Complete Chloroplast Genome Sequence and Phylogenetic Analysis of Aster tataricus.

We sequenced and analyzed the complete chloroplast genome of Aster tataricus (family Asteraceae), a Chinese herb used medicinally to relieve coughs and reduce sputum. The A. tataricus chloroplast genome was 152,992 bp in size, and harbored a pair of inverted repeat regions (IRa and IRb, each 24,850 bp) divided into a large single-copy (LSC, 84,698 bp) and a small single-copy (SSC, 18,250 bp) region. Our annotation revealed that the A. tataricus chloroplast genome contained 115 genes, including 81 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. In addition, 70 simple sequence repeats (SSRs) were detected in the A. tataricus chloroplast genome, including mononucleotides (36), dinucleotides (1), trinucleotides (23), tetranucleotides (1), pentanucleotides (8), and hexanucleotides (1). Comparative chloroplast genome analysis of three Aster species indicated that a higher similarity was preserved in the IR regions than in the LSC and SSC regions, and that the differences in the degree of preservation were slighter between A. tataricus and A. altaicus than between A. tataricus and A. spathulifolius. Phylogenetic analysis revealed that A. tataricus was more closely related to A. altaicus than to A. spathulifolius. Our findings offer valuable information for future research on Aster species identification and selective breeding.

1. Introduction

Aster tataricus is a tall perennial herb of the genus Aster (family Asteraceae). It has been used as a therapeutic traditional medicine for eliminating phlegm and relieving cough for thousands of years [1,2], and cultivated as a high-value medicinal plant. A number of bioactive compounds have been isolated from A. tataricus, such as shionone, epifriedelinol, quercetin, emodin, caffeoylquinic acid, kaempferol, and some other triterpenes or saponins [3,4]. Modern pharmacological studies [5,6,7] have shown that A. tataricus exhibits diverse pharmacological effects, including antibacterial, antiviral, antitumor, and diuretic activities; consequently, it is recorded as a basic clinical herb in the Chinese Pharmacopoeia. Furthermore, as a late-blooming aster, A. tataricus is an appealing ornamental plant in some East Asian countries. However, genomic research on A. tataricus—both nuclear genome sequencing and plastid genome sequencing—has been relatively scarce. This lack of genetic information has hindered the basic research into, and applications of, this valuable plant, such as molecular authentication, breeding, cultivation, and bioactive compound biosynthesis.

The chloroplast, the photosynthetic organelle of most green plants, is involved in both developmental processes and secondary metabolic activities [8], as well as coordination of gene expression between organelle and nuclear genome [9]. The chloroplast (cp) genome is considered to have originated from an ancestral endosymbiotic cyanobacteria [10] and is organized into large clusters of polycistronic transcribed genes [11]. Therefore, it has a highly conserved tetrad structure, with a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverse repeats (IRs) regions. As conserved matrilineal genetic information, chloroplasts play a part in cytoplasmic male sterility (CMS) [12], genetic diversity in populations [13,14,15], and especially, phylogenetic analysis [16,17]. In addition, the comparison of chloroplast sequences has been successfully used to study the evolution [18] and athentication markers to molecular identification at genus levels [19]. Evolutionary events, such as IR contraction and expansion, and re-inversion of SSC, have been found in the comparison of chloroplast genome [20,21]. Moreover, the chloroplast DNA sequences offers adequate information to study the phylogenetics and phylogeography of angiosperms at lower taxonomic levels [8]. In past decades, there have been great advances in our understanding of chloroplasts [22,23,24,25], in terms of their origin, structure, evolution, forward and reverse genetics, and genetic engineering. Moreover, the emergence of second-generation sequencing technology means that there is now less demand for research into chloroplast genomics [26,27]. Therefore, a draft cp genome assembly for A. tataricus is of great significance for exploring molecular identification, phylogenetic relationships, and evolutionary events studies among Asteraceae species.

Here, we reported the first whole chloroplast genome sequence for A. tataricus, together with the characterization of its gene annotations and repeat compositions. We also compared this chloroplast genome with that of other Aster species, which revealed significant variation in genome size and highly divergent regions in intergenic spacers. This comprehensive cp genomic analysis would provide a basis for molecular identification and further understanding of the evolutionary history of Aster species.

