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. 2018 Apr 9;23(4):857. doi: 10.3390/molecules23040857

Chloroplast Genome of the Folk Medicine and Vegetable Plant Talinum paniculatum (Jacq.) Gaertn.: Gene Organization, Comparative and Phylogenetic Analysis

Xia Liu 1,*, Yuan Li 1, Hongyuan Yang 1, Boyang Zhou 1
PMCID: PMC6017404  PMID: 29642545

Abstract

The complete chloroplast (cp) genome of Talinum paniculatum (Caryophyllale), a source of pharmaceutical efficacy similar to ginseng, and a widely distributed and planted edible vegetable, were sequenced and analyzed. The cp genome size of T. paniculatum is 156,929 bp, with a pair of inverted repeats (IRs) of 25,751 bp separated by a large single copy (LSC) region of 86,898 bp and a small single copy (SSC) region of 18,529 bp. The genome contains 83 protein-coding genes, 37 transfer RNA (tRNA) genes, eight ribosomal RNA (rRNA) genes and four pseudogenes. Fifty one (51) repeat units and ninety two (92) simple sequence repeats (SSRs) were found in the genome. The pseudogene rpl23 (Ribosomal protein L23) was insert AATT than other Caryophyllale species by sequence alignment, which located in IRs region. The gene of trnK-UUU (tRNA-Lys) and rpl16 (Ribosomal protein L16) have larger introns in T. paniculatum, and the existence of matK (maturase K) genes, which usually located in the introns of trnK-UUU, rich sequence divergence in Caryophyllale. Complete cp genome comparison with other eight Caryophyllales species indicated that the differences between T. paniculatum and P. oleracea were very slight, and the most highly divergent regions occurred in intergenic spacers. Comparisons of IR boundaries among nine Caryophyllales species showed that T. paniculatum have larger IRs region and the contraction is relatively slight. The phylogenetic analysis among 35 Caryophyllales species and two outgroup species revealed that T. paniculatum and P. oleracea do not belong to the same family. All these results give good opportunities for future identification, barcoding of Talinum species, understanding the evolutionary mode of Caryophyllale cp genome and molecular breeding of T. paniculatum with high pharmaceutical efficacy.

Keywords: Talinum paniculatum, chloroplast genome, medicinal plant, phylogeny

1. Introduction

Chloroplasts are the metabolic centers of our world due to the crucial role of converting sunlight into energy and releasing oxygen. Chloroplast genome encodes many key proteins that are involved in photosynthesis and other important metabolic processes for plant interactions with their environment, such as drought, salt and light [1,2,3,4]. The availability of over 2400 sequenced chloroplast genomes (http://www.ncbi.nlm.nih.gov/genomes/) gave us insights to understand the plant biology diversity, evolution, climatic adaptation, DNA barcoding and expression high-value agricultural or biomedical products by genetic engineering [1,2,5,6,7,8,9].

Java ginseng (Talinum paniculatum (Jacq.) Gaertn.), one of the most popular medicinal plants, belongs to the genus Talinum Adans. in the Portulacaceae family of the Caryophyllales. It is widely used for many ailments, including cancer, diabetes, hepatic disorders, leishmaniasis and reproductive disorders due to its high concentrations of alkaloids, flavonoids, tannins, steroids, and triterpenes [10,11,12,13,14]. The root of T. paniculatum has lots of valuable components, which are similar to those in Panax [15]. Meanwhile, the leaves of T. paniculatum are widely used as a green leafy vegetable for human consumption in many parts of South America, Africa and Asia.

Like Portulaca oleracea, T. paniculatum has high adaptability to drought, salinity, and low nutrient conditions [16]. Few reports have been published on the genetic diversity of chloroplast DNA from the Portulacaceae. The relationship of Portulaca to Anacampserotaceae, Cactaceae Juss, and Talinaceae is still uncertain [17,18,19,20]. The Portulacaeae are composed only a single genus of Portulace [21]. Molecular barcodes based on the chloroplast genome have shown great potential for species discrimination, especially between closely related taxa, but to date, chloroplast genome sequences of only two genus of the Portulacaceae have been analyzed, which are Carnegiea gigantean and Portulaca oleracea [22,23,24]. At both the species and population levels, the complete chloroplast genome sequence might enhance our ability to explore reliable barcoding for accurate plant identification [6,7,25].

Here, we report for the first time the complete cp genome sequence of T. paniculatum. Meanwhile, the gene structure and organization of the cp genome were analyzed. Besides, the cp genome sequences was compared with other genus of Caryophyllales. We also present the results of phylogenetic analyses of DNA sequences for 48 protein-coding genes from T. paniculatum, 35 Caryophyllales cp genomes and two outgroups.

