Sequencing and Analysis of Chrysanthemum carinatum Schousb and Kalimeris indica. The Complete Chloroplast Genomes Reveal Two Inversions and rbcL as Barcoding of the Vegetable.

Chrysanthemum carinatum Schousb and Kalimeris indica are widely distributed edible vegetables and the sources of the Chinese medicine Asteraceae. The complete chloroplast (cp) genome of Asteraceae usually occurs in the inversions of two regions. Hence, the cp genome sequences and structures of Asteraceae species are crucial for the cp genome genetic diversity and evolutionary studies. Hence, in this paper, we have sequenced and analyzed for the first time the cp genome size of C. carinatum Schousb and K. indica, which are 149,752 bp and 152,885 bp, with a pair of inverted repeats (IRs) (24,523 bp and 25,003) separated by a large single copy (LSC) region (82,290 bp and 84,610) and a small single copy (SSC) region (18,416 bp and 18,269), respectively. In total, 79 protein-coding genes, 30 distinct transfer RNA (tRNA) genes, four distinct rRNA genes and two pseudogenes were found not only in C. carinatum Schousb but also in the K. indica cp genome. Fifty-two (52) and fifty-nine (59) repeats, and seventy (70) and ninety (90) simple sequence repeats (SSRs) were found in the C. carinatum Schousb and K. indica cp genomes, respectively. Codon usage analysis showed that leucine, isoleucine, and serine are the most frequent amino acids and that the UAA stop codon was the significantly favorite stop codon in both cp genomes. The two inversions, the LSC region ranging from trnC-GCA to trnG-UCC and the whole SSC region were found in both of them. The complete cp genome comparison with other Asteraceae species showed that the coding area is more conservative than the non-coding area. The phylogenetic analysis revealed that the rbcL gene is a good barcoding marker for identifying different vegetables. These results give an insight into the identification, the barcoding, and the understanding of the evolutionary model of the Asteraceae cp genome.

1. Introduction

Chloroplasts are crucial for sustaining life on Earth. The chloroplast (cp) genome encodes many key proteins for photosynthesis and other important metabolic processes of plants’ interactions with their environment, such as drought, salt, light, and so on, which give us insights to understand the plant biology, diversity, evolution and climatic adaptation, DNA barcoding and genetic engineering [1,2,3,4,5,6]. Although a relatively conserved architecture and core gene set of cp genomes are shown across higher plants due to the absence of sexual recombination or occurrence of cp capture, considerable variation occurs during evolution [7,8,9,10,11].

A lot of research on the evolution and barcoding of the cp genome in Asteraceae was reported [12]. Particularly interestingly, two cp genome regions have a higher inversion frequency in the sunflower family (Asteraceae) compared to most eudicots. One region locates the SSC (small single copy) and the other region locates between the trnC-GCA to trnG-UCC in the LSC (large single copy) region, which maybe help shape our understanding of Compositae evolution and the adaptive versus non-adaptive processes for cellular and genomic complexity [13,14,15,16,17,18,19,20,21,22,23,24,25]. To date, over 2400 sequenced cp genomes (http://www.ncbi.nlm.nih.gov/genomes/) are available. For the Asteraceae family, 129 whole cp genomes were sequenced, of which 16 have no inversions in the SSC compared with most other land plants [24,25,26]. However, all 16 species maintain inversion regions in LSC. If there does turn out to be a relationship between inversion and certain evolutionary factors in certain systems, it would depend on the acquisition of more genome sequence information, and the relationship between the cp genome structure/content and the complexity evolutionary factor [27].

The Asteraceae (Compositae), or sunflower family is the largest clade in the Asterales and the largest family of flowering plants on Earth. They are angiosperms that appeared about 140 million years ago, comprising 1911 genera and 32,913 species. Nearly one-fourth of all the species of flowering plants belong to the Asteracea, Fabacea, and Orchidaceae families [28,29,30]. The Asteraceae family includes a great diversity of species, including annuals, perennials, stem succulents, vines, shrubs, and trees. Commercially important plants in Asteraceae include food crops, ornamental plants for their flowers, and species with medicinal properties. Kalimeris indica (L.) and Chrysanthemum carinatum Schousb both belong to the Asterales family. Kalimeris indica (L.) is a member of Kalimeris in the sunflower family Asteraceae and is variously named as Ma Lan, Ji Er Chang, and Tian Bian Ju in China. Kalimeris indica (L.) is not only a traditional Chinese medicine and Miaos’ medicinal plant employed in China to treat colds, diarrhea, gastric ulcers, acute gastric abscess, conjunctivitis, acute orchitis, blood vomiting, and injuries, but it is also a popular vegetable [31,32,33,34,35]. As well as Kalimeris indica, Chrysanthemum carinatum Schousb, an important species of Chrysanthemum L. in the Asteraceae family, is a popular annual herb foodstuff in China, because of its fragrance, flavor, and abundant nutritional value. Moreover, the aerial parts of Chrysanthemum L. have been used for the protection or remedy of several diseases in oriental medicinal systems [36,37,38,39,40].

Here, we report the complete cp genome sequence of Kalimeris indica (L.) and Chrysanthemum carinatum Schousb for the first time. Meanwhile, their gene structure and organization were analyzed. Furthermore, the whole cp genome sequences were compared with other genii of the Asteraceae family, especially the inversions, which were found and compared. Phylogenetic analyses of DNA sequences for rcbL of 24 plant species indicated that rcbL would be a good molecular barcoding target.

2. Results

2.1. Features of the C. carinatum Schousb and K. indica cp Genome

The complete cp genome of C. carinatum Schousb and K. indica have a typical quadripartite structure and are 149,752 bp and 152,885 bp in size, respectively (Figure 1). C. carinatum Schousb has an LSC region of 82,290 bp and an SSC region of 18,416 bp which are separated by a pair of IR regions of 24,523 bp (Table 1 and Figure 1A). K. indica has an LSC region of 84,610 bp ranging from trnH-GUG to rps19 (Ribosomal protein S19), a SSC region of 18,269 bp from ycf1 (hypothetical protein 1 gene) to ndhF (NAD(P)H dehydrogenase), a pair of IR regions of 25,003 bp from rps19 to ycf1 and ranging from pseudogene ycf1 to pseudogene rps19, respectively (Table 1 and Figure 1B). Both of them have two inversions like most of the sunflower family species, one inversion occurs in the whole SSC region and the other inversion region is located in the LSC from trnC-GCA to trnG-UCC [13,15,41].

Figure 1

The complete cp genome map of C. carinatum Schousb and K. indica. (A) C. carinatum Schousb; (B) K. indica. The genes marked inside the circle are transcribed clockwise, and those outside are counterclockwise. Genes are color-coded according to their function.

Table 1

The base composition in the C. carinatum Schousb and K. indica cp genomes.

