Deciphering tea tree chloroplast and mitochondrial genomes of Camellia sinensis var. assamica.

Tea is the most popular non-alcoholic caffeine-containing and the oldest beverage in the world. In this study, we de novo assembled the chloroplast (cp) and mitochondrial (mt) genomes of C. sinensis var. assamica cv. Yunkang10 into a circular contig of 157,100 bp and two complete circular scaffolds (701719 bp and 177329 bp), respectively. We correspondingly annotated a total of 141 cp genes and 71 mt genes. Comparative analysis suggests repeat-rich nature of the mt genome compared to the cp genome, for example, with the characterization of 37,878 bp and 149 bp of long repeat sequences and 665 and 214 SSRs, respectively. We also detected 478 RNA-editing sites in 42 protein-coding mt genes, which are ~4.4-fold more than 54 RNA-editing sites detected in 21 protein-coding cp genes. The high-quality cp and mt genomes of C. sinensis var. assamica presented in this study will become an important resource for a range of genetic, functional, evolutionary and comparative genomic studies in tea tree and other Camellia species of the Theaceae family.

Background & Summary

Tea is the most popular non-alcoholic caffeine-containing and the oldest beverage in the world since 3000 B. C.[1,2]. The production of tea made from the young leaves of Camellia sinensis var. sinensis and C. sinensis var. assamica, together with ornamentally well-known camellias (e.g., C. japonica, C. reticulata and C. sasanqua) and worldwide renowned wooden oil crop C. oleifera[3] has made the genus Camellia possess huge economic values in Theaceae. Besides its industrial, cultural and medicinal values, botanists and evolutionary biologists have increasingly paid attention to this genus. As a result of frequent hybridization and polyploidization, Camellia is almost commonly regarded as one of the most taxonomically and phylogenetically difficult taxa in flowering plants[4]. Thus, it has long been problematic for the taxonomic classification of the Camellia species based on the morphological characteristics[5]. The chloroplast (cp) genomes are able to provide valuable information for taxonomic classification, tracing source populations[6,7] and the reconstruction of phylogeny to resolve complex evolutionary relationships[8-10] due to the conservation of genomic structure, maternal inheritance and a fairly low recombination rate. Genetically speaking, cp genomes are comparatively conserved than plant mitochondria (mt) genomes which are more heterogeneous in nature. However, the presence of NUPT (nuclear plastid DNA) into cp genomes argues that cp genomes assembled from WGS data may include the heterogeneity due to the nuclear cp DNA transferred to the nucleus, resulting in erroneous phylogenetic inferences[11]. It has long been acknowledged that mtDNA has the propensity to integrate DNA from various sources through intracellular and horizontal transfer[12-14]. Partially due to these reasons, the mt genomes vary from ~200 Kbp to ~11.3 Mbp in some living organisms[15-17]. The dynamic nature of mt genome structure has been recognized, and plant mt genomes can have a variety of different genomic configurations due to the recombination and differences in repeat content[18,19]. These characteristics make the plant mt genome a fascinating genetic system to investigate questions related to evolutionary biology. The first effort has been made to sequence the 13 representative Camellia chloroplast genomes using next-generation Illumina genome sequencing platform, which obtained novel insights into global patterns of structural variation across the Camellia cp genomes[4]. The reconstruction of phylogenetic relationships among these representative species of Camellia suggests that cp genomic resources are able to provide useful data to help to understand their evolutionary relationships and classify the ‘difficult taxa’. Increasing interest in the Camellia plants have made up to thirty-eight of cp genomes be sequenced up to date[20-37]. Recently, we decoded the first nuclear genome of C. sinensis var. assamica cv. Yunkang10, providing novel insights into genomic basis of tea flavors[38]. Besides the lack of the C. sinensis var. assamica cp genome among thirty-eight cp genomes that were sequenced in this genus[4,20-37], up to data, none of mt genome has been determined in the genus Camellia.

In this study, we filtered cpDNA and mtDNA reads from the WGS genome sequence project[38] and de novo assembled the mt genome and cp genome of C. sinensis var. assamica. The information of both cp and mt genomes will help to obtain a comprehensive understanding of the taxonomy and evolution of the genus Camellia. These genome sequences will also facilitate the genetic modification of these economically important plants, for example, through chloroplast genetic engineering technologies.

Methods

Plant materials, DNA extraction and genome sequencing

Young and healthy leaves of an individual plant of cultivar Yunkang10 of C. sinensis var. assamica were collected for genome sequencing in April, 2009, from Menghai County, Yunnan Province, China. Fresh leaves were harvested and immediately frozen in liquid nitrogen after collection, followed by the preservation at −80 °C in the laboratory prior to DNA extraction. High-quality genomic DNA was extracted from leaves using a modified CTAB method[39]. RNase A and proteinase K were separately used to remove RNA and protein contamination. The quality and quantity of the isolated DNA were separately checked by electrophoresis on a 0.8% agarose gel and a NanoDrop D-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). A total of eleven paired-end libraries, including four types of small-insert libraries (180 bp, 260 bp, 300 bp, 500 bp) and seven large-insert libraries (2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 8 Kb, 20 Kb), were prepared following the Illumina’s instructions, and sequenced using Illumina HiSeq. 2000 platform by following the standard Illumina protocols (Illumina, San Diego, CA). We totally generated ~707.88 Gb (~229.31×) of raw sequencing data[38]. Further reads quality control filtering processes yielded a total of ~492.15 Gb (~159.43×) high-quality data retained and used for subsequent genome assembly.

De novo chloroplast and mitochondria genome assemblies

The chloroplast reads were filtered from whole genome Illumina sequencing data of C. sinensis var. assamica, we mapped all the sequencing reads to the reference genomes[4] using bowtie2 (version 2.3.4.3)[40]. The mapped chloroplast reads were assembled into a circular contig of 157,100 bp in length with an overall GC content of 37.29% using CLC Genomics Workbench v. 3.6.1 (CLC Inc., Rarhus, Denmark) (Fig. 1). For mitochondria genome assembly, the PE and MP sequencing reads were used separately. Briefly, we first performed de novo assembly with VELVET v1.2.08[41], which was previously described[42,43]. Scaffolds were constructed using SSPACE v.3.0[44]. False connection was manually removed based on the coverage and distances of paired reads. Gaps between scaffolds were then filled with GapCloser (version 1.12)[45,46] using all pair-end reads. We obtained the two complete circular scaffolds (701719 bp and 177329 bp) of the C. sinensis var. assamica mt genome from the de-novo assembly of the filtered mitochondrial reads (Figs 2–4). The two scaffolds of the mt genome had overall GC contents of 45.63% and 45.81%, respectively. The completed chloroplast and mitochondria genomes are publicly available in NCBI GenBank under accession numbers MH019307, MK574876 and MK574877 and BIG Genome Warehouse WGS000271, WGS000272.

Fig. 1

Genome map of C. sinensis var. assamica cv. Yunkang10. Genes lying outside of the outer circle are transcribed in the clockwise direction whereas genes inside are transcribed in the counterclockwise direction. Genes belonging to different functional groups are color-coded. Area dashed darker gray in the inner circle indicates GC content while the lighter gray corresponds to AT content of the genome.

Fig. 2

The assembly and annotation pipeline of the tea tree mitochondrial genome.