2. Results

2.1. Features of the T. paniculatum cp Genome

The chloroplast genome of T. paniculatum, a typical quadripartite structure, is 156,929 bp in size, including a LSC region of 86,898 bp ranging from trnH-GUG to rps19 (Ribosomal protein S19), a SSC region of 18,529 bp from ndhF (NAD(P)H dehydrogenase) to ycf1 (hypothetical protein 1 gene), a pair of IR regions of 25,751 bp from rps19 to pseudogene ycf1 and ranging from ycf1 to rps19, respectively (Figure 1 and Table 1). The GC content of cp genome in the cp total, LSC (large single copy), SSC (small single copy), IRA (inverted repeat A region) and IRB (inverted repeat B region) is 36.79%, 34.6%, 30.5%, 42.7% and 42.7%, respectively (Table 1), which is similar to the cp genome of other Caryophyllales [23,24] and the higher GC content in IR regions. A higher AT representation at the third codon position and second codon position was significant, which is one of the common characteristics of the chloroplast genome [26,27,28,29]. The overall cp genome of T. paniculatum is made up of 49.98% protein-coding regions, 5.76% rRNAs, 1.78% tRNAs and 50.02% non-coding regions.

Figure 1.

Figure 1

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The complete chloroplast genome map of Talinum paniculatum (Jacp) Gaertn. Genes are color-coded based on functional group. Genes drawn inside the circle are transcribed clockwise, and those outside are transcribed counterclockwise. The genome orientation are the orange arrow.

Table 1.

Chloroplast genome composition of Talinum paniculatum (Jacp) Gaertn.

Region Size (bp) T(U) (%) C (%) A (%) G (%) Genes Protein-Coding Genes tRNA Genes rRNA Genes
LSC 86,898 33.2 17.8 32.2 16.8 83 56 22 0
SSC 18,529 34.6 15.9 34.9 14.6 12 12 1 0
IRA 25,751 29.0 22.3 28.2 20.4 19 7 7 4
IRB 25,751 28.2 20.4 29.0 22.3 19 8 7 4
Total 156,929 31.8 19.0 30.7 18.5 128 83 (4) 37 (7) 8 (4)
CDS 78,438 31.5 17.6 30.8 20.1
1st position 26,146 23.7 18.8 30.7 26.7
2nd position 26,146 32.7 20.2 29.4 17.8
3rd position 26,146 38.1 13.7 32.4 15.7
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CDS: protein-coding regions. The numbers in brackets represent the number of repeated genes.

In total, there are 113 unique functional genes and four pseudogenes in the chloroplast gene of T. paniculatum (Table 1 and Table 2). Among 113 functional genes, 79 protein-coding genes, 30 distinct tRNAs and four distinct rRNAs were contained (Table 2). Remarkably, because all rRNA, seven tRNA, four protein-coding genes and one pseudogene are replicated in the IR regions, the total genes in Figure 1 and Table 2 are 132. Fifty six protein-coding and 22 tRNA genes are located in LSC region, but only 12 protein-coding genes and one tRNA are located in the SSC region. Meanwhile, all eight rRNA are sited in IR regions, which has been found to be common in most cp genomes [23,30]. The ycf1 gene is located at the IR and SSC boundary region, the rps19 gene was located at the IR and LSC boundary region. Pseudogenes of rpl23, rps19, ycf1 are all located in IR regions.

Table 2.

Genes of the Talinum paniculatum (Jacp) Gaertn.

Group of Genes Name of Gene No.
Photosytem I psaA, psaB, psaC, psaI, psaJ 5
Photosytem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ 15
Cytochrome b/f complex petA, petB, petD, petG, petL, petN 6
ATP system atpA, atpB, atpF, atpF, atpH, atpI 6
NADH dehydrogenase ndhA, ndhB *, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK 12
RuBisCO large subnit rbcL 1
RNA polymerase rpoA, rpoB, rpoC1, rpoC2 4
Ribosomal proteins (SSU) rps2, rps3, rps4, rps7 *, rps8, rps11, rps12 *, rps14, rps15, rps16, rps18, rps19 14
Ribosomal proteins (LSU) rpl2, rpl14, rpl16, rpl19, rpl20, rpl22, rpl32, rpl33, rpl36 9
Miscellaneous proteins accD, cemA, ccsA, clpP, infA, matK 6
Hypothetical chloroplast reading frames (ycf) ycf1, ycf2 *, ycf3, ycf4 5
Transfer RNAs trnA-UGC *, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-UCC, trnG-GCC, trnH-GUG, trnI-CAU *, trnIGAU *, trnK-UUU, trnL-UAA, trnL-CAA *, trnL-UAG, trnM-CAU, trnN-GUU *, trnP-UGG, trnQ-UUG, trnR-UCU, trnRACG *, trnS-GCU, trnS-UGA, trnS-GGA, trnT-GGU, trnT-UGU, trnV-UAC, trnV-GAC *, trnW-CCA, trnY-GUA, trnfM-CAU 37
Ribosomal RNAs rrn4.5 *, rrn5 *, rrn16 *, rrn23 * 8
Pseudogene rpl23 *, rps19, ycf1 4
Total 132
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* indicates a duplicated gene.