2. Results and Discussion

2.1. Features of A. tataricus cpDNA

The complete cp genome sequence of A. tataricus was 152,992 bp (GenBank accession number MH669275). The structure of the A. tataricus cp genome was analogous to those from other Aster species [28], and included an LSC region (84,698 bp; covering 55.4%), an SSC region (18,250 bp; covering 11.9%), and a pair of inverted repeats (IRA/IRB, 25,022 bp; covering 16.4%) (Table 1). The content of DNA G + C in the LSC, SSC, and IR regions, and the whole genome, was 35.2%, 31.3%, 43%, and 37.3%, respectively. The DNA G + C content is a very significant indicator when evaluating species affinity, and the cpDNA G + C content of A. tataricus is identical to that of other Aster species [28]. The DNA G + C content of the IR regions in A. tataricus was greater than that of other regions (LSC, SSC); this phenomenon is very common in other plants too [16,17]. The relatively high DNA G + C content of the IR regions is generally attributable to the rRNA genes and tRNA genes [29,30,31].

Table 1

Summary of complete chloroplast genomes for three Aster species.

Species	Aster altaicus	Aster spathulifolius	Aster tataricus
Large single-copy (LSC)
Length (bp)	84,240	81,998	84,698
G + C (%)	35.3	31.4	35.2
Length (%)	55.3	54.9	55.4
Small single-copy (SSC)
Length (bp)	18,196	17,973	18,250
G + C (%)	31.3	35.8	31.3
Length (%)	11.9	12.0	11.9
IR
Length (bp)	25,005	24,751	25,022
G + C (%)	43.0	43.2	43.0
Length (%)	16.4	16.6	16.4
Total
Length (bp)	152,446	149,473	152,992
G + C (%)	37.3	37.7	37.3

In the A. tataricus cp genome, 115 functional genes were observed, including four rRNA genes, 30 tRNA genes, and 81 protein-coding genes (Table 2). Furthermore, 18 genes—seven tRNA, all four rRNA, and seven protein-coding genes—were repeated in the IR regions (Figure 1). The LSC region contained 62 protein-coding and 22 tRNA genes, while the SSC region comprised one tRNA gene and 12 protein-coding genes.

Table 2

Genes in the A. tataricus chloroplast genome.

Category	Gene Group	Gene Names
Self-replication	Large subunit of ribosomal proteins	rpl2 **,a, 14, 16 **, 20, 22, 23 a, 32, 33, 36
	Small subunit of ribosomal proteins	rps2, 3, 4, 7a, 8, 11, 12 **,a, 14, 16 **, 18, 19
	DNA-dependent RNA polymerase	rpoA, B, C1 **, C2
	rRNA genes	rrn16Sa, rrn23Sa, rrn4.5Sa, rrn5Sa
	tRNA genes	trnA-UGC **,a, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-UCC **, trnG-GCC, trnH-GUG, trnI-CAU, trnI-GAU **,a, trnK-UUU **, trnL-CAA, trnL-UAA **, trnL-UAG, trnM-CAU, trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC **, trnW-CCA, trnY-GUA
Photosynthesis	Photosystem I	psaA, B, C, I, J
	Photosystem II	psbA, B, C, D, E, F, H, I, J, K, L, M, N, T, Z,
	NADH oxidoreductase	ndhA **, B **,a, C, D, E, F, G, H, I, J, K
	Cytochrome b6/f complex	petA, B **, D **, G, L, N
	ATP synthase	atpA, B, E, F **, H, I
	Rubisco	rbcL
Other genes	Maturase	matK
	Protease	clpP **
	Envelope membrane protein	cemA
	Subunit acetyl-CoA-carboxylase	AccD
	c-Type cytochrome synthesis gene	CcsA
	Conserved open reading frames	ycf1, 2a, 3 **, 4, 15

** Genes containing introns. a Duplicated gene (genes present in the inverted repeat (IR) regions).

Figure 1

Gene map of the A. tataricus chloroplast genome. Genes drawn inside the circle are transcribed clockwise, and those outside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray in the inner circle corresponds to DNA G + C content, while the lighter gray corresponds to A + T content.