Fig. 4

Circular map of scaffold 2 in the C. sinensis var. assamica cv. Yunkang10 mitochondrial genome. Gene map showing 17 annotated genes with different functional groups that are color-coded on outer circle as transcribed clock-wise (outside) and transcribed counter clock-wise (inside). The inner circle indicates the GC content as dark grey plot.

Genome annotation and visualization

The complete chloroplast genome of C. sinensis var. assamica was preliminarily annotated using the online program DOGMA[47] (Dual Organellar Genome Annotator) followed by manual correction. A total of 141 genes were annotated, of which 87 were protein-coding genes, 46 were tRNA genes and eight were rRNA genes (Table 1). MITOFY[15] was used to characterize the complement of protein-coding and rRNA genes in the mitochondrial genome. A tRNA gene search was carried out using the tRNA scan-SE software (version 1.3.1)[48]. We annotated a total of 71 genes, including 44 protein-coding genes, 24 tRNAs and 3 rRNAs (Table 2). Circular genome maps were drawn with OrganellarGenomeDRAW[49] (Figs 3–4).

Table 1

Gene annotation of the C. sinensis var. assamica cp genome.

Category	Group	Genes
Photosynthesis related genes	Rubisco	rbcL
	Photosystem I	psaA, psaB, psaC, psaI, psaJ
	Assembly/stability of Photosystem I	ycf3
	Photosystem II	psbA, psbB, psbT, psbK, psbI, psbH, psbM, psbN, psbD, psbC, psbZ, psbJ, psbL, psbE, psbF
	ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI
	Cytochrome b/f complex	petA, petB, petD, petN, petL, petG
	Cytochrome csynthesis	ccsA
	NADPH dehydrogenase	ndhA, ndhB (×2), ndhC, ndhD, ndhE, ndhF, ndhH, ndhG, ndhJ, ndhK, ndhI
Transcription and translation related genes	Transcription	rpoA, rpoC2, rpoC1, rpoB
	Ribosomal proteins	rps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12, rps14, rps15, rps16, rps18, rps19, rpl2 (×2), rpl14, rpl16, rpl20, rpl22, rpl23 (×2), rpl32, rpl33, rpl36
	Translation initiation factor	infA
RNA genes	Ribosomal RNA	rrn16S (×2), rrn23S (×2), rrn4.5 (×2), rrn5 (×2)
	Transfer RNA	trnH-GUG, trnK-UUU (×2), trnQ-UUG, trnS-GCU, trnG-UCC (×2), trnR-UCU, trnC-GCA, trnD-GUC, trnY-GUA, trnE-UUC, trnT-GGU, trnS-UGA, trnG-UCC, trnfM-CAU, trnS-GGA, trnT-UGU, trnL-UAA (×2), trnF-GAA, trnV-UAC (×2), trnM-CAU, trnW-CCA, trnP-UGG, trnI-CAU, trnL-CAA (×2), trnV-GAC, trnI-GAU (×3), trnA-UGC (×2), trnR-ACG (×2), trnN-GUU (×2), trnL-UAG, trnN-GUU, trnR-ACG, trnA-UGC (×2), trnV-GAC, trnI-CAU
Other genes	RNA processing	matK
	Carbon metabolism	cemA
	Fatty acid synthesis	accD
	Proteolysis	clpP
Genes of unknown function	Conserved ORFs	ycf1 (×2), cf2, ycf4, ycf2, ycf15 (×2)

Table 2

Gene content of the C. sinensis var. assamica mt genome.

Group of genes	Name of genes
	Scaffold 1	Scaffold 2
Complex I	nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, nad9 (×2)	nad1, nad2
Complex II	sdh3, sdh4	sdh3
Complex III		cob
Complex IV	cox1, cox2, cox3
Complex V	atp1, atp4, atp6, atp8, atp9	atp9
Cytochrome c biogenesis	ccmFn, ccmB, ccmC	ccmFc
Ribosome large subunit	rpl2, rpl10, rpl16	rpl5
Ribosome small subunit	rps1, rps3, rps4, rps7, rps12, rps13, rps19	rps14, rps19
rRNA genes	rrn5, rrn18, rrn16
tRNA genes	trnS(Ser), trnD(Asp), trnK(Lys), trnfM(Met) (×2), trnI(Ile)-cp, trnE(Glu), trnH(His)-cp, trnP(Pro), trnW(Trp)-cp, trnG(Gly), trnQ(Gln), trnC(Cys), trnD(Asp), trnS(Ser), trnV(Val)-cp	trnI(Ile), trnM(Met)-cp, trnC(Cys), trnN(Asn)-cp, trnY(Tyr), trnS(Ser), trnF(Phe), trnP(Pro)
chloroplast-derived genes	trnI(Ile)-cp, trnH(His)-cp, trnW(Trp)-cp, trnV(Val)-cp	trnM(Met)-cp, trnN(Asn)-cp
Other proteins	matR, mttB

Fig. 3

Circular map of scaffold 1 in the C. sinensis var. assamica cv. Yunkang10 mitochondrial genome. Gene map showing 54 annotated genes with different functional groups that are color-coded on outer circle as transcribed clock-wise (outside) and transcribed counter clock-wise (inside). The inner circle indicates the GC content as dark grey plot.

Simple sequence repeats (SSRs) were identified and located using MISA (http://pgrc.ipk-gatersleben.de/misa/). All the annotated SSRs were classified by the size and copy number of their tandemly repeated: monomer (one nucleotide, n ≥ 8), dimer (two nucleotides, n ≥ 4), trimer (three nucleotides, n ≥ 4), tetramer (four nucleotides, n ≥ 3), pentamer (five nucleotides, n ≥ 3), hexamer (six nucleotides, n ≥ 3). A total of 214 SSRs were identified in cp genome with 74.42% of which were monomers, 19.07% of dimers, 0.47% of trimers, 4.65% of tetramers and 0.93% of hexamers (Table 3). There were no pentamers found in the cp genome. In mt genome, we obtained 665 SSRs distributed into monomers, dimers, trimers, pentamers, tetramers and hexamers with 31.53%, 45.35%, 4.95%, 15.17%, 2.70% and 0.15%, respectively (Table 3). Repeat sequences including forward and palindromic repeats, were also searched by REPuter[50] with the following parameters: minimal length 50 nt; mismatch 3 nt. Long repeat sequences (repeat unit > 50 bp) of forward and palindromic repeats were further annotated, resulting in 149 bp from 4 paired repeats in the cp genome (Table 4) and 37,878 bp from 58 paired repeats in the mt genome (Online-only Tables 1–2). Our repeat content analyses indicate that the mt genome is more abundant in repeat sequences and more variable than the cp genome of C. sinensis var. assamica (Table 4; Online-only Tables 1–2).

Table 3

Statistics of SSR motifs in the C. sinensis var. assamica mt and cp genomes.

SSR-Motif	mt Genome		cp Genome
	SSR Number	SSR %	SSR Number	SSR %
Monomer	210	31.53	160	74.42
Dimer	302	45.35	41	19.07
Trimer	33	4.95	1	0.47
Tetramer	101	15.17	10	4.65
Pentamer	18	2.70	0	0.00
Hexamer	1	0.15	2	0.93

Table 4

Long repeats (repeat unit > 50 bp) in the C. sinensis var. assamica cp genome.

Repeat Length	Type*	Start of Copy 1	Start of Copy 2
56	F	93938	93956
56	P	93938	149737
56	P	93956	149755
56	F	149737	149755

*P indicates palindromic repeats; F indicates forward repeats.

Overlapped repeats have been manually removed while calculating total length.