Among the 113 unique genes in T. paniculatum cp genome, 17 genes contain introns, including six tRNA genes and 11 protein-coding genes. Most of them only contain one intron, while like to other angiosperms, ycf3, clpP and rps12 have two introns (Table 3). Twelve genes with introns are located in the LSC, four genes with introns are located in IR regions, and only one gene with intron is located in the SSC region. Interestingly, the 3′ exon of rps12 is duplicated in IRs, but its 5′ exon shared by the 3′ exon was located in the LSC region. Consistent with many research results, the matK gene was inserted into the intron of trnK-UUU, so the intron of trnK-UUU became so maximal [23,30]. Comparing these 17 introns with P. oleracea, most of them is shorter in T. paniculatum, whereas the introns of petB, petD are larger, and rpoC1 intron is the same size [23].

Table 3.

The intron-containing genes in the T. paniculatum cp genome and the lengths of the exons and introns.

No. Gene Location ExonI (bp) IntronI (bp) ExonII (bp) IntronII (bp) ExonIII (bp)
1 trnK-UUU LSC 35 2502 37
2 rps16 LSC 202 867 41
3 trnG-UCC LSC 23 707 48
4 atpF LSC 410 745 145
5 rpoC1 LSC 1611 794 432
6 ycf3 LSC 153 773 229 769 125
7 trnL-UAA LSC 37 599 50
8 trnV-UAC LSC 35 586 38
9 clpP LSC 229 590 291 894 71
10 petB LSC 6 768 642
11 petD LSC 8 792 475
12 rpl16 LSC 399 1102 9
13 ndhB * IR 756 668 777
14 rps12 * IR 114 - 232 533 26
15 trnI-GAU * IR 37 947 35
16 trnA-UGC * IR 38 818 35
17 ndhA SSC 539 1087 553
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* indicates duplicated gene.

2.2. Codon Usage of the T. paniculatum cp Genome

As shown in Table 4, total 26,146 codons involved in the protein-coding in T. paniculatum. Among 26,146 codons, the amino acids used most frequently were leucine and isoleucine, which encode in 2780 (10.63%) and 2258 (8.63%) codons, respectively. While only 303 (1.50%) codons encode cysteine as the least frequent universal amino acid (Table 4).

Table 4.

Codon usage in the T. paniculatum cp genome.

Amino Acid Codon Count RSCU Amino Acid Codon Count RSCU Amino Acid Codon Count RSCU Amino Acid Codon Count RSCU
Phe UUU(F) 975 1.3 Ser UCU(S) 556 1.67 Tyr UAU(Y) 779 1.61 Stop UGA(*) 20 0.72
Phe UUC(F) 527 0.7 Ser UCC(S) 331 0.99 Tyr UAC(Y) 190 0.39 Trp UGG(W) 460 1
Leu UUA(L) 854 1.84 Ser UCA(S) 408 1.22 Stop UAA(*) 46 1.66 Ala GCU(A) 615 1.75
Leu UUG(L) 554 1.2 Ser UCG(S) 187 0.56 Stop UAG(*) 17 0.61 Ala GCC(A) 234 0.66
Leu CUU(L) 601 1.3 Ser AGU(S) 393 1.18 His CAU(H) 448 1.49 Ala GCA(A) 416 1.18
Leu CUC(L) 179 0.39 Ser AGC(S) 127 0.38 His CAC(H) 155 0.51 Ala GCG(A) 144 0.41
Leu CUA(L) 408 0.88 Pro CCU(P) 426 1.6 Gln CAA(Q) 729 1.54 Arg CGU(R) 360 1.36
Leu CUG(L) 184 0.4 Pro CCC(P) 200 0.75 Gln CAG(Q) 216 0.46 Arg CGC(R) 92 0.35
Ile AUU(I) 1130 1.5 Pro CCA(P) 299 1.12 Asn AAU(N) 960 1.53 Arg CGA(R) 366 1.38
Ile AUC(I) 410 0.54 Pro CCG(P) 143 0.54 Asn AAC(N) 297 0.47 Arg CGG(R) 120 0.45
Ile AUA(I) 718 0.95 Thr ACU(T) 531 1.61 Lys AAA(K) 1061 1.5 Arg AGA(R) 475 1.79
Met AUG(M) 607 1 Thr ACC(T) 250 0.76 Lys AAG(K) 357 0.5 Arg AGG(R) 175 0.66
Val GUU(V) 514 1.48 Thr ACA(T) 402 1.22 Asp GAU(D) 883 1.66 Gly GGU(G) 552 1.26
Val GUC(V) 160 0.46 Thr ACG(T) 134 0.41 Asp GAC(D) 184 0.34 Gly GGC(G) 190 0.43
Val GUA(V) 522 1.5 Cys UGU(C) 235 1.55 Glu GAA(E) 1041 1.53 Gly GGA(G) 712 1.62
Val GUG(V) 194 0.56 Cys UGC(C) 68 0.45 Glu GAG(E) 323 0.47 Gly GGG(G) 302 0.69
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RSCU: Relative synonymous codon usage. RSCU > 1 are highlighted in bold. * indicates stop codon.