The sequences of the tRNA and protein-coding genes were studied, and the frequency of codon usage was inferred and summarized for the A. tataricus chloroplast genome. Our study revealed that 23,441 codons characterize the coding capacity of 81 protein-coding and 30 tRNA genes in A. tataricus (Table 3). Of these codons, 4065 (17.34%) were found to code for leucine and 204 (0.87%) for tryptophan, which represented the maximum and minimum prevalent number of amino acids in the A. tataricus chloroplast genome, respectively. A- and U-ending codons were ordinary.

Table 3

Codon–anticodon recognition patterns and codon usage for the A. tataricus chloroplast genome.

Amino Acid	Codon	No.	RSCU *	tRNA	Amino Acid	Codon	No.	RSCU *	tRNA
Phe	UUU	1064	1.34		Tyr	UAU	793	1.39
Phe	UUC	528	0.66	trnF-GAA	Tyr	UAC	348	0.61	trnY-GUA
Leu	UUA	554	1.55	trnL-UAA	Stop	UAA	508	1.09
Leu	UUG	492	1.37	trnL-CAA	Stop	UAG	368	0.79
Leu	CUU	459	1.28		His	CAU	374	1.36
Leu	CUC	205	0.57		His	CAC	175	0.64	trnH-GUG
Leu	CUA	272	0.76	trnL-UAG	Gln	CAA	482	1.4	trnQ-UUG
Leu	CUG	169	0.47		Gln	CAG	206	0.6
Ile	AUU	798	1.46		Asn	AAU	792	1.39
Ile	AUC	397	0.73	trnI-GAU	Asn	AAC	347	0.61	trnN-GUU
Ile	AUA	441	0.81	trnI-UAU	Lys	AAA	914	1.42	trnK-UUU
Met	AUG	415	1	trn(f)M-CAU	Lys	AAG	376	0.58
Val	GUU	364	1.45		Asp	GAU	471	1.43
Val	GUC	176	0.7	trnV-GAC	Asp	GAC	187	0.57	trnD-GUC
Val	GUA	318	1.26	trnV-UAC	Glu	GAA	550	1.39	trnE-UUC
Val	GUG	148	0.59		Glu	GAG	241	0.61
Ser	UCU	509	1.37		Cys	UGU	345	1.15
Ser	UCC	329	0.89	trnS-GGA	Cys	UGC	253	0.85	trnC-GCA
Ser	UCA	493	1.33	trnS-UGA	Stop	UGA	524	1.12
Ser	UCG	292	0.79		Trp	UGG	491	1	trnW-CCA
Pro	CCU	259	1.29		Arg	CGU	204	0.73	trnR-ACG
Pro	CCC	156	0.78	trnP-GGG	Arg	CGC	108	0.39
Pro	CCA	224	1.12	trnP-UGG	Arg	CGA	282	1.01
Pro	CCG	164	0.82		Arg	CGG	173	0.62
Thr	ACU	321	1.22		Arg	AGA	329	0.89	trnR-UCU
Thr	ACC	246	0.93	trnT-GGU	Arg	AGG	277	0.75
Thr	ACA	314	1.19	trnT-UGU	Ser	AGU	578	2.06
Thr	ACG	174	0.66		Ser	AGC	337	1.2	trnS-GCU
Ala	GCU	250	1.25		Gly	GGU	315	0.95
Ala	GCC	169	0.85		Gly	GGC	205	0.62	trnG-GCC
Ala	GCA	242	1.21	trnA-UGC	Gly	GGA	466	1.4	trnG-UCC
Ala	GCG	138	0.69		Gly	GGG	342	1.03

* RSCU: relative synonymous codon usage.

