Biochemistry & Molecular Biology Journal Open Access

Keywords

Coptis teeta Wallich; Chloroplast genome; Endangered species; IUCN; Phylogenetic

Introduction

Coptis teeta Wallich, a perennial herb of Ranunculaceae, was endemic to Eastern Himalaya with narrow distribution range. It is a shade-tolerant species, mainly distributed in the moist temperate, evergreen, broad-leaved forests in northwest Yunnan, China, and northeast India and it occupied highly specialized niches in temperate oak - rhododendron forests and restricted to elevations between 2350 and 3100 m [1,2]. The rhizome of this species, known as Yunnan goldthread (Yunlian in Chinese), is important Chinese herbology since the period of Sheng-Nong (3000 B.C.) [1]. It has excellent pharmacological activity and was used to treat various diseases such as diarrhea, disorder of glucose metabolism, hypertension, cardiovascular and cerebral vessel diseases [3]. The previous study revealed that the species have highly specific microsite requirements that cannot be met in other habitats. Owing to the over-exploitation, several anthropogenic factors, and environmental disruption, the wild population of C. teeta decreased rapidly in recent years [4]. C. teeta has been listed in IUCN Red List of Threatened Species (https://www.iucnredlist.org/) as endangered species with status “A2cd”. And it is also included in Category II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [5]. Therefore, it is necessary to protect this endangered plant for its highly economic and ecological values, and for the conservation of biodiversity.

To date, few studies of this species have been performed due to lack of genomic data of C. teeta. The previous studies mainly focus on phylogenetic analysis and biogeographic pattern of Coptis by using two plastid and one nuclear markers including psbA-trnH, trnL-trnF and ITS, and six markers, including five plastid and one nuclear markers, respectively [6,7]. In this study, as a part of the genome sequencing project of C. teeta, we assemble and annotate its complete plastid genome and describing its characteristics.

Materials and Methods

Plant material and DNA extraction

Fresh leaves of C. teeta were collected from Gongshan County (27°73′E, 98°66′N), Yunnan province and voucher specimens were deposited in Yunnan University of Traditional Chinese Medicine. Total genomic DNA was extracted using the modified plant genome kit (Bioteke, Beijing, China). DNA quality was detected by electrophoresis on 1% agarose gel (Figure 1) and 1 μL of DNA sample to test concentration using to the NanoDrop spectrophotometers (ThermoFisher Scientific, Wilmington, Delaware, USA), the result showed that its value is 62.6 ng/μL>50 ng/μL.

Figure 1: 1% Agarose gel electrophoretic separation mapping of total DNA.

Genome sequencing, assembly and annotation

A sequence library was constructed and sequencing was performed using the Illumina HiSeq 2500-PE150 platform (Illumina, CA, USA). All raw reads were filtered by using NGSQC Toolkit_v2.3.3 with default parameters to obtain clean reads that has discard low quality regions [8]. The plastome was de novo assembled using GetOrganelle pipeline (https:// github.com/Kinggerm/GetOrganelle). The complete chloroplast genome was annotated with the online annotation tool GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) [9], using the published cp genome of C. chinensis (NCBI accession number: NC036485) as a reference sequence, then manual correction was performed with Geneious R11software [10]. The plastid genome map was drawn using OGDRAW program (https:// ogdraw.mpimp-golm.mpg.de/) [11]. The annotated cp genome of C. teeta has been deposited into GenBank with the Accession Number MH359096.

Repeats and simple sequence repeats (SSRs) analysis

REPuter [12] was used to find forward and reversed tandem repeats≥15 bp with minimum alignment score and maximum period size at 100 and 500, respectively. IMEx [13] was used to visualize the SSRs with the minimum repeat numbers set to 10, 5, 4, 3, 3 and 3 for mono-, di-, tri-, tetra-, penta- and hexanucleotides, respectively.

Phylogenetic analysis

The phylogenetic analysis was conducted based on 31 published chloroplast genomes to infer phylogenetic position of C. teeta within the family of Ranunculaceae. The cp genome of Nandina domestica (GenBank: DQ923117) was included as outgroup. The LSC, SSC and one IR region of the total 32 chloroplast genomes were aligned using MAFFT 7.308 [14]. The maximum likelihood (ML) tree was reconstructed by RAxML 8.2.11 [15] with the nucleotide substitution model of GTR+G and node support was estimated by means of bootstrap analysis with 1000 replicates.

