomic analyses of Camellia in Theaceae. Although Zhuyeqi shared similarities in genome structure, gene content, and gene sequences with other Camellia genus, some hotspots were identified in the LSC and SSC regions of the Zhuyeqi genome, providing molecular markers for its systematic evolutionary analysis. Ka/Ks analysis showed that the Protein-encoding genes of Zhuyeqi chloroplast genome underwent strong purifying selection during evolution. Phylogenetic analysis showed that Zhuyeqi was most closely related to Camellia sinensis (KJ996106.1), indicating that Zhuyeqi belonged to the genus Camellia in the family Theaceae.

Authors' contributions

Zhiyin CHEN: Investigation, Data curation, Methodology, Software, Validation, Writing - original draft, Funding acquisition. Qing Liu and Ying XIAO and Guihua ZHOU: Investigation, Data curation, Software, Methodology, Validation. Penghui YU and Jing BAI: Investigation, Data curation, Software, Methodology, Validation. Hua Huang and Yihui GONG: Conceptualization, Investigation, Data curation, Methodology, Writing - review & editing, Visualization, Validation, Project administration, Supervision, Resources, Funding acquisition.

Funding

This work was funded by various sources, including the Outstanding Youth Project Supported by Scientific Research Fund of Hunan Provincial Education Department, grant number: 21B0782. Additionally, it received support from the Scientific Research Fund of Hunan Provincial Education Department, grant number: 22A0608.

Competing Interest

The authors declare that they have no conflicts of interest.

Data Availability Statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under the accession OL450428.1. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA779759, SRR16922592, and SAMN23075201, respectively.