There were 18 intron-containing genes in total: 12 protein-coding genes and six tRNA genes (Table 4). Fifteen genes (nine protein-coding and six tRNA genes) comprised one intron, and two genes (ycf3 and clpP) comprised two introns (Table 4). The intron of the trnK-UUU gene contains the matK gene, and the size of the intron was 2497 bp. The rps12 gene was a trans-spliced gene, with the 5′ end located in the LSC region and the copied 3′ end located in the IR regions. Earlier studies have reported that ycf3 is essential for the constant accumulation of the photosystem I compound [32]. Therefore, we suppose that the intron gain in ycf3 of A. tataricus may be valuable information in terms of further studies of the mechanism of photosynthesis evolution. We compared the length of exons and introns in genes with introns in the A. tataricus and A. spathulifolius chloroplast genomes (Table 4). While these were found to be broadly similar, some differences were noted: (1) in the A. spathulifolius chloroplast genome, the rpl16 gene had no intron; (2) there was significant variation in rps12 and rps16 intron length between the two species; and (3) in A. spathulifolius, the rpl12 gene had no intron.

Table 4

A comparison of exon and intron length in genes with introns in the A. tataricus and A. spathulifolius chloroplast genomes.

Gene	Location	Exon I (bp)	Intron I (bp)	Exon II (bp)	Intron II (bp)	Exon III (bp)
trnK-UUU	LSC	37	2497	38
		37	2502	35
trnG-UCC	LSC	23	732	48
		23	723	47
trnL-UAA	LSC	34	441	50
		37	423	50
trnV-UAC	LSC	36	575	37
		38	573	37
trnI-GAU	IR	38	781	35
		43	776	35
trnA-UGC	IR	38	820	35
		38	820	35
rps12 *	LSC	234	535	25	——	114
		114	——	243	——	243
rps16	LSC	234	820	40
		39	826	216
rpl16	LSC	402	1008	10
		——	——	——
rpl2	IR	391	671	434
		393	668	435
rpoC1	LSC	431	709	1639
		429	721	1641
ndhA	SSC	552	1055	540
		553	1105	540
ndhB	IR	777	674	756
		777	670	756
ycf3	LSC	124	690	228	739	155
		124	697	230	739	153
petB	LSC	6	754	658
		6	745	642
atpF	LSC	144	718	411
		145	699	410
clpP	LSC	71	812	291	614	229
		71	800	291	623	229
petD	LSC	9	645	526
		9	724	474

Exon and intron lengths in genes with introns in the A. tataricus chloroplast genome (gray background), and in the A. spathulifolius chloroplast genome (normal background) * The rps12 gene is a trans-spliced gene with the 5′ end located in the LSC region and the duplicated 3′ ends located in the IR regions.

Advances in phylogenetic research have revealed that chloroplast genome evolution encompasses both structural changes and nucleotide substitutions [33,34,35]. A few examples of these changes, including intron or gene losses [21,36], have been discovered in chloroplast genomes. Introns play an important role in regulating gene expression. They can increase gene expression at a particular position and at a specific time [37]. Intron regulation mechanisms have been reported in other species. More experimental work is required to study the relationship between intron loss and gene expression introns in A. tataricus.

2.2. Simple Sequence Repeat (SSR) Analysis

Simple sequence repeats (SSRs) of 10 bp or longer are inclined toward slipped-strand mispairing, which is known to be the main mutational mechanism utilized in SSR polymorphisms. SSRs in the chloroplast genome can be extremely variable at the intra-specific level and are often used as genetic markers in population genetics and evolutionary studies [38,39,40,41]. In this research, we investigated the SSRs in the chloroplast genome of A. tataricus and in that of two other Aster species (Figure 2). The cp genome of Aster tataricus, Aster altaicus, and Aster spathulifolius contained 70, 58, and 36 SSRs, respectively. The level of mononucleotide repeat content was high (A. tataricus, 51.4%; A. altaicus, 62.1%; A. spathulifolius, 77.8%) in all the above species. These results will provide chloroplast SSR markers that can be used to study genetics, select germplasm for breeding, and facilitate the molecular identification of species.

Figure 2

Analysis of simple sequence repeats (SSRs) in the three Aster chloroplast genomes.