Region	C. carinatum Schousb					K. indica
	T(U) (%)	C (%)	A (%)	G (%)	Length (bp)	T(U) (%)	C (%)	A (%)	G (%)	Length (bp)
LSC	32.4	17.6	32.0	18.1	82,290	32.6	17.3	32.2	17.9	84,610
SSC	35.1	14.7	34.1	16.1	18,416	34.9	14.8	33.9	16.4	18,269
IRa	28.3	22.3	28.6	20.8	24,523	28.3	22.2	28.7	20.8	25,003
IRb	28.6	20.8	28.3	22.3	24,523	28.7	20.8	28.3	22.2	25,003
Total	31.8	19.0	30.7	18.5	149,752	31.8	19.0	30.7	18.5	152,885
CDS	31.5	17.7	30.7	20.1	77,289	31.5	17.7	30.6	20.3	78,372
1st position	23.7	18.9	30.6	26.7	25,763	23.9	18.8	30.6	26.8	26,124
2nd position	32.6	20.4	29.3	17.7	25,763	32.6	20.3	29.3	17.8	26,124
3rd position	38.3	13.7	32.1	15.9	25,763	38.0	13.9	31.8	16.3	26,124

From Table 1, the overall GC content of the C. carinatum Schousb and K. indica cp genome is the same (37.5%), with a higher GC content (43.1% and 43%) in the IR regions than in the LSC (35.7% and 35.2%) and SSC regions (30.8% and 31.2%), which is similar to other Asteraceaes [11,12,13,14,15,17,18,25,26,42].

The AT content in the third, second and first codon position of the C. carinatum Schousb cp genome are 70.4%, 61.9%, and 54.3%, and the AT content in the third, second, and first codon position of K. indica are 69.8%, 61.9%, and 54.5% (Table 1). The results showed that the third codon position and the second codon position have significantly higher AT representation, which is a common feature of the cp genome [43,44,45,46].

2.2. Functional Genes of the C. carinatum Schousb and K. indica cp Genome

There are 113 unique functional genes and two pseudogenes in the C. carinatum Schousb or K. indica cp genome (Table 2). Among the 113 functional genes, 79 protein-coding genes, 30 distinct transfer RNA (tRNA) genes, and four distinct rRNA genes were found not only in the C. carinatum Schousb genome but also in the K. indica cp genome (Table 2). Notably, seven protein-coding, seven tRNA, and all the rRNA genes are duplicated in the IR regions, which is common in most cp genomes [47,48]. Meanwhile, the coding regions constitute 51.6% and 51.3% of the genome in the C. carinatum Schousb and K. indica cp genome, respectively. The non-coding regions constitute 48.4% and 48.7% including the introns, pseudogenes, and intergenic spacers, respectively. Two pseudogenes (ycf1 and rps19) are both found in both C. carinatum Schousb and K. indica cp genomes, a common feature shared with cp genomes from other plants [25,47,48].

Table 2

The genes present in the C. carinatum Schousb and K. indica cp genomes.

Group of Genes	C. carinatum Schousb Gene Names	K. indica Gene Names
Photosystem I	psaA, psaB, psaC, psaI, psaJ	psaA, psaB, psaC, psaI, psaJ
Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
Cytochrome b/f complex	petA, petB *, petD *, petG, petL, petN	petA, petB *, petD *, petG, petL, petN
ATP synthase	atpA, atpB, atpE, atpF *, atpH, atpI	atpA, atpB, atpE, atpF *, atpH, atpI
NADH dehydrogenase	ndhA *, ndhB * (×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK	ndhA *, ndhB * (×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
RuBisCO large subunit	rbcL	rbcL
RNA polymerase	rpoA, rpoB, rpoC1 *, rpoC2	rpoA, rpoB, rpoC1 *, rpoC2
Ribosomal proteins (SSU)	rps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12 ** (×2), rps14, rps15, rps16 *, rps18, rps19	rps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12 ** (×2), rps14, rps15, rps16 *, rps18, rps19
Ribosomal proteins (LSU)	rpl2 * (×2), rpl14, rpl16 *, rpl20, rpl22, rpl23 (×2), rpl32, rpl33, rpl36	rpl2 * (×2), rpl14, rpl16 *, rpl20, rpl22, rpl23 (×2), rpl32, rpl33, rpl36
Miscellaneous proteins	accD, clpP **, matK, ccsA, cemA, infA	accD, clpP **, matK, ccsA, cemA, infA
Hypothetical chloroplast reading frames (ycf)	ycf1, ycf2 (×2), ycf3 **, ycf4	ycf1, ycf2 (×2), ycf3 **, ycf4
Transfer RNAs	trnA-UGC (×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC, trnH-GUG, trnI-CAU (×2), trnI-GAU (×2), trnK-UUU, trnL-CAA(×2), trnL-UAA, trnL-UAG, trnfM-CAU, trnM-CAU, trnN-GUU (×2), trnP-UGG, trnQ-UUG, trnR-ACG (×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (×2), trnV-UAC, trnW-CCA, trnY-GUA	trnA-UGC (×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC, trnH-GUG, trnI-CAU (×2), trnI-GAU (×2), trnK-UUU, trnL-CAA(×2), trnL-UAA, trnL-UAG, trnfM-CAU, trnM-CAU, trnN-GUU (×2), trnP-UGG, trnQ-UUG, trnR-ACG (×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (×2), trnV-UAC, trnW-CCA, trnY-GUA
Ribosomal RNAs	rrn4.5 (×2), rrn5 (×2), rrn16 (×2), rrn23 (×2)	rrn4.5 (×2), rrn5 (×2), rrn16 (×2), rrn23 (×2)
Pseudogenes	ycf1, rps19	ycf1, rps19
Total	132	132

(×2) indicates a duplicated gene; * represents the introns that the gene contains; ** indicates there are two introns that the gene contains.

Among the C. carinatum Schousb or K. indica cp genomes, a total of 18 genes (six tRNA genes and 12 protein-coding genes) contain introns (Table 3). They are mostly located in the LSC region (13 genes), with only one located in the SSC region, and four genes locate in the IR regions. Like other angiosperms, clpP, rps12, and ycf3 contain two introns [48]. Interestingly, rps12 was found to be a trans-spliced gene, whose 5′ end is located in the LSC region and the duplicated 3′ end is located in the IR region. Consistent with many research results, the largest intron is in the trnK-UUU gene, which contains the matk gene in the intron. Moreover, trnL-UAA has the smallest intron in both of them. Comparing these 18 introns between C. carinatum Schousb and K. indica, most of them are in K. indica and are longer than those in C. carinatum Schousb, whereas the introns of rpl16, trnK-UUU, and the introII rps12 are a bit shorter, and the trnV-UAC intron is the same size, which may be one reason why the whole cp genome of K. indica is larger than C. carinatum Schousb.

Table 3

The intron-containing genes of the C. carinatum Schousb and K. indica cp genomes and the lengths of the introns and exons.