Online-only Table 1

Long repeats (repeat unit > 50 bp) in Scaffold 1 of the C. sinensis var. assamica mt genome.

Repeat Length	Type*	Start of Copy 2	Start of Copy 1
5119	F	207173	443366
2191	F	389017	391244
1963	F	210330	212292
1962	F	212292	446523
1930	F	383226	385188
1650	F	205522	207173
1650	F	205522	443366
1469	F	538290	539780
814	F	496567	498047
705	F	619432	621461
665	F	497382	498862
255	P	151984	200526
228	P	448476	544136
204	F	277002	363807
131	P	73675	482324
125	F	301855	468834
104	F	297204	623713
88	F	228824	559689
87	F	594334	641398
84	F	530415	646532
82	P	224027	395044
82	F	509347	623862
81	P	152363	200041
80	F	304361	306020
78	P	299987	587603
74	F	165777	570981
70	F	165878	571083
69	F	123050	384677
69	F	123050	386639
67	F	18495	27472
66	F	299782	537227
66	P	364849	599005
66	F	684228	684285
65	P	508609	683320
64	F	542385	560020
63	F	605770	619261
62	P	70098	424512
62	F	151516	524252
62	P	156839	486845
61	F	123120	384747
61	F	123120	386709
61	P	142673	486240
60	F	302012	395122
59	P	265260	472040
58	F	285626	402303
57	P	152478	199950
57	F	276881	363698
56	F	402376	658389
55	P	41703	667438
55	F	258578	486959

*P indicates palindromic repeats; F indicates forward repeats. Overlapped repeats have been manually removed while calculating total length.

Online-only Table 2

Long repeats (repeat unit > 50 bp) in Scaffold 2 of the C. sinensis var. assamica mt genome.

Repeat Length	Type*	Start of Copy 1	Start of Copy 2
704	F	30739	32294
156	P	29085	67620
86	F	67291	136332
67	P	4255	17574
67	P	23998	45730
62	F	67282	135282
55	F	120664	129253
53	F	135291	136332

*P indicates palindromic repeats; F indicates forward repeats. Overlapped repeats have been manually removed while calculating total length.

Prediction of RNA-editing sites

Putative RNA editing sites in protein-coding genes were predicted using the PREP-cp and PREP-mt Web-based program (http://prep.unl.edu/)[51,52]. To achieve a balanced trade-off between the number of false positive and false negative sites, the cutoff score (C-value) was set to 0.8 and 0.6, respectively[53].

Almost all transcripts of protein encoding genes in the plant mitochondria are subject to RNA editing except the T-urf13 gene[54]. Our results showed that the extent of RNA editing varied by gene for both cp and mt genomes of C. sinensis var. assamica. In the C. sinensis var. assamica cp genome, we detected 54 RNA-editing sites in 21 protein-coding genes, ranging from one editing site in atpF, atpI, petB, psaI, psbE, psbF, rpoA, rps2 and rps8 to 8 editing sites in ndhB (Online-only Table 3). In the C. sinensis var. assamica mt genome, we predicted 478 RNA-editing sites in 42 protein-coding genes; they varied from two editing site in atp9 (of scaffold2), sdh3 (of scaffold1 and scaffold2, respectively) and rps14 (of scaffold2) to 35 editing sites in ccmFn (of scaffold1) (Online-only Table 4–5).

Online-only Table 3

Predicted RNA-editing sites in the C. sinensis var. assamica cp genome. (*The cutoff score (C-value) was set to 0.8).

No.	Gene	Nucleotide Pos	AA Pos	Effect	Score*
1	accD	64	22	CGG (R) => TGG (W)	1
2	accD	1469	490	CCT (P) => CTT (L)	1
3	atpA	791	264	CCA (P) => CTA (L)	1
4	atpA	914	305	TCA (S) => TTA (L)	1
5	atpF	92	31	CCA (P) => CTA (L)	0.86
6	atpI	134	45	GCT (A) => GTT (V)	1
7	matK	445	149	CAC (H) => TAC (Y)	1
8	matK	467	156	TCG (S) => TTG (L)	1
9	matK	631	211	CAT (H) => TAT (Y)	1
10	matK	1234	412	CAT (H) => TAT (Y)	1
11	ndhA	341	114	TCA (S) => TTA (L)	1
12	ndhA	566	189	TCA (S) => TTA (L)	1
13	ndhA	1028	343	TCT (S) => TTT (F)	1
14	ndhA	1073	358	TCT (S) => TTT (F)	1
15	ndhB	149	50	TCA (S) => TTA (L)	1
16	ndhB	467	156	CCA (P) => CTA (L)	1
17	ndhB	586	196	CAT (H) => TAT (Y)	1
18	ndhB	611	204	TCA (S) => TTA (L)	0.8
19	ndhB	737	246	CCA (P) => CTA (L)	1
20	ndhB	746	249	TCT (S) => TTT (F)	1
21	ndhB	830	277	TCA (S) => TTA (L)	1
22	ndhB	1481	494	CCA (P) => CTA (L)	1
23	ndhD	20	7	ACG (T) => ATG (M)	1
24	ndhD	401	134	TCA (S) => TTA (L)	1
25	ndhD	692	231	TCA (S) => TTA (L)	1
26	ndhD	896	299	TCA (S) => TTA (L)	1
27	ndhD	905	302	CCT (P) => CTT (L)	1
28	ndhD	1328	443	TCA (S) => TTA (L)	0.8
29	ndhF	205	69	CAT (H) => TAT (Y)	0.8
30	ndhF	290	97	TCA (S) => TTA (L)	1
31	ndhG	166	56	CAT (H) => TAT (Y)	0.8
32	ndhG	314	105	ACA (T) => ATA (I)	0.8
33	petB	641	214	CCA (P) => CTA (L)	1
34	psaI	80	27	TCT (S) => TTT (F)	0.86
35	psbE	214	72	CCT (P) => TCT (S)	1
36	psbF	77	26	TCT (S) => TTT (F)	1
37	rpoA	368	123	TCG (S) => TTG (L)	1
38	rpoB	338	113	TCT (S) => TTT (F)	1
39	rpoB	473	158	TCA (S) => TTA (L)	0.86
40	rpoB	551	184	TCA (S) => TTA (L)	1
41	rpoB	566	189	TCG (S) => TTG (L)	1
42	rpoB	973	325	CTT (L) => TTT (F)	0.86
43	rpoB	2000	667	TCT (S) => TTT (F)	1
44	rpoB	2336	779	ACA (T) => ATA (I)	1
45	rpoC1	41	14	TCA (S) => TTA (L)	1
46	rpoC1	1556	519	TCG (S) => TTG (L)	1
47	rpoC2	1505	502	ACG (T) => ATG (M)	0.86
48	rpoC2	2290	764	CGG (R) => TGG (W)	1
49	rpoC2	2726	909	ACT (T) => ATT (I)	1
50	rpoC2	3728	1243	TCA (S) => TTA (L)	0.86
51	rps2	248	83	TCA (S) => TTA (L)	1
52	rps8	182	61	TCA (S) => TTA (L)	0.86
53	rps14	80	27	TCA (S) => TTA (L)	1
54	rps14	149	50	CCA (P) => CTA (L)	1

Online-only Table 4

Predicted RNA-editing sites in Scaffold 1 of the C. sinensis var. assamica mt genome.