Results and Discussion

Characteristics of chloroplast genome of C. teeta

The complete chloroplast genome of C. teeta is a circular DNA with 154,280 bp in length, comprising four subunits: one large singe copy (LSC) (87,519 bp), one small singe copy (SSC) (17,595 bp) and two inverted repeat regions (IRs) (24,583 bp for each) (Figure 2). The overall GC content was 38.3 %. The IR regions had a higher GC content (43.3%) than LSC (36.7%) and SSC regions (32.2%). That was caused by the high GC content of the four ribosomal RNA (rRNA) genes (55.5%) presented in the IR regions, similar to that of C. chinensis Franchet [16].

Figure 2: Plastome map of Coptis teeta. The darker gray in the inner circle corresponds to GC content, while the lighter gray corresponds to AT content.

The chloroplast genome of C. teeta contains 125 genes, comprising 81 protein-coding genes (PCGs), 36 transfer RNA (tRNA) genes and 8 rRNA genes. Among these genes, ndhA, ndhB, rpl2, rpoc1, atpF, rps16, trnA-UGC, trnI-GAU, trnV-UAC, trnL-UAA, trnG-UCC, trnK-UUU contain one intron, while the clpP and ycf3 genes contain two introns. The trnK-UUU gene has a larger intron of 2,853 bp compared with other introns. The IR regions include seven tRNAs (trnN-GUU, trnR-ACG, trnA-UGC, trnL-GAU, trnV-GAC, trnL-CAA, trnI-CAU ), four rRNAs without intron (rrn16, rrn23, rrn4.5 and rrn5) and four PCGs (rcf1, rcf2, rps7, ndhB, rps15) and all of these genes are totally duplicated. Additionally, one tRNA (trnL-UAG) and ten PGGs (rpl32, rps15, ccsA, ndhD, Psac, ndhE, ndhG, ndhI, ndhA, ndhH) are contained in SSC region of C. teeta chloroplast genome.

Repeat and SSR analysis

For repeat structure analysis, 42 forward and 23 reverted repeats with minimal repeat size of 15 bp were detected in cp genome of C. teeta (Table 1). Most of these repeats were between 15 and 20 bp. The longest forward repeats were of 39 bp, one sequence of which located in the intergenic region between trnV-GAC and rps7 of inverted repeated regions (IR), the other sequence located in ycf3 of LSC. There are 31 repeats with two sequences started in the same region. Among them, 21 repeats located in the LSC region, 7 located in the IR regions, and 3 located in SSC region. Other 34 repeats with two sequences started in separated regions.