C. Gene	Location	Exon I (bp)	Intron I (bp)	Exon II (bp)	Intron II (bp)	Exon III (bp)	K. Gene	Location	Exon I (bp)	Intron I (bp)	Exon II (bp)	Intron II (bp)	Exon III (bp)
atpF	LSC	145	699	410			atpF	LSC	145	709	410
clpP	LSC	71	796	291	611	229	clpP	LSC	71	814	291	615	229
ndhA	SSC	539	1045	553			ndhA	SSC	553	1064	539
ndhB	IR	777	670	756			ndhB	IR	777	674	756
petB	LSC	6	751	642			petB	LSC	6	823	642
petD	LSC	8	675	475			petD	LSC	8	809	475
rpl16	LSC	9	1029	399			rpl16	LSC	9	1098	399
rpl2	IR	391	664	434			rpl2	IR	391	671	434
rpoC1	LSC	432	733	1641			rpoC1	LSC	432	742	1638
rps12 *	LSC	114	-	232	536	26	rps12 *	LSC	114		232	535	26
rps16	LSC	41	891	184			rps16	LSC	41	876	226
trnA-UGC	IR	38	812	35			trnA-UGC	IR	35	820	38
trnG-UCC	LSC	23	722	47			trnG-UCC	LSC	23	732	48
trnI-GAU	IR	43	775	35			trnI-GAU	IR	38	780	35
trnK-UUU	LSC	37	2562	30			trnK-UUU	LSC	37	2539	35
trnL-UAA	LSC	37	422	50			trnL-UAA	LSC	37	438	50
trnV-UAC	LSC	38	573	37			trnV-UAC	LSC	38	573	37
ycf3	LSC	125	698	229	735	153	ycf3	LSC	125	702	229	739	153

* The rps12 gene is a trans-spliced gene with the 5′ end located in the LSC region and a duplicated 3′ end in the IR region.

2.3. Codon Usage of the K. indica and C. carinatum Schousb cp Genome

As shown in Table 4 and Table 5, a total of 25,415 and 26,124 codons are involved in the protein-coding in C. carinatum Schousb and K. indica, respectively. Of these codons, leucine, isoleucine, and serine are the most frequent amino acids in C. carinatum Schousb, which encode in 2778 (10.93%), 2195 (8.64%), and 2080 (8.18%) codons, respectively. Leucine, isoleucine, and serine are the most frequent amino acids in K. indica, which encode in 2795 (10.70%), 2190 (8.38%) and 2131 (8.16%) codons, respectively. Both of them contain 85 stop codons, which is the least frequent codon. The cysteine codons are the least frequent universal amino acid, which has 286 (1.13%) and 294 (1.13%) codons in C. carinatum Schousb and K. indica, respectively.

Table 4

The codon-anticodon recognition pattern and codon usage for the C. carinatum Schousb cp genome.

Amino Acid	Codon	No.	RSCU	tRNA	Amino Acid	Codon	No.	RSCU	tRNA
Phe	UUU	956	1.32		Stop	UAA	49	1.73
Phe	UUC	496	0.68	trnF-GAA	Stop	UAG	21	0.74
Leu	UUA	874	1.89	trnL-UAA	Stop	UGA	15	0.53
Leu	UUG	553	1.19	trnL-CAA	His	CAU	452	1.52
Leu	CUU	620	1.34		His	CAC	143	0.48	trnH-GUG
Leu	CUC	183	0.40		Gln	CAA	703	1.51	trnQ-UUG
Leu	CUA	358	0.77	trnL-UAG	Gln	CAG	227	0.49
Leu	CUG	190	0.41		Asn	AAU	978	1.56
Ile	AUU	1075	1.47		Asn	AAC	277	0.44	trnN-GUU
Ile	AUC	428	0.58	trnI-GAU	Lys	AAA	1026	1.50	trnK-UUU
Ile	AUA	692	0.95	trnI-CAU	Lys	AAG	345	0.50
Met	AUG	613	1.00	trn(f)M-CAU	Asp	GAU	831	1.59
Val	GUU	490	1.44		Asp	GAC	214	0.41	trnD-GUC
Val	GUC	166	0.49	trnV-GAC	Glu	GAA	986	1.50	trnE-UUC
Val	GUA	523	1.54	trnV-UAC	Glu	GAG	331	0.50
Val	GUG	178	0.52		Cys	UGU	198	1.38
Ser	UCU	580	1.77		Cys	UGC	88	0.62	trnC-GCA
Ser	UCC	313	0.96	trnS-GGA	Trp	UGG	448	1.00	trnW-CCA
Ser	UCA	394	1.20	trnS-UGA	Arg	CGU	336	1.32	trnR-ACG
Ser	UCG	154	0.47		Arg	CGC	100	0.39
Ser	AGA	474	1.86		Arg	CGA	339	1.33
Ser	AGG	165	0.65	trnS-GCU	Arg	CGG	118	0.46
Tyr	UAU	799	1.64		Arg	AGU	406	1.24	trnR-UCU
Tyr	UAC	175	0.36	trnY-GUA	Arg	AGC	118	0.36
Pro	CCA	322	1.17	trnP-UGG	Gly	GGU	581	1.32
Pro	CCG	159	0.58		Gly	GGC	188	0.43	trnG-GCC
Pro	CCU	434	1.58		Gly	GGA	686	1.56	trnG-UCC
Pro	CCC	184	0.67		Gly	GGG	305	0.69
Thr	ACU	529	1.64		Ala	GCA	416	1.18	trnA-UGC
Thr	ACC	236	0.73	trnT-GGU	Ala	GCG	162	0.46
Thr	ACA	404	1.25	trnT-UGU	Ala	GCU	611	1.73
Thr	ACG	125	0.39		Ala	GCC	223	0.63

RSCU: Relative synonymous codon usage. RSCU > 1 are highlighted in bold.

Table 5

The codon-anticodon recognition pattern and codon usage for the K. indica cp genome.

Amino Acid	Codon	No.	RSCU	tRNA	Amino Acid	Codon	No.	RSCU	tRNA
Phe	UUU	982	1.31		Stop	UAA	49	1.73
Phe	UUC	515	0.69	trnF-GAA	Stop	UAG	21	0.74
Leu	UUA	870	1.87	trnL-UAA	Stop	UGA	15	0.53
Leu	UUG	578	1.24	trnL-CAA	His	CAU	452	1.51
Leu	CUU	607	1.30		His	CAC	148	0.49	trnH-GUG
Leu	CUC	186	0.4		Gln	CAA	723	1.53	trnQ-UUG
Leu	CUA	375	0.81	trnL-UAG	Gln	CAG	220	0.47
Leu	CUG	179	0.38		Asn	AAU	969	1.53
Ile	AUU	1072	1.47		Asn	AAC	295	0.47	trnN-GUU
Ile	AUC	427	0.58	trnI-GAU	Lys	AAA	1036	1.48	trnK-UUU
Ile	AUA	691	0.95	trnI-CAU	Lys	AAG	360	0.52
Met	AUG	633	1	trn(f)M-CAU	Asp	GAU	847	1.61
Val	GUU	503	1.45		Asp	GAC	205	0.39	trnD-GUC
Val	GUC	180	0.52	trnV-GAC	Glu	GAA	987	1.47	trnE-UUC
Val	GUA	517	1.49	trnV-UAC	Glu	GAG	359	0.53
Val	GUG	190	0.55		Cys	UGU	209	1.42
Ser	UCU	585	1.76		Cys	UGC	85	0.58	trnC-GCA
Ser	UCC	308	0.93	trnS-GGA	Trp	UGG	463	1	trnW-CCA
Ser	UCA	401	1.21	trnS-UGA	Arg	CGU	351	1.33	trnR-ACG
Ser	AGA	488	1.85		Arg	CGC	108	0.41
Ser	AGG	177	0.67	trnS-GCU	Arg	CGA	346	1.31
Ser	UCG	172	0.52		Arg	CGG	114	0.43
Tyr	UAU	804	1.64		Arg	AGU	405	1.22	trnR-UCU
Tyr	UAC	175	0.36	trnY-GUA	Arg	AGC	121	0.36
Pro	CCU	414	1.50		Gly	GGU	571	1.28
Pro	CCC	209	0.76		Gly	GGC	199	0.45	trnG-GCC
Pro	CCA	314	1.14	trnP-UGG	Gly	GGA	681	1.53	trnG-UCC
Pro	CCG	167	0.61		Gly	GGG	328	0.74
Thr	ACU	531	1.62		Ala	GCU	622	1.74
Thr	ACC	239	0.73	trnT-GGU	Ala	GCC	233	0.65
Thr	ACA	405	1.23	trnT-UGU	Ala	GCA	410	1.15	trnA-UGC
Thr	ACG	137	0.42		Ala	GCG	161	0.45