No.	Gene	Nucleotide Position	AA Pos	Effect	Score*
1	matR	32	11	TCC (S) => TTC (F)	0.62
2	matR	236	79	TCC (S) => TTC (F)	0.62
3	matR	326	109	CCA (P) => CTA (L)	1
4	matR	917	306	TCA (S) => TTA (L)	1
5	matR	1442	481	GCC (A) => GTC (V)	0.62
6	matR	1667	556	TCC (S) => TTC (F)	1
7	matR	1688	563	CCT (P) => CTT (L)	1
8	matR	1708	570	CGC (R) => TGC (C)	1
9	matR	1744	582	CAC (H) => TAC (Y)	1
10	matR	1775	592	CCG (P) => CTG (L)	1
11	matR	1814	605	CCA (P) => CTA (L)	0.88
12	matR	1832	611	TCA (S) => TTA (L)	0.88
13	ccmFn	38	13	CCG (P) => CTG (L)	1
14	ccmFn	98	33	CCT (P) => CTT (L)	1
15	ccmFn	137	46	TCG (S) => TTG (L)	1
16	ccmFn	142	48	CGT (R) => TGT (C)	1
17	ccmFn	151	51	CCT (P) => TCT (S)	0.83
18	ccmFn	248	83	TCA (S) => TTA (L)	1
19	ccmFn	256	86	CGG (R) => TGG (W)	1
20	ccmFn	283	95	CTT (L) => TTT (F)	0.83
21	ccmFn	334	112	CAT (H) => TAT (Y)	0.67
22	ccmFn	356	119	TCC (S) => TTC (F)	0.67
23	ccmFn	391	131	CCT (P) => TCT (S)	1
24	ccmFn	478	160	CGT (R) => TGT (C)	0.83
25	ccmFn	706	236	CCT (P) => TTT (F)	0.67
26	ccmFn	707	236	CCT (P) => TTT (F)	0.67
27	ccmFn	716	239	TCA (S) => TTA (L)	0.83
28	ccmFn	754	252	CGT (R) => TGT (C)	1
29	ccmFn	776	259	TCA (S) => TTA (L)	1
30	ccmFn	788	263	CCA (P) => CTA (L)	1
31	ccmFn	803	268	TCA (S) => TTA (L)	1
32	ccmFn	893	298	GCG (A) => GTG (V)	1
33	ccmFn	952	318	CGC (R) => TGC (C)	1
34	ccmFn	1270	424	CGG (R) => TGG (W)	1
35	ccmFn	1298	433	CCA (P) => CTA (L)	1
36	ccmFn	1315	439	CAT (H) => TAT (Y)	1
37	ccmFn	1330	444	CGG (R) => TGG (W)	1
38	ccmFn	1348	450	CGG (R) => TGG (W)	1
39	ccmFn	1381	461	CGG (R) => TGG (W)	1
40	ccmFn	1399	467	CGT (R) => TGT (C)	1
41	ccmFn	1442	481	TCG (S) => TTG (L)	1
42	ccmFn	1462	488	CTT (L) => TTT (F)	1
43	ccmFn	1466	489	CCA (P) => CTA (L)	1
44	ccmFn	1478	493	TCA (S) => TTA (L)	1
45	ccmFn	1487	496	TCT (S) => TTT (F)	1
46	ccmFn	1513	505	CCC (P) => TCC (S)	1
47	ccmFn	1561	521	CGG (R) => TGG (W)	0.67
48	nad5	155	52	CCG (P) => CTG (L)	1
49	nad5	238	80	CCG (P) => TCG (S)	0.8
50	nad5	269	90	TCC (S) => TTC (F)	0.7
51	nad5	355	119	CCT (P) => TTT (F)	1
52	nad5	356	119	CCT (P) => TTT (F)	1
53	nad5	371	124	CCA (P) => CTA (L)	0.9
54	nad5	395	132	TCT (S) => TTT (F)	0.9
55	nad5	503	168	CCT (P) => CTT (L)	1
56	nad5	536	179	CCT (P) => CTT (L)	1
57	nad5	626	209	TCT (S) => TTT (F)	0.9
58	nad5	628	210	CGC (R) => TGC (C)	0.9
59	nad5	673	225	CTT (L) => TTT (F)	0.9
60	nad5	710	237	TCG (S) => TTG (L)	1
61	nad5	722	241	TCA (S) => TTA (L)	1
62	nad5	832	278	CCA (P) => TCA (S)	0.9
63	nad5	872	291	ACG (T) => ATG (M)	1
64	nad5	1307	436	TCA (S) => TTA (L)	1
65	nad4	29	10	TCC (S) => TTC (F)	0.67
66	nad4	74	25	ACT (T) => ATT (I)	0.89
67	nad4	77	26	CCT (P) => CTT (L)	0.78
68	nad4	107	36	CCG (P) => CTG (L)	1
69	nad4	154	52	CCC (P) => TCC (S)	1
70	nad4	158	53	CCT (P) => CTT (L)	1
71	nad4	166	56	CGG (R) => TGG (W)	1
72	nad4	197	66	TCT (S) => TTT (F)	1
73	nad4	362	121	ACA (T) => ATA (I)	0.89
74	nad4	368	123	TCT (S) => TTT (F)	1
75	nad4	376	126	CGT (R) => TGT (C)	0.78
76	nad4	403	135	CGC (R) => TGC (C)	1
77	nad4	416	139	CCT (P) => CTT (L)	0.89
78	nad4	433	145	CTT (L) => TTT (F)	1
79	nad4	436	146	CCC (P) => TTC (F)	0.89
80	nad4	437	146	CCC (P) => TTC (F)	0.89
81	nad4	449	150	CCA (P) => CTA (L)	1
82	nad4	547	183	CTC (L) => TTC (F)	0.67
83	nad4	1336	446	CAC (H) => TAC (Y)	1
84	nad4	1352	451	CCG (P) => CTG (L)	1
85	nad4	1357	453	CGC (R) => TGC (C)	1
86	atp6	37	13	CCA (P) => TCA (S)	0.75
87	atp6	116	39	TCA (S) => TTA (L)	1
88	atp6	167	56	CCG (P) => CTG (L)	1
89	atp6	173	58	CCG (P) => CTG (L)	1
90	atp6	224	75	TCC (S) => TTC (F)	1
91	atp6	229	77	CGC (R) => TGC (C)	0.75
92	atp6	236	79	TCG (S) => TTG (L)	0.67
93	atp6	254	85	TCG (S) => TTG (L)	1
94	atp6	262	88	CGT (R) => TGT (C)	1
95	atp6	269	90	CCC (P) => CTC (L)	1
96	atp6	401	134	TCA (S) => TTA (L)	1
97	atp6	460	154	CCT (P) => TCT (S)	1
98	atp6	463	155	CAT (H) => TAT (Y)	1
99	atp6	485	162	CCA (P) => CTA (L)	1
100	atp6	527	176	TCA (S) => TTA (L)	1
101	atp6	548	183	TCC (S) => TTC (F)	1
102	atp6	635	212	CCG (P) => CTG (L)	1
103	atp6	656	219	TCA (S) => TTA (L)	1