2.3. Comparative Chloroplast Genomic Analysis

Comparative analysis of genomes is a tremendously important step in genomics [42,43]. Comparing the structural changes amongst Aster chloroplast genomes revealed that the chloroplast genome A. spathulifolius was the smallest of the three whole Aster chloroplast genomes (Table 1). A. spathulifolius had the fewest IR regions (17,973 bp) among these sequenced Aster chloroplast genomes. We supposed that the dissimilar length of the IR regions was the principal reason for the change in sequence length. To explicate the level of genome differences, the sequence identity of the Aster chloroplast DNAs was computed using mVISTA software, with A. tataricus as a reference (Figure 3). The results of this comparison showed that the IR (A/B) regions exhibited fewer differences than the LSC and SSC regions. Moreover, the non-coding regions showed more variability than the coding regions, and the marked differences in regions among the three chloroplast genomes were evident in the intergenic spacers. Of the three Aster chloroplast genomes, A. tataricus and A. altaicus exhibited the fewest differences.

Figure 3

Comparison of three chloroplast genomes using mVISTA. Gray arrows and thick black lines above the alignment indicate gene orientation. Purple bars represent exons, blue bars represent untranslated regions (UTRs), pink bars represent conserved non-coding sequences (CNS), and gray bars represent mRNA. The y-axis represents the percentage identity (shown: 50–100%).

2.4. Inverted Repeat (IR) Contraction and Expansion in the A. tataricus Chloroplast Genome

Contractions and expansions of the IR regions at the borders are ordinary evolutionary events and represent the main reasons for changes in the size of chloroplast genomes; they play a significant role in evolution [44,45,46]. For A. altaicus, A. spathulifolius and A. tataricus, we conducted an exhaustive comparison of four junctions, LSC-IRA (JLA), LSC-IRB (JLB), SSC-IRA (JSA), and SSC-IRB (JSB), between the two IRs (IRA and IRB) and the two single-copy regions (LSC and SSC) (Figure 4). The JSA junction was placed in the ycf1 pseudogene region in all the Aster species chloroplast genomes and outspread to different lengths (A. altaicus, 563 bp; A. spathulifolius, 608 bp; A. tataricus, 563 bp) within the IRA region of all the genomes; the IRB region contained 563, 567, and 565 bp of the ycf1 gene, respectively. Recently, it was reported that ycf1 is required for plant viability and codes Tic214, a significant component of the ArabidopsisTic complex member [47,48]. Correspondingly, the trnH gene was placed in the LSC region, 3, 22, and 3 bp away from the IRB/LSC border in the three Aster chloroplast genomes, respectively. The JLA in the Aster species was overlapped by rps19. The ndhF gene was found to be 22, 5, and 22 bp away from the IRA/SSC border in the Aster species.

Figure 4

Comparison of border distance between adjacent genes and junctions of the LSC, SSC, and two IR regions among the chloroplast genomes of three Aster species. Boxes below the main line indicate the adjacent border genes. The figure is not to scale with respect to sequence length and only shows relative changes at or near the IR/SC borders.

Although the gene order in chloroplasts is generally conserved in most green plants, it has been reported that many sequences are rearranged in chloroplast genomes from an extensive variety of different plant species, including inversions in the LSC region, IR contractions or expansions with inversions, and re-inversion in the SSC region [49,50,51,52,53]. Sequence rearrangements that convert chloroplast genome structure in related species may also reveal information about genetic diversity that could be used for molecular classification and evolution studies.

2.5. Phylogenetic Analysis

The availability of a completed A. tataricus cp genome provided us with sequence information that can be used to study the phylogeny of A. tataricus among Asteraceae. We performed multiple sequence alignments using the whole cp genome sequences in 16 Asteraceae species. One additional cp genome, Paeonia ostii (Paeoniaceae), was included as an outgroup (Figure 5). The method of maximum likelihood (ML) was used to construct a phylogenetic tree. The results strongly supported the finding that A. altaicus and A. tataricus are sister species, and A. tataricus is closer to A. altaicus than to A. spathulifolius.

Figure 5

Maximum likelihood (ML) phylogenetic tree reconstruction including 17 species based on concatenated sequences from all chloroplast genomes. The position of A. tataricus is indicated in red text. Paeonia ostii was used as the outgroup.