Usually, relative synonymous codon usage (RSCU) can be divided into four types, including lack of bias (RSCU < 1.0), low bias (1.0 < RSCU< 1.2), moderately biased (1.2 < RSCU< 1.3) and highly biased (RSCU > 1.3) [49,50]. As shown in Table 4 and Table 5, it is uncannily similar that there are 32 lack of bias codons, two no-bias codons with RSCU = 1 (tryptophan and methionine), and 21 highly biased codons, with the exception of five low bias codons in C. carinatum Schousb but two low bias codons in K. indica, and three moderately biased codons in C. carinatum Schousb but six moderately biased codons in K. indica, respectively. The UAA stop codon was the significantly favorite stop codon in the cp genomes. The results showed that the RSCU was significantly biased except for tryptophan and methionine in C. carinatum Schousb and K. indica and that the A/T ending is very rich, which is popular in the cp genomes of higher plants [51,52].

2.4. Repeats Structure and SSRs

Analysis of the repeat structure showed that the total of 42 repeats of C. carinatum Schousb is significantly less than K. indica (59), which may be one reason for the larger cp genome of K. indica. There are 18 forward repeats, 20 palindromic repeats, three reverse repeats and one complement repeats in the cp genome of the C. carinatum Schousb (Table 6) and 17 forward repeats, 28 palindromic repeats, 11 reverse repeats, and eight complement repeats in the K. indica cp genome (Table 7). All of them range from 30 to 60 bp in length and are mostly located in the intergenic spacer (IGS) and intron sequences. Comparison analysis of the repeats of six other species of Asteraceae showed that H. annuus contained the most repeats (572) and T. mongolicum contained the fewest repetitive sequences (28) (Figure 2). Among the eight species of Asteraceae, we also found that the base fragment with the most repeats was between 30–39 bp in length. Complement repeats are relatively richer than in other families, although A. annua, E. paradoxa, T. mongolicum and C. indicum do not contain complement repeats [48].

Table 6

The repeats of the C. carinatum Schousb cp genome and their distribution.

No.	Size (bp)	Type	Repeat 1 Location	Repeat 2 Location	Region	No.	Size (bp)	Type	Repeat 1 Location	Repeat 2 Location	Region
1	60	F	ycf2 (CDS)	ycf2 (CDS)	IRb	22	31	R	IGS (trnT-GGU, psbD)	IGS (trnT-GGU, psbD)	LSC
2	60	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	23	30	P	IGS (psbI, trnS-GUC)	trnS-GGA (CDS)	LSC
3	60	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	24	30	F	ycf2 (CDS)	ycf2 (CDS)	IRb
4	60	F	ycf2 (CDS)	ycf2 (CDS)	IRa, IRa	25	30	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa
5	51	F	IGS (rps11, rpl36)	IGS (rps11, rpl36)	LSC	26	30	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa
6	48	P	IGS (psbT, psbN)	IGS (psbT, psbN)	LSC	27	35	F	psaB (CDS)	psaA (CDS)	LSC
7	46	F	IGS (accD, psaI)	IGS (accD, psaI)	LSC	28	35	F	ycf3 (intron2)	ndhB (intron)	LSC, IRb
8	45	F	ycf2 (CDS)	ycf2 (CDS)	IRb	29	35	P	ycf3 (intron2)	ndhB (intron)	LSC, IRa
9	45	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	30	32	P	IGS (trnT-GGU, psbD)	IGS (trnT-GGU, psbD)	LSC
10	45	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	31	31	F	IGS (psbI, trnS-GUC)	IGS (psbI, trnS-GUC)	LSC
11	46	P	IGS (petN, psbM)	IGS (petN, psbM)	LSC	32	30	P	IGS (psbC, trnS-UGA)	trnS-GGA (CDS)	LSC
12	39	P	IGS (rps12, trnV-GAC)	ndhA (intron)	IRb, SSC	33	30	F	rbcL (CDS)	IGS (rbcL, accD)	LSC
13	39	F	ndhA (intron)	IGS (trnV-GAC, rps12)	SSC, IRa	34	32	F	IGS (psbI, trnS-GUC)	IGS (psbC, trnS-UGA)	LSC
14	41	F	ycf3 (intron2)	IGS (rps12, trnV-GAC)	LSC, IRb	35	31	R	IGS (trnL-UAG, rpl32)	IGS (trnL-UAG, rpl32)	SSC
15	41	P	ycf3 (intron2)	IGS (trnV-GAC, rps12)	LSC, IRa	36	30	C	IGS (trnT-GGU, psbD)	IGS (trnT-GGU, psbD)	LSC
16	39	P	ycf3 (intron2)	ndhA (intron)	LSC, SSC	37	30	F	psaB (CDS)	psaA (CDS)	LSC
17	42	F	ycf2 (CDS)	ycf2 (CDS)	IRb	38	30	F	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa
18	42	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	39	30	P	ycf2 (CDS)	ycf2 (CDS)	IRb
19	42	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	40	30	F	IGS (trnL-UAG, rpl32)	IGS (trnL-UAG, rpl32)	SSC
20	42	F	ycf2 (CDS)	ycf2 (CDS)	IRa	41	30	R	IGS (trnL-UAG, rpl32)	IGS (trnL-UAG, rpl32)	SSC
21	41	P	IGS (ndhE, psaC)	IGS (psaC, ndhD)	SSC	42	30	P	ycf2 (CDS)	ycf2 (CDS)	IRa

F = forward, P = palindrome, R = reverse, C = complement, IGS = intergenic spacer.

Table 7

The repeats of the K. indica cp genome and their distribution.