ID	Repeat Start 1	Type	Size (bp)	Repeat Start 2	E-Value	Region	Gene
1	161	F	15	94911	6.23	IRb/LSC	ycf1; IGS
2	1319	F	15	146257	6.23	IRb/SSC	ycf1; ndhA
3	1686	F	18	8903	0.0974	IRb	trnN-GUU; trnl-GAU
4	1729	F	15	22897	6.23	IRb	trnN-GUU; ycf2
5	2352	F	15	34195	6.23	IRb/LSC	trnR-ACG; trnS-GCU
6	2352	F	15	61944	6.23	IRb/LSC	trnR-ACG; trnS-UGA
7	3964	F	15	7706	6.23	IRb	rrn23; trnl-GAU
8	6084	F	15	91596	6.23	IRb/LSC	IGS
9	6598	F	19	7684	0.0244	IRb	trnA-UGC; trnl-GAU
10	7003	F	15	95317	6.23	IRb/LSC	trnA-UGC;IGS
11	7017	F	18	74997	0.0244	IRb/LSC	trnA-UGC; trnF-GAA
12	8184	F	18	8215	0.0974	IRb	IGS
13	9475	F	15	148599	6.23	IRb/SSC	rrn16; ndhH
14	10179	F	17	57871	0.39	IRb/LSC	trnV-GAC; trnT-GGU
15	11638	F	16	38957	1.56	IRb/LSC	IGS
16	11991	F	39	70172	2.21E-14	IRb/LSC	IGS; ycf3
17	19923	F	16	20059	1.56	IRb	ycf2
18	20113	F	16	71145	1.56	IRb/LSC	ycf2; IGS
19	24047	F	16	94901	1.56	IRb/LSC	ycf2; IGS
20	30432	F	20	62669	0.00609	LSC	IGS
21	32229	F	16	81798	1.56	LSC	IGS
22	34192	F	21	61941	0.00152	LSC	trnS-GCU; trnS-UGA
23	35625	F	19	62985	0.0244	LSC	trnG-GCC; trnG-UCC
24	38000	F	16	38051	1.56	LSC	atpF
25	39908	F	17	101565	0.39	LSC	IGS; petB
26	45078	F	16	94306	1.56	LSC	rpoC2; IGS
27	46933	F	16	98952	1.56	LSC	rpoC1; psbB
28	54520	F	16	102145	1.56	LSC	IGS; petB
29	55783	F	16	77512	1.56	LSC	IGS
30	57577	F	17	139324	0.39	LSC/SSC	IGS
31	58768	F	16	92720	1.56	LSC	IGS
32	62639	F	17	71507	0.39	LSC	IGS
33	62772	F	16	152783	1.56	LSC/SSC	IGS
34	63190	F	21	92511	0.00152	LSC	trnfM-CAU; trnP-UGG
35	65213	F	21	67437	0.00152	LSC	psaB; psaA
36	70175	F	37	146807	3.54E-13	LSC/SSC	ycf3; ndhA
37	77524	F	16	95015	1.56	LSC	IGS
38	106539	F	16	139969	1.56	LSC/SSC	rpl14; IGS
39	108087	F	16	138701	1.56	LSC/SSC	IGS
40	138345	F	16	153380	1.56	SSC	IGS
41	139327	F	16	150849	1.56	SSC	IGS
42	139331	F	17	153983	0.39	SSC	IGS
43	412	R	15	52362	6.23	SSC/LSC	IGS; petB
44	1320	R	15	17771	6.23	SSC/IRa	IGS; ycf1
45	10547	R	15	19893	6.23	SSC/IRa	psaC; IGS
46	11639	R	15	101219	6.23	SSC/LSC	ndhD; IGS
47	12190	R	15	118155	6.23	SSC/LSC	ndhD; atpA
48	30692	R	16	74169	1.56	IRa/LSC	rps7; atpB
49	32228	R	17	77513	0.39	IRa/LSC	ndhB; ndhK
50	38958	R	18	101216	0.0974	IRa/LSC	ycf2; IGS
51	39215	R	16	81430	1.56	IRa/LSC	ycf2; rps14
52	39633	R	16	90680	1.56	IRa/LSC	ycf2; rps14
53	40216	R	16	62753	1.56	IRa/LSC	ycf2; petL
54	44548	R	17	139326	0.39	LSC/IRb	rpl22; ndhB
55	44548	R	16	150849	1.56	LSC/IRb	rpl22; rrn23
56	55782	R	17	81799	0.39	LSC	psbB; rps4
57	55869	R	17	73441	0.39	LSC	psbB; atpB
58	71197	R	16	147968	1.56	LSC/IRb	rbcL; trnA-UGC
59	73193	R	16	108492	1.56	LSC	atpB; rpoC2
60	77510	R	16	93741	1.56	LSC	ndhK; psbC
61	77512	R	17	81798	0.39	LSC	ndhK; rps4
62	81801	R	16	93738	1.56	LSC	rps4; psbC;
63	83628	R	16	139655	1.56	LSC/IRb	ycf1; ndhB
64	85488	R	16	149157	1.56	SSC/IRb	IGS; rrn23
65	139356	R	16	139960	1.56	IRb	ndhB
F: Forward; R: Reverted; IGS: intergenic space

Table 1: Repeat sequences in C. teeta chloroplast genome.

cpSSRs markers are widely used to study the population genetics and evolutionary processes of wild plants [17,18]. There were totally 62 SSRs in cp genome of C. teeta, most of which were in LSC (Table 2). Among them, 31 (50.0%) were mononucleotide SSRs, fifteen (24.2%) were dinucleotide SSRs, six (9.7%) were tri-nucleotide SSRs, eight (12.9%) were tetranucleotide SSRs, one (0.2%) was penta-nucleotide SSR, and one (0.2%) was hexa-nucleotide SSRs. Only twelve SSRs were located in genes and the others were in intergenic regions. 30 (96.8%) of the mononucleotide SSRs belonged to the A/T type, which were consistent with the hypothesis that cpSSRs were generally composed of short polyadenine (poly A) or polythymine (poly T) repeats and rarely contained tandem guanine (G) or cytosine (C) repeats. These cpSSR markers can be used in the conservation genetics of C. teeta.