104	atp6	664	222	CAT (H) => TAT (Y)	1
105	atp6	671	224	TCT (S) => TTT (F)	1
106	atp6	680	227	TCA (S) => TTA (L)	1
107	atp6	707	236	ACA (T) => ATA (I)	0.92
108	atp6	718	240	CAA (Q) => TAA (X)	1
109	mttB	58	20	CAT (H) => TAT (Y)	0.88
110	mttB	83	28	TCG (S) => TTG (L)	0.88
111	mttB	91	31	CCA (P) => TCA (S)	1
112	mttB	127	43	CGT (R) => TGT (C)	0.88
113	mttB	134	45	CCA (P) => CTA (L)	0.62
114	mttB	164	55	TCC (S) => TTC (F)	0.75
115	mttB	196	66	CCG (P) => TCG (S)	1
116	mttB	253	85	CGT (R) => TGT (C)	0.62
117	mttB	290	97	TCT (S) => TTT (F)	1
118	mttB	299	100	TCG (S) => TTG (L)	0.75
119	ccmB	28	10	CAT (H) => TAT (Y)	0.89
120	ccmB	43	15	CCC (P) => TCC (S)	0.67
121	ccmB	71	24	CCA (P) => CTA (L)	1
122	ccmB	80	27	TCG (S) => TTG (L)	1
123	ccmB	128	43	TCA (S) => TTA (L)	1
124	ccmB	137	46	TCC (S) => TTC (F)	1
125	ccmB	149	50	CCG (P) => CTG (L)	1
126	ccmB	154	52	CGG (R) => TGG (W)	1
127	ccmB	160	54	CCT (P) => TCT (S)	0.67
128	ccmB	164	55	CCG (P) =>=> CTG (L)	0.89
129	ccmB	172	58	CCT (P) => TCT (S)	0.89
130	ccmB	179	60	CCT (P) => CTT (L)	1
131	ccmB	193	65	CCT (P) => TTT (F)	0.89
132	ccmB	194	65	CCT (P) => TTT (F)	0.89
133	ccmB	286	96	CGG (R) => TGG (W)	1
134	ccmB	304	102	CGT (R) => TGT (C)	0.78
135	ccmB	313	105	CGT (R) => TGT (C)	0.89
136	ccmB	338	113	CCG (P) => CTG (L)	1
137	ccmB	367	123	CGG (R) => TGG (W)	0.78
138	ccmB	424	142	CGT (R) => TGT (C)	0.89
139	ccmB	428	143	TCG (S) => TTG (L)	1
140	ccmB	467	156	TCG (S) => TTG (L)	0.89
141	ccmB	476	159	CCA (P) => CTA (L)	0.89
142	ccmB	485	162	TCA (S) => TTA (L)	1
143	ccmB	494	165	TCA (S) => TTA (L)	1
144	ccmB	503	168	CCA (P) => CTA (L)	1
145	ccmB	512	171	TCT (S) => TTT (F)	1
146	ccmB	514	172	CGT (R) => TGT (C)	1
147	ccmB	551	184	TCA (S) => TTA (L)	1
148	ccmB	554	185	TCG (S) => TTG (L)	0.89
149	ccmB	566	189	TCC (S) => TTC (F)	0.78
150	ccmB	569	190	TCT (S) => TTT (F)	0.78
151	ccmB	572	191	CCG (P) => CTG (L)	1
152	ccmB	596	199	TCG (S) => TTG (L)	0.89
153	rpl10	101	34	TCG (S) => TTG (L)	0.83
154	rpl10	239	80	TCG (S) => TTG (L)	0.83
155	rpl10	314	105	TCA (S) => TTA (L)	0.83
156	rps7	152	51	CCA (P) => CTA (L)	0.75
157	rps7	343	115	CAC (H) => TAC (Y)	0.62
158	rps7	368	123	TCA (S) => TTA (L)	0.88
159	atp1	1039	347	CCC (P) => TCC (S)	1
160	atp1	1064	355	TCG (S) => TTG (L)	1
161	atp1	1178	393	TCA (S) => TTA (L)	0.9
162	atp1	1216	406	CTT (L) => TTT (F)	1
163	atp1	1292	431	CCG (P) => CTG (L)	0.8
164	atp1	1415	472	CCA (P) => CTA (L)	1
165	atp1	1490	497	CCA (P) => CTA (L)	0.9
166	atp9	20	7	TCA (S) => TTA (L)	1
167	atp9	50	17	TCA (S) => TTA (L)	1
168	atp9	82	28	CTT (L) => TTT (F)	1
169	atp9	92	31	TCG (S) => TTG (L)	1
170	atp9	134	45	TCA (S) => TTA (L)	1
171	atp9	182	61	TCG (S) => TTG (L)	1
172	atp9	191	64	CCA (P) => CTA (L)	1
173	atp9	212	71	TCA (S) => TTA (L)	1
174	atp9	215	72	TCC (S) => TTC (F)	1
175	atp9	223	75	CGA (R) => TGA (X)	1
176	sdh3	67	23	CCC (P) => TCC (S)	1
177	sdh3	376	126	CTC (L) => TTC (F)	0.83
178	rpl16	79	27	CAG (Q) => TAG (X)	1
179	rpl16	227	76	ACT (T) => ATT (I)	1
180	rpl16	355	119	CTC (L) => TTC (F)	0.89
181	rpl16	524	175	CCA (P) => CTA (L)	1
182	rpl16	530	177	TCG (S) => TTG (L)	0.75
183	rps3	314	105	CCA (P) => CTA (L)	0.86
184	rps3	647	216	CCG (P) => CTG (L)	1
185	rps3	674	225	CCG (P) => CTG (L)	0.86
186	rps3	785	262	TCA (S) => TTA (L)	1
187	rps3	838	280	CGT (R) => TGT (C)	1
188	rps3	902	301	TCA (S) => TTA (L)	0.86
189	rps19	62	21	TCG (S) => TTG (L)	1
190	rps19	109	37	CCT (P) => TTT (F)	1
191	rps19	110	37	CCT (P) => TTT (F)	1
192	rpl2	215	72	CCA (P) => CTA (L)	0.75
193	rpl2	329	110	CCA (P) => CTA (L)	1
194	rpl2	494	165	GCG (A) => GTG (V)	0.67
195	rpl2	517	173	CTC (L) => TTC (F)	1
196	rpl2	550	184	CCC (P) => TCC (S)	1
197	atp8	47	16	TCA (S) => TTA (L)	1
198	atp8	58	20	CTC (L) => TTC (F)	1
199	atp8	452	151	CCA (P) => CTA (L)	0.75
200	cox3	289	97	CTT (L) => TTT (F)	0.92
201	cox3	304	102	CGG (R) => TGG (W)	1
202	cox3	311	104	TCT (S) => TTT (F)	0.92
203	cox3	314	105	TCT (S) => TTT (F)	0.92
204	cox3	419	140	CCC (P) => CTC (L)	1