No.	Size (bp)	Type	Repeat 1 Location	Repeat 2 Location	Region	No.	Size (bp)	Type	Repeat 1 Location	Repeat 2 Location	Region
1	48	P	IGS (psbT, psbN)	IGS (psbT, psbN)	LSC	31	32	F	IGS (rrn5, rrn4.5)	IGS (rrn5, rrn4.5)	IRa
2	39	P	ycf3 (intron2)	ndhA (intron)	LSC, SSC	32	34	R	IGS (trnT-GGU, psbD)	IGS (trnT-GGU, psbD)	LSC
3	41	F	ycf3 (intron2)	IGS (rps12, trnV-GAC)	LSC, IRb	33	31	R	IGS (ndhC, trnV-UAC)	petB (intron)	LSC
4	41	P	ycf3 (intron2)	IGS (trnV-GAC, rps12)	LSC, IRa	34	33	C	IGS (accD, psaI)	petD (CDS)	LSC
5	39	P	IGS (rps12, trnV-GAC)	ndhA (intron)	IRb, SSC	35	33	C	IGS (accD, psaI)	IGS (accD, psaI)	LSC
6	39	F	ndhA (intron)	IGS (trnV-GAC, rps12)	SSC, IRa	36	30	P	IGS (psbC, trnS-UGA)	trnS-GGA (CDS)	LSC
7	42	F	ycf2 (CDS)	ycf2 (CDS)	IRb	37	32	C	IGS (trnH-GUG, psbA)	IGS (accD, psaI)	LSC
8	42	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	38	32	F	IGS (psbI, trnS-GCU)	IGS (psbC, trnS-UGA)	LSC
9	42	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	39	32	P	IGS (petN, psbM)	IGS (petN, psbM)	LSC
10	42	F	ycf2 (CDS)	ycf2 (CDS)	IRa	40	32	C	IGS (trnG-UCC, trnT-GGU)	petD (intron)	LSC
11	41	R	petD (intron)	petD (intron)	LSC	41	32	F	psaB (CDS)	psaA (CDS)	LSC
12	43	P	IGS (psaC, ndhD)	IGS (psaC, ndhD)	SSC	42	32	R	IGS (accD, psaI)	IGS (accD, psaI)	LSC
13	39	R	IGS (accD, psaI)	IGS (accD, psaI)	LSC	43	31	P	IGS (trnG-UCC, trnT-GGU)	IGS (ndhC, trnV-UAC)	LSC
14	31	F	IGS (rps12, trnV-GAC)	IGS (rps12, trnV-GAC)	IRb	44	31	P	IGS (trnG-UCC, trnT-GGU)	IGS (trnG-UCC, trnT-GGU)	LSC
15	31	P	IGS (rps12, trnV-GAC)	IGS (trnV-GAC, rps12)	IRb, IRa	45	31	P	IGS (trnT-GGU, psbD)	IGS (trnT-UGU, trnL-UAA)	LSC
16	31	P	IGS (rps12, trnV-GAC)	IGS (trnV-GAC, rps12)	IRb, IRa	46	31	F	IGS (psaA, ycf3)	IGS (psaA, ycf3)	LSC
17	31	F	IGS (trnV-GAC, rps12)	IGS (trnV-GAC, rps12)	IRa	47	31	R	IGS (accD, psaI)	IGS (accD, psaI)	LSC
18	37	P	IGS (rpl22, rps19)	IGS (rpl22, rps19)	LSC	48	31	F	IGS (accD, psaI)	petD (intron)	LSC
19	31	R	petD (intron)	petD (intron)	LSC	49	31	C	petD (intron)	petD (intron)	LSC
20	30	P	IGS (psbI, trnS-GCU)	trnS-GGA (CDS)	LSC	50	31	F	ndhF (CDS)	IGS (ndhF, ycf1)	SSC
21	30	F	ycf2 (CDS)	ycf2 (CDS)	IRb	51	30	C	IGS (trnG-UCC, trnT-GGU)	IGS (trnT-GGU, psbD)	LSC
22	30	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	52	30	R	IGS (trnT-GGU, psbD)	IGS (trnT-GGU, psbD)	LSC
23	30	P	ycf2 (CDS)	ycf2 (CDS)	IRb, IRa	53	30	C	ycf3 (intron2)	IGS (ndhC, trnV-UAC)	LSC
24	32	P	IGS (trnG-UCC, trnT-GGU)	IGS (trnG-UCC, trnT-GGU)	LSC	54	30	C	IGS (ndhC, trnV-UAC)	IGS (accD, psaI)	LSC
25	32	P	IGS (psbZ, trnG-GCC)	IGS (psbZ, trnG-GCC)	LSC	55	30	F	IGS (ndhC, trnV-UAC)	IGS (ndhC, trnV-UAC)	LSC
26	32	P	IGS (accD, psaI)	petD (CDS)	LSC	56	30	F	IGS (accD, psaI)	IGS (accD, psaI)	LSC
27	32	R	petD (CDS)	petD (CDS)	LSC	57	30	R	IGS (accD, psaI)	IGS (accD, psaI)	LSC
28	32	F	IGS (rrn4.5, rrn5)	IGS (rrn4.5, rrn5)	IRb	58	30	R	IGS (accD, psaI)	petD (intron)	LSC
29	32	P	IGS (rrn4.5, rrn5)	IGS (rrn5, rrn4.5)	IRb, IRa	59	30	F	petD (intron)	petD (intron)	LSC
30	32	P	IGS (rrn4.5, rrn5)	IGS (rrn5, rrn4.5)	IRb, IRa

F = forward, P = palindrome, R = reverse, C = complement, IGS = intergenic spacer.

Figure 2

The repeat sequences of eight Asteraceae cp genomes. F (forward), P (palindrome), R (reverse), and C (complement) represent the repeat types. Different colours represent the repeats in different lengths.

There are 70 and 90 simple sequence repeats (SSRs) in the cp genome of C. carinatum Schousb and K. indica, respectively (Table 8 and Table 9). Most of them are mononuclear repeats; 43 in C. carinatum Schousb and 30 in K. indica, respectively. Eight dinucleotide repeats, five trinucleotide repeats, thirteen tetranucleotide repeats, and one pentanucleotide repeats were also found in the C. carinatum Schousb cp genome. Eighteen dinucleotide repeats, twenty-eight trinucleotide repeats, seven tetranucleotide repeats, and seven pentanucleotide repeats were also discovered in K. indica. The majority of SSRs are located in the LSC region, 81.43% (C. carinatum Schousb) and 78.89% (K. indica) of SSRs are located in the LSC (Table 8 and Table 9) whereas, only twelve (C. carinatum Schousb) and 9 (K. indica) SSRs are located in the CDSs (Table 10). Comparing with the six other species of Asteraceae, the most SSRs and the least SSRs located in CDS were found in the K. indica cp genome. Furthermore, most of the SSRs are AT repeats, which is consistent with the AT richness [48,51].

Table 8

The simple sequence repeats in the C. carinatum Schousb cp genome.