ID	Repeat Motif	Length (bp)	Start	End	Region	Gene
1	(T) 10	10	2610	2619	IRb
2	(AATA) 3	12	11645	11656	IRb
3	(ATCT) 3	12	29571	29582	LSC	trnK-UUU
4	(A) 10	10	30435	30444	LSC
5	(C) 11	11	31241	31251	LSC	rps16
6	(AT) 7	14	32232	32245	LSC
7	(A) 11	11	33582	33592	LSC
8	(T) 11	11	33825	33835	LSC
9	(T) 10	10	35187	35196	LSC	trnG-UCC
10	(T) 10	10	35734	35743	LSC
11	(CTGT) 3	12	36989	37000	LSC	atpA
12	(T) 10	10	40322	40331	LSC
13	(T) 10	10	42352	42361	LSC
14	(T) 14	14	44550	44563	LSC	rpoC2
15	(AT) 5	10	45302	45311	LSC	rpoC2
16	(TA) 5	10	45923	45932	LSC	rpoC2
17	(TA) 5	10	53640	53649	LSC
18	(A) 10	10	54985	54994	LSC
19	(TA) 7	14	55785	55798	LSC
20	(TA) 5	10	55828	55837	LSC
21	(T) 13	13	57582	57594	LSC
22	(TTATA) 3	15	58077	58091	LSC
23	(TA) 6	12	58573	58584	LSC
24	(AAAG) 3	12	59161	59172	LSC
25	(A) 10	10	62672	62681	LSC
26	(ATA) 4	12	68971	68982	LSC
27	(A) 11	11	72877	72887	LSC
28	(A) 14	14	73196	73209	LSC
29	(AT) 5	10	73741	73750	LSC
30	(T) 10	10	75418	75427	LSC
31	(TA) 7	14	77514	77527	LSC
32	(ATA) 4	12	77525	77536	LSC
33	(ACCA) 3	12	78836	78847	LSC	trnV-UAC
34	(ATA) 4	12	81391	81402	LSC	atpB
35	(AT) 7	14	81801	81814	LSC
36	(AT) 6	12	86612	86623	LSC
37	(T) 11	11	86623	86633	LSC
38	(A) 12	12	86710	86721	LSC
39	(T) 10	10	89930	89939	LSC
40	(TA) 6	12	93742	93753	LSC
41	(T) 10	10	94315	94324	LSC
42	(ATA) 4	12	95016	95027	LSC
43	(T) 10	10	97412	97421	LSC	clpP
44	(ATT) 4	12	100305	100316	LSC	psbN
45	(TTCT) 3	12	108128	108139	LSC
46	(A) 16	16	108491	108506	LSC
47	(TTAT) 3	12	125025	125036	IRa
48	(A) 10	10	134063	134072	IRa
49	(T) 14	14	139329	139342	SSC
50	(AT) 5	10	139695	139704	SSC
51	(AT) 5	10	143541	143550	SSC
52	(AT) 5	10	145476	145485	SSC
53	(ATA) 4	12	146996	147007	SSC	ndhA
54	(T) 14	14	150851	150864	SSC
55	(T) 11	11	151012	151022	SSC
56	(CATT) 3	12	151870	151881	SSC
57	(T) 12	12	152189	152200	SSC
58	(T) 11	11	152711	152721	SSC
59	(CTTTTA) 3	18	152750	152767	SSC
60	(A) 11	11	153416	153426	SSC
61	(T) 11	11	153836	153846	SSC
62	(T) 11	11	153984	153994	SSC

Table 2: Simple sequence repeats (SSRs) in the C. teeta chloroplast genome.

Phylogenetic analysis

The phylogenetic tree showed that species from the Ranunculaceae formed a monophyletic clade (Figure 3) and the intra-family topology was consistent with previous studies [6,16,19]. The result strongly supported C. teeta and its congeneric species, C. chinensis, as sister group with 100% bootstrap value. This newly reported chloroplast genome will provide new insights into phylogenetic studies within the Ranunculaceae and facilitate future conservation of C. teeta.

Figure 3: The plastome phylogeny of Ranunculaceae. Bootstraps were shown next to the node.

Conclusion

In this study, we reported and analyzed the first complete chloroplast genome of C. teeta, which are an endemic and endangered plant and a source for famous traditional Chinese medicine.

The circular chloroplast genome exhibits typical quadripartite regions with 154,280 bp in size, including two inverted repeat (IR, 24,583 bp) regions, one large singe copy region (LSC) and one small singe copy region (SSC) of 87,519 bp and 17,595 bp, respectively.

The cp genome of C. teeta was rich in SSRs, which will be informative sources for developing new molecular markers to evaluate genetic diversity and provide effective strategies for conservation of this species. The phylogenetic analysis showed that C. teeta and C. chinensis form one clade as sister group. This information will be useful on phylogenetic analysis of genus Coptis and will also enhance our understanding on the evolutionary relationships among Ranunculaceae.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 81560613), Special subsidies for public health services of TCM “the national survey of TCM resources” (DSS, MOF. No 66/2017), and the Key laboratory training program in Yunnan (2017DG006).

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