205	cox3	422	141	CCT (P) => CTT (L)	0.92
206	cox3	512	171	TCA (S) => TTA (L)	0.75
207	cox3	653	218	TCG (S) => TTG (L)	1
208	cox3	754	252	CGG (R) => TGG (W)	0.92
209	cox3	764	255	CCA (P) => CTA (L)	0.92
210	sdh4	155	52	CCA (P) => CTA (L)	0.88
211	sdh4	203	68	CCA (P) => CTA (L)	0.75
212	sdh4	259	87	CAT (H) => TAT (Y)	0.88
213	cox1	155	52	TCT (S) => TTT (F)	1
214	cox1	167	56	TCT (S) => TTT (F)	1
215	cox1	265	89	CCA (P) => TCA (S)	1
216	cox1	356	119	TCA (S) => TTA (L)	1
217	cox1	365	122	TCT (S) => TTT (F)	1
218	cox1	428	143	TCC (S) => TTC (F)	1
219	cox1	464	155	TCA (S) => TTA (L)	1
220	cox1	503	168	CCA (P) => CTA (L)	1
221	cox1	581	194	TCT (S) => TTT (F)	1
222	cox1	628	210	CGG (R) => TGG (W)	1
223	cox1	659	220	CCC (P) => CTC (L)	1
224	cox1	674	225	TCC (S) => TTC (F)	1
225	cox1	758	253	ACA (T) => ATA (I)	1
226	cox1	773	258	TCT (S) => TTT (F)	1
227	cox1	950	317	TCC (S) => TTC (F)	1
228	cox1	1099	367	CAC (H) => TAC (Y)	1
229	cox1	1187	396	CCG (P) => CTG (L)	0.89
230	cox1	1318	440	CGT (R) => TGT (C)	0.78
231	cox1	1346	449	TCA (S) => TTA (L)	1
232	cox1	1402	468	CCA (P) => TCA (S)	1
233	cox1	1412	471	TCG (S) => TTG (L)	1
234	nad7	38	13	TCG (S) => TTG (L)	0.75
235	nad7	77	26	TCA (S) => TTA (L)	1
236	nad7	83	28	TCA (S) => TTA (L)	1
237	nad7	137	46	TCA (S) => TTA (L)	1
238	nad7	205	69	CAT (H) => TAT (Y)	1
239	nad7	212	71	TCA (S) => TTA (L)	1
240	nad7	277	93	CGT (R) => TGT (C)	1
241	nad7	296	99	TCA (S) => TTA (L)	0.88
242	nad7	305	102	TCA (S) => TTA (L)	1
243	nad7	344	115	TCA (S) => TTA (L)	1
244	nad7	494	165	TCC (S) => TTC (F)	1
245	nad7	539	180	TCA (S) => TTA (L)	0.88
246	nad7	812	271	TCA (S) => TTA (L)	0.88
247	nad7	859	287	CCT (P) => TCT (S)	0.88
248	nad7	943	315	CGT (R) => TGT (C)	1
249	nad7	965	322	TCT (S) => TTT (F)	1
250	nad7	989	330	TCT (S) => TTT (F)	1
251	nad7	1010	337	CCA (P) => CTA (L)	1
252	nad7	1052	351	TCT (S) => TTT (F)	1
253	nad9	428	143	TCC (S) => TTC (F)	0.73
254	nad9	506	169	TCT (S) => TTT (F)	0.75
255	nad9	527	176	CCA (P) => CTA (L)	0.92
256	nad9	581	194	TCG (S) => TTG (L)	0.92
257	nad9	604	202	CAT (H) => TAT (Y)	1
258	nad9	712	238	CCG (P) => TCG (S)	0.83
259	nad9	742	248	CGG (R) => TGG (W)	1
260	nad9	782	261	TCC (S) => TTC (F)	1
261	nad9	812	271	TCA (S) => TTA (L)	1
262	nad9	853	285	CTT (L) => TTT (F)	1
263	nad9	953	318	TCT (S) => TTT (F)	1
264	nad4L	11	4	TCT (S) => TTT (F)	1
265	nad4L	17	6	TCA (S) => TTA (L)	1
266	nad4L	25	9	CGG (R) => TGG (W)	1
267	nad4L	56	19	CCT (P) => CTT (L)	1
268	nad4L	65	22	TCA (S) => TTA (L)	1
269	nad4L	70	24	CCA (P) => TCA (S)	1
270	nad4L	80	27	TCA (S) => TTA (L)	1
271	nad4L	101	34	TCG (S) => TTG (L)	0.88
272	nad4L	128	43	TCG (S) => TTG (L)	1
273	nad4L	149	50	TCA (S) => TTA (L)	0.75
274	nad4L	158	53	TCA (S) => TTA (L)	0.88
275	nad4L	167	56	CCA (P) => CTA (L)	0.88
276	nad4L	200	67	TCA (S) => TTA (L)	1
277	nad4L	251	84	TCT (S) => TTT (F)	0.88
278	atp4	71	24	TCA (S) => TTA (L)	1
279	atp4	89	30	TCA (S) => TTA (L)	1
280	atp4	118	40	CGT (R) => TGT (C)	0.71
281	atp4	215	72	TCG (S) => TTG (L)	1
282	atp4	248	83	CCT (P) => CTT (L)	1
283	atp4	395	132	TCA (S) => TTA (L)	1
284	atp4	407	136	CCA (P) => CTA (L)	0.71
285	atp4	416	139	ACT (T) => ATT (I)	0.86
286	ccmC	76	26	CGG (R) => TGG (W)	0.78
287	ccmC	103	35	CAT (H) => TAT (Y)	1
288	ccmC	115	39	CGG (R) => TGG (W)	0.78
289	ccmC	133	45	CTT (L) => TTT (F)	0.67
290	ccmC	161	54	CCG (P) => CTG (L)	0.78
291	ccmC	179	60	GCG (A) => GTG (V)	0.78
292	ccmC	184	62	CGG (R) => TGG (W)	1
293	ccmC	299	100	TCT (S) => TTT (F)	1
294	ccmC	331	111	CGG (R) => TGG (W)	1
295	ccmC	395	132	TCG (S) => TTG (L)	1
296	ccmC	400	134	CTT (L) => TTT (F)	0.89
297	ccmC	421	141	CGT (R) => TGT (C)	0.78
298	ccmC	436	146	CCT (P) => TCT (S)	0.89
299	ccmC	446	149	CCG (P) => CTG (L)	0.78
300	ccmC	451	151	CCT (P) => TCT (S)	1
301	ccmC	458	153	TCA (S) => TTA (L)	0.78
302	ccmC	463	155	CGT (R) => TGT (C)	1
303	ccmC	467	156	GCT (A) => GTT (V)	0.78
304	ccmC	473	158	CCG (P) => CTG (L)	1
305	ccmC	497	166	TCT (S) => TTT (F)	1
306	ccmC	521	174	TCG (S) => TTG (L)	1