Unit	Length	No.	Location	Region	Unit	Length	No.	Location	Region	Unit	Length	No.	Location	Region
A	19	1	IGS (rpl32, ndhF)	SSC	T	22	1	IGS (atpF, atpA)	LSC	TA	14	1	IGS (trnH-GUG, psbA)	LSC
	13	1	IGS (psbE, petL)	LSC		16	1	rps16 (intron)	LSC		12	2	IGS (trnT-GGU, psbD)	LSC
	12	1	IGS (ycf1, rps15)	SSC		14	3	IGS (trnE-UUC, rpoB)	LSC				IGS (rpl33, rps18)	LSC
	11	6	IGS (trnK-UUU, rps16)	LSC				IGS (atpB, rbcL)	LSC		10	1	rpoC1 (exonII)	LSC
			IGS (trnR-UCU, trnG-UCC)	LSC				IGS (rps8, rpl14)	LSC	TG	10	1	IGS (rps16, trnQ-UUG)	LSC
			IGS (psbZ, trnG-GCC)	LSC		13	3	IGS (ndhC, trnV-UAC)	LSC	ATT	12	2	IGS (cemA, petA)	LSC
			IGS (psbE, petL)	LSC				IGS (rbcL, accD)	LSC				IGS (trnL-UAG, rpl32)	SSC
			IGS (rps18, rpl20)	LSC				IGS (psbB, psbT)	LSC	GAA	15	1	ycf1	SSC
			IGS (petD, rpoA)	LSC		12	5	IGS (trnH-GUG, psbA)	LSC	TTA	12	1	ndhA (intron)	SSC
	10	10	trnk-UUU (intron)	LSC				IGS (trnD-GUC, trnY-GUA)	LSC	TTC	12	1	psbC	LSC
			rpoB	LSC				ycf3 (intronII)	LSC	AATA	12	2	IGS (trnK-UUU, rps16)	LSC
			rpoC1 (intron)	LSC				clpP (intronI)	LSC				ndhA (intron)	SSC
			rpoC1 (exonII)	LSC				ycf1	LSC	ATAC	12	1	IGS (trnF-GAA, ndhJ)	LSC
			rpoC1 (exonII)	LSC		11	1	IGS (petA, psbJ)	LSC	ATTG	12	1	ycf2	IRa
			IGS (psaA, ycf3)	LSC		10	9	trnk-UUU (intron)	LSC	ATTT	22	1	IGS (atpF, atpA)	LSC
			IGS (ycf3, trnS-GGA)	LSC				IGS (psbM, trnD-GUC)	LSC	CAAT	12	1	IGS (ycf2, trnL-CAA)	IRb
			IGS (trnT-UGU, rnL-UAA)	LSC				rpoC1 (intron)	LSC	GATT	12	1	ndhA (intron)	SSC
			psbT	LSC				IGS (atpI, atpH)	LSC	TAGA	12	1	IGS (rbcL, accD)	LSC
			IGS (rrn5, trnR-ACG)	IRb				IGS (atpI, atpH)	LSC	TAAA	12	1	IGS (atpI, atpH)	LSC
C	12	1	rps16 (intron)	LSC				IGS (atpA, trnR-UCU)	LSC	TAAT	12	1	IGS (petN, psbM)	LSC
AT	14	1	rps16 (intron)	LSC				rpoA	LSC	TATT	12	2	IGS (rpl33, rps18)	LSC
	12	1	IGS (psbZ, trnG-GCC)	LSC				IGS (rpl14, rpl16)	LSC				ndhD	SSC
	10	1	rpoC2	LSC				IGS (trnR-ACG, rrn5)	IRa	TTTC	16	1	rpl16 (intron)	LSC
										AATTT	15	1	IGS (ccsA, trnL-UAG)	SSC

Table 9

The simple sequence repeats in the K. indica cp genome.

Unit	Length	No.	Location	Region	Unit	Length	No.	Location	Region	Unit	Length	No.	Location	Region
A	18	1	IGS (trnS-UGA, psbZ)	LSC	AT	16	1	IGS (psbZ, trnG-GCC)	LSC	GAA	15	1	ycf1	SSC
	12	1	IGS (psbZ, trnG-GCC)	LSC		10	7	rps16 (intron)	LSC		12	1	ycf1	SSC
	11	3	IGS (trnQ-UUG, psbK)	LSC		10		rpoC2	LSC	TAT	21	1	IGS (rps12, trnV-GAC)	IRb
	11		IGS (psbM, trnD-GUC)	LSC		10		IGS (psbZ, trnG-GCC)	LSC		12	2	IGS (trnT-UGU, trnL-UAA)	LSC
	11		IGS (ycf4, cemA)	LSC		10		IGS (psbZ, trnG-GCC)	LSC		12		IGS (trnF-GAA, ndhJ)	LSC
	10	7	rpoB	LSC		10		IGS (psbE, petL)	LSC	TTA	15	2	IGS (ndhC, trnV-UAC)	LSC
	10		rpoC1 (exonII)	LSC		10		IGS (petD, rpoA)	LSC		15		IGS (clpP, psbB)	LSC
	10		IGS (petB, petD)	LSC		10		rpl16 (intron)	LSC		12	2	petB (intron)	LSC
	10		ndhB (intron)	IRb	TA	23	1	petD (intron)	LSC		12		petD (intron)	LSC
	10		ndhA (intron)	SSC		20	1	IGS (accD, psaI)	LSC		12	1	psbC	LSC
	10		IGS (trnL-UAG, rpl32)	SSC		18	1	petD (intron)	LSC		12	1	IGS (trnK-UUU, rps16)	LSC
	10		IGS (rpl2, rps19)	IRa		14	1	IGS (accD, psaI)	LSC		12	1	IGS (trnG-UCC, trnT-GGU)	LSC
T	18	1	IGS (trnE-UUC, rpoB)	LSC		12	3	IGS (trnT-GGU, psbD)	LSC		12	1	IGS (trnE-UUC, rpoB)	LSC
	17	1	rpoA	LSC		12		petD (intron)	LSC		12	1	IGS (clpP, psbB)	LSC
	14	2	IGS (rpl20, rps12)	LSC		12		IGS (rpl22, rps19)	LSC		12	1	IGS (trnS-GCU, trnC-GCA)	LSC
	14		ndhA (intron)	SSC		10	3	rpoC1 (exonII)	LSC		12	1	IGS (ndhI, ndhG)	SSC
	13	1	IGS (psbE, petL)	LSC		10		IGS (psaJ, rpl33)	LSC	TATC	12	1	IGS (psbA, trnK-UUU)	LSC
	12	2	IGS (atpF, atpA)	LSC		10		ndhA (intron)	SSC	TATT	12	2	IGS (rpl33, rps18)	LSC
	12		clpP (intronII)	LSC	AAT	12	2	IGS (trnR-UCU, trnG-UCC)	LSC		12		IGS (psaC, ndhD)	SSC
	11	2	IGS (atpB, rbcL)	LSC		12		IGS (trnT-GGU, psbD)	LSC	TCTA	12	1	ndhA (intron)	SSC
	11		IGS (psaI, ycf4)	LSC	ATA	21	1	IGS (trnV-GAC, rps12)	IRa	TTCT	12	1	IGS (trnG-UCC, trnT-GGU)	LSC
	10	9	IGS (trnC-GCA, petN)	LSC		15	3	ycf3 (intron)	LSC	TTTA	12	1	petD (intron)	LSC
	10		IGS (rpoC2, rps2)	LSC		15		IGS (trnP-UGG, psaJ)	LSC	TTTC	12	1	trnK-UUU (intron)	LSC
	10		IGS (atpI, atpH)	LSC		15		IGS (trnL-UAG, rpl32)	SSC	ATTAG	15	1	IGS (rbcL, accD)	LSC
	10		IGS (ndhC, trnV-UAC)	LSC		12	2	IGS (trnG-UCC, trnT-GGU)	LSC	TATAT	23	1	petD (intron)	LSC
	10		clpP (intronI)	LSC		12		rpl16 (intron)	LSC		20	1	IGS (accD, psaI)	LSC
	10		IGS (rps8, rpl14)	LSC	ATT	15	1	IGS (trnG-UCC, trnT-GGU)	LSC		15	1	IGS (accD, psaI)	LSC
	10		IGS (rps19, rpl2)	IRb	TAA	15	1	IGS (accD, psaI)	LSC	TATTA	21	2	IGS (rps12, trnV-GAC)	IRb
	10		IGS (psaC, ndhD)	SSC		12	2	IGS (trnE-UUC, rpoB)	LSC		21		IGS (trnV-GAC, rps12)	IRa
	10		ndhB (intron)	IRa		12		IGS (trnT-GGU, psbD)	LSC	TCCTA	15	1	IGS (rps4, trnT-UGU)	LSC