3. Materials and Methods

3.1. DNA Sequencing, Chloroplast Genome Assembly, and Validation

The A. tataricus was planted in the China Academy of Chinese Medical Sciences (N 39°56′, E 116°25′, Beijing, China). Fresh leaves were gathered and covered with tin foil, frozen in liquid nitrogen, and maintained at −80 °C. An improved cetyltrimethylammonium bromide (CTAB) method was used to obtain the whole genomic DNA of A. tataricus [54]. The concentration of DNA was estimated using an ND-2000 spectrometer (Nanodrop Technologies, Wilmington, DE, USA) [55]. A 250-bp shotgun library was constructed according to the manufacturer’s instructions (Vazyme Biotech Co. Ltd., Nanjing, China). The library was sequenced using an Illumina X Ten platform (Illumina, San Diego, CA, USA) double terminal sequencing method (150 pair-ends). The sample contained 5 G of raw data, and over 34 million paired-end reads (SRA accession: SRP154896) were obtained.

The raw data was filtered using Skewer-0.2.2 (Institute of Plant Quarantine Research, Chinese Academy of Inspection and Quarantine, Beijing, China, https://sourceforge.net/projects/skewer/) [56]. BLAST searches were used to abstract chloroplast-like reads from clean-reads in comparison with reference sequences (A. altaicus). Lastly, we used the chloroplast-like reads to assemble sequences using SOAPdenovo-2.04 (BGI·tech, Shenzhen, China, https://sourceforge.net/projects/soapdenovo2/files/SOAPdenovo2/) [57]. SSPACE-3.0 (Leiden University, Leiden, The Netherlands https://www.baseclear.com/services/bioinformatics/basetools/sspace-standard/) [58] and GapCloser-1.12 (BGI·tech, Shenzhen, China, https://sourceforge.net/projects/ oapdenovo2/files/GapCloser/) [59] were used to outspread sequences and fill gaps. PCR amplification and Sanger sequencing were used to confirm the four junction regions between the IR regions and the LSC/SSC regions, to confirm the assembly (Table S1).

3.2. Gene Annotation and Sequence Analyses

CpGAVAS [60] was used to annotate the sequences; DOGMA [61] and BLAST were used to check the annotation findings. tRNAscanSEv1.21 [62], with default settings, was used to identify all tRNA genes. OGDRAWv1.2 [63] was used to show the structural features of the chloroplast genomes. Relative synonymous codon usage (RSCU) values were defined using MEGA5.2 [64].

3.3. Genome Comparison

mVISTA [65] (Shuffle-LAGAN mode) was used to compare the whole chloroplast genome of A. tataricus, A. altaicus (KX352465), and A. spathulifolius (KF279514), with the annotation of A. tataricus as the reference. Phobos version 3.3.12 [66] was employed to detect SSRs within the cp genome, with the search parameters set at 10 repeat units for mononucleotides, _8 repeat units for dinucleotides,_4 repeat units for trinucleotides and tetranucleotides, and _3 repeat units for pentanucleotide and hexanucleotide SSRs.

3.4. Phylogenetic Analysis

We downloaded 16 whole chloroplast genome sequences of Asteraceae species from the National Center for Biotechnology Information (NCBI) Organelle Genome and Nucleotide Resources database. The whole chloroplast genome sequences were used to analyze the phylogenetics. The software clustalw2 (The Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland) was used to align sequences. MEGA5.2 was used to analyze and plot the phylogenetic tree with ML (maximum likelihood). We used 1000 replicates and TBR (tree bisection and reconnection) branch exchange to complete the bootstrap analysis. Furthermore, Paeonia ostii was set as the outgroup.

4. Conclusions

To our knowledge, we were the first to complete the sequencing and analysis of the whole chloroplast genome of A. tataricus, showing that the quadruple structure, gene order, DNA G + C content, and codon usage features were similar to those of the other Aster chloroplast genomes studied. Compared with the chloroplast genomes of the other two Aster species, the chloroplast genome of A. tataricus was the largest, while the genome structure and composition were found to be similar. Of the three Aster chloroplast genomes, A. tataricus and A. altaicus exhibited the fewest differences. Examination of the phylogenetic relationships among the three Aster species revealed that A. tataricus was more closely related to A. altaicus than to A. spathulifolius. The findings of this study offer an assembly of a whole chloroplast genome of A. tataricus, which would be valuable for molecular identification, breeding, and further biological discoveries.