307	ccmC	548	183	TCT (S) => TTT (F)	1
308	ccmC	568	190	CCT (P) => TCT (S)	1
309	ccmC	575	192	CCC (P) => CTC (L)	1
310	ccmC	605	202	TCC (S) => TTC (F)	1
311	ccmC	608	203	CCC (P) => CTC (L)	0.89
312	ccmC	614	205	TCA (S) => TTA (L)	0.78
313	ccmC	619	207	CGT (R) => TGT (C)	0.78
314	ccmC	650	217	CCT (P) => CTT (L)	0.78
315	ccmC	656	219	CCA (P) => CTA (L)	0.89
316	ccmC	673	225	CCT (P) => TCT (S)	0.78
317	cox2	71	24	TCT (S) => TTT (F)	1
318	cox2	161	54	TCA (S) => TTA (L)	0.95
319	cox2	163	55	CGG (R) => TGG (W)	1
320	cox2	253	85	CGG (R) => TGG (W)	1
321	cox2	278	93	CCG (P) => CTG (L)	1
322	cox2	379	127	CGG (R) => TGG (W)	1
323	cox2	443	148	ACG (T) => ATG (M)	1
324	cox2	461	154	CCA (P) => CTA (L)	1
325	cox2	476	159	TCA (S) => TTA (L)	1
326	cox2	544	182	CCT (P) => TCT (S)	1
327	cox2	557	186	CCT (P) => CTT (L)	1
328	cox2	581	194	TCA (S) => TTA (L)	1
329	cox2	632	211	TCG (S) => TTG (L)	0.84
330	cox2	698	233	ACG (T) => ATG (M)	1
331	cox2	742	248	CGG (R) => TGG (W)	1
332	rps13	5	2	TCA (S) => TTA (L)	0.6
333	rps13	26	9	TCA (S) => TTA (L)	0.9
334	rps13	56	19	TCA (S) => TTA (L)	0.9
335	rps13	100	34	CGT (R) => TGT (C)	0.9
336	rps13	287	96	TCG (S) => TTG (L)	1
337	rps4	133	45	CCG (P) => TCG (S)	0.67
338	rps4	164	55	TCA (S) => TTA (L)	1
339	rps4	184	62	CCC (P) => TCC (S)	0.83
340	rps4	193	65	CAT (H) => TAT (Y)	1
341	rps4	257	86	CCA (P) => CTA (L)	1
342	rps4	266	89	CCA (P) => CTA (L)	0.83
343	rps4	278	93	TCG (S) => TTG (L)	0.67
344	rps4	290	97	CCG (P) => CTG (L)	0.83
345	rps4	335	112	CCG (P) => CTG (L)	1
346	rps4	482	161	TCA (S) => TTA (L)	1
347	rps4	914	305	TCG (S) => TTG (L)	0.83
348	rps4	925	309	CAT (H) => TAT (Y)	0.83
349	rps4	935	312	CCA (P) => CTA (L)	0.67
350	rps4	950	317	TCT (S) => TTT (F)	1
351	rps4	1001	334	CCA (P) => CTA (L)	0.83
352	rps4	1010	337	CCT (P) => CTT (L)	1
353	rps4	1015	339	CGG (R) => TGG (W)	1
354	nad1	8	3	CCT (P) => CTT (L)	0.9
355	nad1	65	22	TCC (S) => TTC (F)	1
356	nad1	100	34	CCT (P) => TCT (S)	0.9
357	nad1	149	50	GCG (A) => GTG (V)	0.9
358	nad1	209	70	TCC (S) => TTC (F)	1
359	nad1	308	103	TCA (S) => TTA (L)	1
360	nad1	434	145	ACT (T) => ATT (I)	1
361	nad6	7	3	CTT (L) => TTT (F)	1
362	nad6	83	28	TCG (S) => TTG (L)	1
363	nad6	88	30	CCC (P) => TTC (F)	0.7
364	nad6	89	30	CCC (P) => TTC (F)	0.7
365	nad6	95	32	CCA (P) => CTA (L)	1
366	nad6	103	35	CGC (R) => TGC (C)	1
367	nad6	161	54	CCA (P) => CTA (L)	1
368	nad6	169	57	CAT (H) => TAT (Y)	1
369	nad6	191	64	TCA (S) => TTA (L)	1
370	nad6	446	149	TCC (S) => TTC (F)	1
371	nad6	463	155	CCT (P) => TCT (S)	0.8
372	nad6	569	190	TCT (S) => TTT (F)	1
373	nad2	26	9	TCC (S) => TTC (F)	0.89
374	nad2	203	68	TCT (S) => TTT (F)	0.67
375	nad2	206	69	TCC (S) => TTC (F)	1
376	nad2	230	77	TCT (S) => TTT (F)	1
377	nad2	236	79	TCC (S) => TTC (F)	0.67
378	nad2	251	84	CCA (P) => CTA (L)	1
379	nad2	262	88	CGC (R) => TGC (C)	1
380	nad2	289	97	CAT (H) => TAT (Y)	1
381	nad2	296	99	TCA (S) => TTA (L)	1
382	nad2	323	108	CCT (P) => CTT (L)	1
383	nad2	392	131	TCG (S) => TTG (L)	1
384	rps12	71	24	TCG (S) => TTG (L)	0.94
385	rps12	100	34	CGC (R) => TGC (C)	1
386	rps12	104	35	CCG (P) => CTG (L)	1
387	rps12	196	66	CAC (H) => TAC (Y)	0.94
388	rps12	221	74	TCG (S) => TTG (L)	0.88
389	rps12	269	90	TCG (S) => TTG (L)	0.94
390	rps12	284	95	TCC (S) => TTC (F)	0.76
391	nad3	5	2	TCA (S) => TTA (L)	0.79
392	nad3	44	15	CCG (P) => CTG (L)	1
393	nad3	62	21	CCA (P) => CTA (L)	0.95
394	nad3	80	27	CCA (P) => CTA (L)	1
395	nad3	146	49	TCC (S) => TTC (F)	1
396	nad3	208	70	CCT (P) => TTT (F)	0.95
397	nad3	209	70	CCT (P) => TTT (F)	0.95
398	nad3	215	72	CCG (P) => CTG (L)	1
399	nad3	230	77	TCC (S) => TTC (F)	0.86
400	nad3	247	83	CCT (P) => TCT (S)	1
401	nad3	251	84	CCC (P) => CTC (L)	0.91
402	nad3	266	89	CCG (P) => CTG (L)	1
403	nad3	275	92	TCT (S) => TTT (F)	1
404	nad3	317	106	TCT (S) => TTT (F)	0.95
405	nad3	344	115	TCG (S) => TTG (L)	1
406	nad3	349	117	CGG (R) => TGG (W)	1
407	rps1	23	8	CCT (P) => CTT (L)	0.67
408	rps1	56	19	CCT (P) => CTT (L)	0.67
409	rps1	380	127	TCA (S) => TTA (L)	0.67