Table 10

The distribution of SSRs present in the Asteraceae cp genomes.

Taxon	Genome Size (bp)	GC (%)	SSR Type							CDS
			Mono	Di	Tri	Tetra	Penta	Hexa	Total	% a	No. b	% c
C . carinatum Schousb	149,752	37.47	43	8	5	13	1	0	70	51.6	12	17.1
K . indica	152,885	37.25	30	18	22	13	7	0	90	51.3	9	10
D . alveolatum	152,265	37.37	34	14	11	14	2	0	75	51.4	8	10.7
A . annua	150,952	37.48	39	10	4	15	3	0	71	52.1	10	14.1
E . paradoxa	151,837	37.59	37	4	4	5	0	1	51	51.5	14	27.5
H . annuus	151,104	37.62	40	4	4	4	0	0	52	51.2	14	26.9
T . mongolicum	151,451	37.67	14	5	3	6	1	1	30	51.3	11	36.7
C . indicum	150,972	37.48	38	10	4	14	1	0	67	48.8	7	10.4

CDS: protein-coding regions. a the percentage ratio of the total length of the CDS to the genome size. b the total number of SSRs in CDS. c the percentage ratio of the total number of SSRs in CDS to the total number of SSRs in the whole genome.

2.5. Comparison of the Whole cp Genome of Asteraceae

Using C. carinatum Schousb as a reference, mVISTA was used to compare the overall sequence of the cp genome of the eight Asteraceae species (Figure 3). It was found that the cp genome sequence of C. carinatum Schousb had the shortest size, whereas the K. indica cp genome had the longest size among the eight Astareace species. The difference of the sequence length was mainly due to the difference of length of the LSC region and there was no significant differences in the SSC and IRs regions’ length. It is important to note that the SSC regions of A. annua and T. mongolicum are completely different from C. carinatum Schousb because of the inversion in most of the Asteraceae species, whereas the inversion region in LSC compared to other eudicots is relatively constant [13,14,15,16,17,18,19,20,21,22,23,24,25]. In order to illustrate this, we compared the cp genomes between 129 species of Asteraceae and some cp genomes of non-Asteraceae species using the mVISTA software to confirm the structural changes (Data not shown). Among them, only 16 species of Asteraceae (Artemisia argyi, Artemisia capillaries, Artemisia frigida, Artemisia gmelinii, Artemisia montana, Aster altaicus, Carthamus tinctorius, Centaurea diffusa, Eclipta prostrate, Lactuca sativa, Saussurea chabyoungsanica, Saussurea involucrate, Saussurea polylepis, Taraxacum mongolicum, Taraxacum officinale, Taraxacum platycarpum) did not invert in the SSC region (Supplemental Table S1). However, the LSC region was generally inverted. In addition, the coding area is more conservative than the non-coding area [53,54].

Figure 3

The comparison of eight Asteraceae cp genomes by using mVISTA. The grey arrows above the contrast indicate the direction of the gene translation.The y-axis represents the percent identity between 50% and 100%. Protein codes (exon), rRNA, tRNA and conserved non-coding sequence (CNS) are shown in different colors, respectively.

2.6. IR Contraction and Expansion

The IR contraction and expansion of C. carinatum Schousb and K. indica were analyzed by comparing the LSC/IRB/SSC/IRA boundary among the eight species of Asteraceae (Figure 4). For all of them, the junction of the IRB and LSC regions was connected by the rps19 gene such that pseudogenes rps19 in the IRA/LSC boundaries were usually found in the duplication of the 3′end of the rps19 in IRA, in which the pseudogene rps19 lengths ranged from 60 to 99 bp. The ycf1 gene was located in both IRB/SSC and IRA/LSC boundaries. Additionally, 477 to 581 bp pseudogenes ycf1 at the border of IRA/SSC were produced. Among these eight cp genomes, C. carinatum Schousb had the largest ycf1 pseudogene (581 bp in length), whereas the inversion of the SSC region in A. annua and T. mongolicum caused the pseudogene ycf1 to appear at the border of IRB/SSC. T. mongolicum and C. indicum showed the absence of the ycf1 pseudogene and the rps19 pseudogene. With the exception of T. mongolicum, all ndhF genes were located in the SSC region, which has a distance between 35–77 bp from the IRA border, and the ndhF gene of C. carinatum Schousb has the largest distance to IRA border. Furthermore, the ndhF gene of T. mongolicum is 10 bp across the IRB region and intersects with the pseudogene ycf1. The trnH-GUG genes are located in the LSC region between 0–13 bp from the IRA border and C. carinatum Schousb has the largest one. Oddly, a relatively smaller IR size but longer pseudogene ycf1 length were found in C. carinatum Schousb, which may be due to the occurrence of the contraction in the intergenic regions in C. carinatum Schousb [48].

Figure 4

The comparison of the borders of the LSC, SSC, and IR regions among the eight Asteraceae cp genomes.

2.7. Phylogenetic Analysis and Barcoding

The cp genome is a useful resource and tool for taxonomy, determining evolutionary relationships within families, and DNA barcoding [42,55,56,57,58,59]. Here, for obtaining a useful barcoding marker and a reasonable phylogenetic status of C. carinatum Schousb and K. indica, we established the phylogenetic tree of 24 sample species using the Maximum Likelihood (ML) method by the alignment of 18 genes (atpF, clpP, matK, ndhA, ndhB, ndhF, petB, petD, psaB, psbA, rbcL, rpl2, rpl16, rpoB, rpoC1, rps12, rps16, rps19), respectively (Supplemental Figure S1). These 24 species include 18 Asteraceae species, four Orchidaceae species, one Chenopodiaceae species and one Cruciferae species. We compared six non-Asteraceae species with 18 Asteraceae species because the former are morphologically similar to C. carinatum Schousb and K. indica. From the results of the alignment, the matK, ndhF and rbcL genes are better than the others. There are eight nodes that had bootstrap values >90% when matK and ndhF were used for gene alignment. However, they both had three nodes with bootstrap values <40% and one node even had a bootstrap value <30%. As for the rbcL gene, 10 out of the 19 nodes had bootstrap values >90%. The minimum bootstrap value is 46% and the rest of the nodes had bootstrap values >50%. We show the phylogenetic tree by rbcL gene alignment in Figure 5, which shows that C. carinatum Schousb is a closely related species with Chrysanthemum indicum and that K. indica is closely related with the Conyza bonariensisspecies, which is consistent with their classification and morphology. Hence, the rbcL gene could be a good candidate gene to be used as a barcoding marker [51,52,54].