Trofimov D, Cadar D, Schmidt-Chanasit J, et al. A comparative analysis of complete chloroplast genomes of seven Ocotea species (Lauraceae) confirms low sequence divergence within the Ocotea complex[J]. Scientific Reports, 2022, 12(1): 1-13.
Birky C W. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution[J]. Proceedings of the National Academy of Sciences, 1995, 92(25): 11331-11338.
Zhai W, Duan X H, Zhang R, et al. Chloroplast genomic data provide new and robust insights into the phylogeny and evolution of the Ranunculaceae[J]. Molecular Phylogenetics and Evolution, 2019, 135: 12-21.
Chen Q, Wu X B, Zhang D Q. Phylogenetic analysis of Fritillaria cirrhosa D. Don and its closely related species based on complete chloroplast genomes[J]. Peer J, 2019, 7: e7480.
Hong S Y, Cheon K S, Yoo K O, et al. Comparative analysis of the complete chloroplast genome sequences of three Amaranthus species[J]. Plant Genetic Resources, 2019, 17(3): 245-254.
Yan M, Zhao X Q, Zhou J Q, et al. The complete chloroplast genome of cultivated apple (Malus domestica Cv.‘Yantai Fuji 8’)[J]. Mitochondrial DNA Part B, 2019, 4(1): 1213-1216.
Azim M K, Khan I A, Zhang Y. Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome. Plant Molecular Biology, 2014, 85(1): 193-208.
Cheng H, Li J F, Zhang H, et al. The complete chloroplast genome sequence of strawberry (Fragaria× ananassa Duch.) and comparison with related species of Rosaceae[J]. Peer J, 2017, 5: e3919.
Kim Y, Shin J, Oh D R, et al. Comparative analysis of complete chloroplast genome sequences and insertion-deletion (Indel) polymorphisms to distinguish five vaccinium species[J]. Forests, 2020, 11(9): 927.
Guo D H, Li D M, Li H, et al. The complete chloroplast genome sequence of Vitis vinifera Muscat Hamburg[J]. Mitochondrial DNA Part B, 2020, 5(1): 117-1.
Raveendar S, Na Y W, Lee J R, et al. The complete chloroplast genome of Capsicum annuum var. glabriusculum using Illumina sequencing[J]. Molecules, 2015, 20(7): 13080-13088.
Liu H, Ye H, Zhang N, et al. Comparative analyses of chloroplast genomes provide comprehensive insights into the adaptive evolution of Paphiopedilum (Orchidaceae)[J]. Horticulturae, 2022, 8(5): 391.
Zhao Y, Qu D, Ma Y. Characterization of the Chloroplast Genome of Argyranthemum frutescens and a Comparison with Other Species in Anthemideae[J]. Genes, 2022, 13(10): 1720.
Cao D L, Zhang X J, Xie S Q, et al. Application of chloroplast genome in the identification of Traditional Chinese Medicine Viola philippica[J]. BMC genomics, 2022, 23(1): 1-19.
Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi S K, Ohto C, Torazawa K, Meng B Y, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M. The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression[J]. Plant Molecular Biology Reporter, 1986, 5(9): 2043–2049.
[16] Cho K S, Cheon K S, Hong S Y, et al. Complete chloroplast genome sequences of Solanum commersonii and its application to chloroplast genotype in somatic hybrids with Solanum tuberosum[J]. Plant Cell Reports, 2016, 35: 2113-2123.
[17] Kawakami S, Ebana K, Nishikawa T, et al. Genetic variation in the chloroplast genome suggests multiple domestication of cultivated Asian rice (Oryza sativa L.)[J]. Genome, 2007, 50(2): 180-187.
[18]Ogihara Y, Isono K, Kojima T, et al. Chinese spring wheat (Triticum aestivum L.) chloroplast genome: complete sequence and contig clones[J]. Plant Molecular Biology Reporter, 2000, 18: 243-253.
[19]Alzohairy A M. BioEdit: An important software for molecular biology[J]. Gerf Bulletin of Biosciences, 2011, 2(1): 60–61.
[20] Peden J F. Analysis of codon usage[J]. Biosystems, 1999, 5: 45–50.
[21] Thiel T, Michalek W, Varshney R, Graner A. Exploiting est databases for the development and characterization of gene-derived ssr-markers in barley (Hordeum vulgarel.)[J].Theoretical & Applied Genetics, 2003, 106(3): 411–422.
[22] Faircloth B C. MSATCOMMANDER: Detection of microsatellite repeat arrays and automated, locus‐specific primer design[J]. Molecular ecology resources, 2008, 8(1): 92-94.
[23] Kurtz S, Choudhuri J V, Ohlebusch E, Schleiermacher C, Stoye J, Giegerich R. REPuter: the manifold applications of repeat analysis on a genomic scale[J]. Nucleic Acids Research, 2001, 29(22): 4633–4642.
[24] Amiryousefi A, Hyvönen J, Poczai P. IRscope: an online program to visualize the junction sites of chloroplast genomes[J].Bioinformatics,2018, 34(17): 3030–3031.
[25] Zhang Z, Li J, Zhao X Q, Wang J, Wong G K S, Yu J. KaKs_Calculator: Calculating Ka and Ks Through Model Selection and Model Averaging[J]. Genomics, Proteomics & Bioinformatics, 2006, 4(4): 259–263.
[26]Li Y, Sylvester S P, Li M, et al. The complete plastid genome of Magnolia zenii and genetic comparison to Magnoliaceae species[J]. Molecules, 2019, 24(2): 261.
[27]Zhou M, Long W, Li X. Patterns of synonymous codon usage bias in chloroplast genomes of seed plants[J]. Forestry Studies in China, 2008, 10: 235-242.