*The cutoff score (C-value) was set to 0.6.

Online-only Table 5

Predicted RNA-editing sites in Scaffold 2 of the C. sinensis var. assamica mt genome.

No.	Gene	Nucleotide Position	AA Pos	Effect	Score*
1	rps19	116	39	TCG (S) => TTG (L)	1
2	rps19	163	55	CCT (P) => TTT (F)	1
3	rps19	164	55	CCT (P) => TTT (F)	1
4	atp9	53	18	TCA (S) => TTA (L)	1
5	atp9	83	28	TCA (S) => TTA (L)	1
6	cob	118	40	CCG (P) => TCG (S)	0.92
7	cob	178	60	CAC (H) => TAC (Y)	1
8	cob	286	96	CTC (L) => TTC (F)	1
9	cob	298	100	CAC (H) => TAC (Y)	1
10	cob	325	109	CAT (H) => TAT (Y)	1
11	cob	358	120	CGG (R) => TGG (W)	1
12	cob	419	140	CCA (P) => CTA (L)	1
13	cob	568	190	CAT (H) => TAT (Y)	0.92
14	cob	680	227	TCT (S) => TTT (F)	1
15	cob	808	270	CCC (P) => TCC (S)	1
16	cob	853	285	CAT (H) => TAT (Y)	1
17	cob	908	303	CCA (P) => CTA (L)	1
18	cob	914	305	TCT (S) => TTT (F)	1
19	cob	982	328	CAC (H) => TAC (Y)	0.85
20	cob	1015	339	CGC (R) => TGC (C)	1
21	cob	1084	362	CCT (P) => TCT (S)	1
22	cob	1124	375	CCG (P) => CTG (L)	1
23	rps14	47	16	GCG (A) => GTG (V)	0.6
24	rps14	271	91	CCT (P) => TCT (S)	0.6
25	rpl5	35	12	TCA (S) => TTA (L)	0.78
26	rpl5	47	16	CCG (P) => CTG (L)	1
27	rpl5	59	20	CCG (P) => CTG (L)	0.89
28	rpl5	64	22	CAC (H) => TAC (Y)	1
29	rpl5	92	31	TCG (S) => TTG (L)	1
30	rpl5	172	58	CGC (R) => TGC (C)	0.89
31	rpl5	518	173	CCA (P) => CTA (L)	0.89
32	rpl5	521	174	CCG (P) => CTG (L)	1
33	nad2	110	37	TCT (S) => TTT (F)	1
34	nad2	125	42	TCC (S) => TTC (F)	1
35	nad2	272	91	TCT (S) => TTT (F)	0.67
36	nad2	284	95	TCA (S) => TTA (L)	1
37	nad2	293	98	TCT (S) => TTT (F)	1
38	nad2	412	138	CAT (H) => TAT (Y)	1
39	nad2	442	148	CGT (R) => TGT (C)	0.78
40	nad2	446	149	ACT (T) => ATT (I)	1
41	nad2	512	171	TCA (S) => TTA (L)	0.78
42	nad2	542	181	TCA (S) => TTA (L)	1
43	nad2	611	204	TCG (S) => TTG (L)	1
44	nad2	731	244	CCA (P) => CTA (L)	0.67
45	nad2	760	254	CGT (R) => TGT (C)	1
46	nad2	932	311	TCA (S) => TTA (L)	0.67
47	nad2	941	314	CCA (P) => CTA (L)	1
48	nad2	989	330	TCA (S) => TTA (L)	1
49	sdh3	67	23	CCA (P) => TCA (S)	1
50	sdh3	74	25	TCC (S) => TTC (F)	1
51	ccmFc	38	13	TCC (S) => TTC (F)	0.83
52	ccmFc	50	17	CCT (P) => CTT (L)	1
53	ccmFc	52	18	CGT (R) => TGT (C)	1
54	ccmFc	103	35	CCC (P) => TCC (S)	1
55	ccmFc	119	40	TCT (S) => TTT (F)	1
56	ccmFc	122	41	TCC (S) => TTC (F)	1
57	ccmFc	146	49	CCT (P) => CTT (L)	1
58	ccmFc	151	51	CCT (P) => TCT (S)	0.83
59	ccmFc	155	52	TCA (S) => TTA (L)	1
60	ccmFc	160	54	CCT (P) => TCT (S)	0.67
61	ccmFc	203	68	ACG (T) => ATG (M)	1
62	ccmFc	305	102	TCA (S) => TTA (L)	0.83
63	ccmFc	391	131	CGT (R) => TGT (C)	1
64	ccmFc	406	136	CGT (R) => TGT (C)	0.83
65	ccmFc	620	207	GCG (A) => GTG (V)	1
66	ccmFc	704	235	GCT (A) => GTT (V)	0.83
67	ccmFc	1100	367	CCA (P) => CTA (L)	1
68	ccmFc	1121	374	TCG (S) => TTG (L)	1
69	ccmFc	1276	426	CGA (R) => TGA (X)	1

*The cutoff score (C-value) was set to 0.6.

Phylogenetic analyses

To further determine the phylogenetic position of C. sinensis var. assamica we performed phylogenomic analysis of 20 complete cp genomes using the GTR + R + I model under the maximum likelihood (ML) inference in MEGA v.7.0[55]. Besides C. sinensis var. assamica cv. Yunkang 10, we selected cp genomes from the eighteen Camelia species (C. oleifera, C. crapnelliana, C. szechuanensis, C. mairei, C. elongata, C. grandibracteata, C. leptophylla, C. petelotii, C. pubicosta, C. reticulata, C. azalea, C. japonica, C. cuspidata, C. danzaiensis, C. impressinervis, C. pitardii, C. yunnanensis and C. taliensis) using Apterosperm oblata as outgroup. Our results showed that C. sinensis var. assamica was grouped with C. grandibracteata with 100% bootstrap support (Fig. 5).

Fig. 5

Phylogenetic relationships of 20 complete chloroplast genomes. Maximum likelihood phylogenetic tree of C. sinensis var. assamica cv. Yunkang 10 with 18 species in the genus Camellia based on complete chloroplast genome sequences. The chloroplast sequence of Apterosperma oblata was set as outgroup. The position of C. sinensis var. assamica cv. Yunkang 10 is shown in bold and bootstrap values are shown for each node.

The same method was used for phylogenetic analysis with mt genome. A total of thirteen conserved mt protein-coding genes among C. sinensis var. assamica and 14 other plant species were individually aligned with ClustalW[56], and then concatenated to construct a contiguous sequence in the order of cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7 and nad9. The selected 14 species includes Cycas taitungensis, Ginkgo biloba, Triticum aestivum, Oryza sativa, Sorghum bicolor, Zea mays, Gossypium arboretum, G. barbadense, Carica papaya, Vitis vinifera, Hevea brasiliensis, Bupleurum falcatum, Glycine max and Salvia miltiorrhiza. The alignment file was used for the construction of Neighbor-Joining Tree at 1000 bootstrap replicates with MEGA 7.0.26[55]. Our results showed that C. sinensis var. assamica is clearly grouped with other dicots that were separated from monocots of the angiosperms while the two gymnosperms (Cycas taitungensis and Ginkgo biloba) were formed the basal clade (Fig. 6).

Fig. 6

Phylogeny inferred from 13 genes common in the 15 plant mitochondrial genomes. Neighbor-joining tree of C. sinensis var. assamica cv. Yunkang 10 with other 14 species based on 13 conserved protein-coding gene sequences with bootstrap support values on each node. The mt sequence of Cycas taitungensis and Ginkgo biloba were set as outgroup.

Data Records

Raw reads from Illumina are deposited in the NCBI Sequence Read Archive (SRA)[57-62] and BIG Genome Warehouse[63]. Assembled cp genome sequences and accompanying gene annotations of C. sinensis var. assamica are deposited in the NCBI GenBank[64] and BIG Genome Warehouse[65]. The mt genome final assembly and accompanying gene annotations are deposited at NCBI GenBank[66,67] and BIG Genome Warehouse[68]. The alignment and tree files of the chloroplast genome and mitochondrial genome form the Camellia genus were deposited in Figshare database[69].

Technical Validation

Quality filtering of raw reads

The initially generated raw sequencing reads were evaluated in terms of the average quality score at each position, GC content distribution, quality distribution, base composition, and other metrics. Furthermore, the sequencing reads with low quality were also filtered out before the genome assembly and annotation of gene structure.

Assembly and validation

The chloroplast reads were filtered from whole genome Illumina sequencing data of C. sinensis var. assamica. We mapped all the cleaned reads to the reference chloroplast sequence[4] using bowtie2 (version 2.3.4.3)[40] with default parameters. The mapped chloroplast reads were de novo assembled into the complete chloroplast genome.

For mitochondria genome assembly, the PE and MP sequencing reads were used separately. Briefly, we first performed de novo assembly with VELVET v1.2.08[41], which was previously described[42,43]. Scaffolds were constructed using SSPACE v.3.0[44]. False connection was manually removed based on the coverage and distances of paired reads. Gaps between scaffolds were then filled with GapCloser (version 1.12)[45,46] using all pair-end reads.

Measurement(s)	genome assembly
Technology Type(s)	DNA sequencing
Sample Characteristic - Organism	Camellia sinensis