Figure 5

The molecular phylogenetic analysis of the cp protein-coding gene rcbL for 24 samples using the Maximum Likelihood method. The tree was constructed by using MEGA7. The stability of each tree node was tested by bootstrap analysis with 1000 replicates.

3. Discussion

We reported on the two genome sequence of C. carinatum Schousb and K. indica, which provides an important resource to study the evolutionary and inversion mechanism as well as the molecular barcoding of vegetables in the Asteraceae family. Although cp genomes of Angiosperms are well-conserved in the genomic structure, the inversion and IR expansion contraction occur frequently [7,8,9,10,11]. These results showed that the inversion of trnC-GCA to trnG-UCC in the LSC and the whole SSC occurred in these two species, which are congruent with most of the cp genomes of Asteraceae family [41,60,61,62]. Oddly, though some no-inversion occurs in the SSC region, the inversion of trnC-GCA to trnG-UCC in the LSC does not occur, which maybe could make amplification of trnC-GCA or trnG-UCC boundary regions a good resource for the molecular taxonomy of Asteraceae. Meanwhile, the SSC/IRA and LSC/IRB border extends into the ycf1 and rps19 with the subsequent formation of the ycf1 and rps19 pseudogenes, respectively [63,64]. The ycf1 and rps19 pseudogenes occur in most of the cp genomes, with deletion regions of similar size in species of the same family, but different deletion size between different families (Figure 4) [48].

Here, the analysis of the codon usage frequency and RSCU showed that leucine and isoleucine are the most popular amino acids in the cp genomes in the C. carinatum Schousb and K. indica as Angiosperms [23,52,65,66,67]. Meanwhile, a significantly higher T bases appearance was indicated in the 3th CDS position than in the 2nd position or 1st position. The T bases appear at the end of most favorite synonymous codons (RSCU > 1.0), but the reverse occurs for the G base (Table 1, Table 4 and Table 5). Consistent with most earlier reports about repeats and SSRs, the mononucleotide repeats with A/T repeats are more abundant, which may lead to AT richness in the Angiosperm cp genomes [68,69,70].

Here, we compared the whole cp genome of six species of Asteraceae, which revealed that the inversion is usual in most Asteraceae species, with some no-inversion occuring only in the SSC regions’ inversions, but not in the inversions of trnC-GCA to trnG-UCC in the LSC. Among the 129 Asteraceae species that have been sequenced, inversion occurs in the LSC region. Whether or not the SSC region is inverted is closely related to the genus. For example, Diplostephium has the most sequenced species and all of them have inverted SSC regions. Inconsistent inversions within the genus only occurred in the Aster genus and the Taraxacum genus (Supplemental Table S1). This phenomenon may be a key feature of the cp genome of Asteraceae. It provides an insight into future evolution research.

Here, the rbcL genes represents a good diversity among some vegetables. The closer the relationship between species, the higher the sequence similarity of the genes. The phylogenetic tree of the rbcL gene among the Asteraceae species can obtain a better discrimination result. More importantly, it also could differentiate different family species, which illustrate that the rbcL gene is not only highly distinguished within the Asteraceae family, but also highly distinguished within most of vegetables. So, rbcL would be one of the best choices for DNA barcoding to distinguish between vegetables [71,72,73].

4. Materials and Methods

4.1. DNA Extraction and Sequencing

The total DNA was extracted from about 100 g fresh leaves of C. carinatum Schousb and K. indica using the CTAB method [74]. The total DNA quantity was evaluated by the value of the ratio of absorbance measurements at 260 nm and 280 nm (A260/A280) using Nanodrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), whereas visual assessment of the DNA size and integrity was performed using gel electrophoresis. The DNA was sheared to fragments of 300~500 bp. Paired-end libraries were prepared wih the TruSeqTM DNA Sample Prep Kit and the TruSeq PE Cluster Kit. The genome was then sequenced using the HiSeq4000 platform (Illumina, Santiago, CA, USA).

4.2. Genome Assembly

The assembly of the cp genome of C. carinatum Schousb and K. indica were first carried out through the error correction and production of initial contigs by the GS FLX De Novo Assembler Software (Newbler V2.6). PCR amplification and Sanger sequencing were performed to verify the four-f- junction regions between the IRs and the LSC/SSC. The final cp genome of C. carinatum Schousb and K. indica were submitted to the Genbank with the accession number MG710386 and MG710387, respectively.

4.3. Gene Annotation and Codon Usage Analysis

The cp genome was annotated using BLAST [75] and DOGMA with manual corrections [76]. The tRNAscan-SE was used to identify the tRNA genes [77]. OGDRAW was used to draw the circular genome map [78]. MEGA5 was used to analyze the characteristics of the variations in the synonymous codon [79]. The relative synonymous codon usage values (RSCU), codon usage, and GC content were also determined by MEGA7 [80].

4.4. Repeat Structure and Single Sequence Repeats (SSRs) Analysis

Analysis of tandem repeats with more than 30 bp and a minimum of 90% sequence (forward, palindromic, reverse, and complement) and single sequence repeats (SSRs) was performed by REPuter [81] and MISA [82], respectively, with the same parameters as described in Ni et al. [53].

4.5. Comparative Genome Analysis of the C. carinatum Schousb and K. indica Genomes

Comparison of the overall cp genome of C. carinatum Schousb and K. indica with six cp genomes of Asteraceae was performed by mVISTA [83,84] using the annotation of C. carinatum Schousb as a reference.

4.6. Phylogenetic Analysis

A total of 24 complete cp genome sequences were downloaded from the NCBI Organelle Genome Resources database. MEGA7 [80] was used to construct the evolutionary tree of the cp protein-coding gene rcbL of 24 samples by the Maximum Likelihood method.

5. Conclusions

In this study, the complete cp genome of C. carinatum Schousb and K. indica were reported and analyzed for the first time. Both of them are key traditional Chinese medicines and edible vegetables. Comparing them with other Asteraceae species, the cp genome of these two species display two inversions, one is trnC-GCA to trnG-UCC in the LSC and other is the whole SSC region. We found that most of the inversions of trnC-GCA to trnG-UCC almost happened in every cp genome of the Asteraceae species. Seventy and ninety simple sequence repeats (SSRs) were present in the cp genome of C. carinatum Schousb and K. indica, respectively. Certainly, these results provide good chances for developing barcoding molecular markers for different families distinguish, which can be obtained through combination by different barcoding markers or through the boundary region marker. The rbcL is a good barcoding marker. Meanwhile, these results would be useful for the evolutionary study of C. carinatum Schousb and K. indica, and might also contribute to the genetics and barcoding of easily-confused leafy vegetables.