[28]Necşulea A, Lobry J R. A new method for assessing the effect of replication on DNA base composition asymmetry[J]. Molecular biology and evolution, 2007, 24(10): 2169-2179.
[29]Li L, Hu Y, He M, et al. Comparative chloroplast genomes: insights into the evolution of the chloroplast genome of Camellia sinensis and the phylogeny of Camellia[J]. BMC genomics, 2021, 22(1): 1-22.
[30]Lu R S, Li P, Qiu Y X. The complete chloroplast genomes of three Cardiocrinum (Liliaceae) species: comparative genomic and phylogenetic analyses[J]. Frontiers in Plant Science, 2017, (7): 2054.
[31]Yuan C, Zhong W, Mou F, Gong Y, Pu D, Ji P, Huang H, Yang Z, Zhang C. The complete chloroplast genome sequence and phylogenetic analysis of Chuanminshen(Chuanminshenviolaceum Sheh et Shan)[J]. Physiology & Molecular Biology of Plants, 2017, 23(1): 17.
Takahashi D, Sakaguchi S, Isagi Y, et al. Comparative chloroplast genomics of series Sakawanum in genus Asarum (Aristolochiaceae) to develop single nucleotide polymorphisms (SNPs) and simple sequence repeat (SSR) markers[J]. Journal of Forest Research, 2018, 23(6): 387-392.
Yamane K, Kawahara T. Size homoplasy and mutational behavior of chloroplast simple sequence repeats (cpSSRs) inferred from intra-and interspecific variations in four chloroplast regions of diploid and polyploid Triticum and Aegilops species[J]. Genetic resources and crop evolution, 2018, 65: 727-743.
Alanazi K M, Ali M A, Kim S Y, et al. The cp genome characterization of Adenium obesum: Gene content, repeat organization and phylogeny[J]. Saudi Journal of Biological Sciences, 2021, 28(7): 3768-3775.
Wu L, Cui Y, Wang Q, et al. Identification and phylogenetic analysis of five Crataegus species (Rosaceae) based on complete chloroplast genomes[J]. Planta, 2021, 254: 1-12.
Li D M, Zhao C Y, Liu X F. Complete chloroplast genome sequences of Kaempferia galanga and Kaempferia elegans: molecular structures and comparative analysis[J]. Molecules, 2019, 24(3): 474.
Mo Z, Lou W, Chen Y, et al. The chloroplast genome of Carya illinoinensis: genome structure, adaptive evolution, and phylogenetic analysis[J]. Forests, 2020, 11(2): 207.
Loewe L, Charlesworth B, Bartolomé C, et al. Estimating selection on nonsynonymous mutations[J]. Genetics, 2006, 172(2): 1079-1092.
Yang Z, Nielsen R. Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models[J]. Molecular biology and evolution, 2000, 17(1): 32-43.
Ivanova Z, Sablok G, Daskalova E, Zahmanova G, Apostolova E, Yahubyan G, Baev V. Chloroplast genome analysis of resurrection tertiary relict Haberlea rhodopensis highlights genes important for desiccation stress response[J]. Frontiers in Plant Science, 2017, 8(1): 204.
Kimura M. The neutral theory of molecular evolution and the world view of the neutralists[J]. Genome, 1989, 31(1): 24–31.
Perkins A J. Molecular phylogenetics and species delimitation in annual species of Hydrocotyle (Araliaceae) from South Western Australia[J]. Molecular Phylogenetics and Evolution, 2019, 134(1): 129–141.
Van De Wiel C C M, Van Der Schoot J, Van Valkenburg J L C H, Duistermaat H, Smulders M J M. DNA barcoding discriminates the noxious invasive plant species, floating pennywort (Hydrocotyle ranunculoides Lf), from non‐invasive relatives[J]. Molecular Ecology Resources, 2009, 9(4): 1086–1091
Zhang X, Zhou T, Kanwal N, Zhao Y, Bai G, Zhao G. Completion of eight Gynostemma BL. (Cucurbitaceae) chloroplast genomes: characterization, comparative analysis, and phylogenetic relationships[J]. Frontiers in plant science, 2017, 8(1): 1583.
Zhao M L, Song Y, Ni J, Yao X, Tan Y H, Xu Z F. Comparative chloroplast genomics and phylogenetics of nine Lindera species (Lauraceae)[J]. Scientific Reports, 2018, 8(1): 1–11.
Ding P, Shao Y H, Li Q, Gao J L, Zhang R J, Lai X P,Wang D P, Zhang H Y. The complete chloroplast genome sequence of the medicinal plant Andrographis paniculata[J]. Mitochondrial DNA Part A, 2016, 27(4): 2347-2348.

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published 28 Jun, 2023

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Complete Chloroplast Genome Sequence of Camellia sinensis: Genome Structure, Adaptive Evolution, and Phylogenetic Relationships

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 DNA Extraction and Genome Sequencing

2.2 Genome structure analysis

2.3 Codon preference analysis

2.4 Repeat sequence analysis

2.5 IR/SC boundary comparison analysis

2.6 Nucleotide substitution rate (Ka/Ks) analysis

2.7 Phylogenetic analysis

3 Results

3.1 Genome Structure of Zhuyeqi Chloroplast Genome

3.2 Analysis of codon preference

3.3. Detection of Repeat Sequences and SSRs in the cp genome of Zhuyeqi

3.4. IR Expansion and Contraction

3.6 Ka/Ks analysis in the cp of Zhuyeqi

3.7 Phylogenetic analysis

4. Discussion

5. Conclusions

Declarations

References

Status:

Journal Publication

Version 1