The relative synonymous codon usage (RSCU) has been divided into four models, 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) [30,31]. As shown in Table 4, there are 32 lack of bias codons with values < 1.0, except tryptophan and methionine, four low bias codons, four moderately biased codons and 21 highly biased codons, respectively. The TAA stop codon was found to be preferred. Meanwhile, due to 29 preferred synonymous codons (RSCU > 1.0) end with A or T, the third position of CDS contains 70.5% A or T bases, a significantly higher percentage than in the 2nd position (62.1%) or the 1st position (54.4%) (Table 1 and Table 4). The results showed that the bias was significant in usage of synonymous codons except tryptophan and methionine in T. paniculatum as well as A/T ending rich in cp genome. These results revealed that the RSCU and codon usage exsited biased codon usage, which was consistent with previous reports in cp genomes of higher plants [32,33].

2.3. Repeats Structure and SSR in T. paniculatum cp Genome

Analysis of the repeat structure analysis using REPuter detected a total of 51 repeat sequences, including 20 forward repeats, 21 palindromic repeats, one complement repeats and nine reverse repeats in the cp genome of T. paniculatum (Table 5). The repeats range from 30 to 61 bp in length and are mostly located in the intergenic spacer (IGS) and intron sequences. Twenty five repeats are located in exons of matK, trnS-GGA, trnG-UCC, psaB, psaA and ycf2 genes, respectively. A comparative analysis revealed that 30–39 bp were the most repetitive in cp genome of seven species in Caryophyllales (Figure 2). Complement repeats were rare, as only T. paniculatum and C. gigantea contain them [24]. The reverse repeats of T. paniculatum are the most in the seven cp genomes.

Table 5.

Repeat sequences distribution in the T. paniculatum (Jacp) Gaertn chloroplast genome.

No. Size (bp) Type Repeat 1 Start Repeat 1 Location Repeat 2 Start Repeat 2 Location Location
1 35 F 3144 matK 6441 IGS (rps16, trnQ-UUG) LSC
2 30 P 4153 IGS (trnK-UUU (exon), rps16) 4211 IGS (trnK-UUU (exon), rps16) LSC
3 30 R 4578 IGS (trnK-UUU (exon), rps16) 4581 IGS (trnK-UUU (exon), rps16) LSC
4 30 C 4581 IGS (trnK-UUU (exon), rps16) 4582 IGS (trnK-UUU (exon), rps16) LSC
5 30 R 6862 IGS (trnQ-UUG, psbK) 8268 IGS (trnS-GCU, trnG-UCC) LSC
6 30 F 7743 IGS (psbI, trnS-GCU) 7770 IGS (psbI, trnS-GCU) LSC
7 32 F 7896 IGS (psbI, trnS-GCU) 36,017 IGS (psbC, trnS-UGA) LSC
8 30 P 7898 IGS (psbI, trnS-GCU) 46,268 trnS-GGA LSC
9 37 R 8258 IGS (trnS-GCU, trnG-UCC) 8261 IGS (trnS-GCU, trnG-UCC) LSC
10 37 R 8258 IGS (trnS-GCU, trnG-UCC) 8264 IGS (trnS-GCU, trnG-UCC) LSC
11 35 F 8258 IGS (trnS-GCU, trnG-UCC) 8277 IGS (trnS-GCU, trnG-UCC) LSC
12 35 R 8266 IGS (trnS-GCU, trnG-UCC) 8277 IGS (trnS-GCU, trnG-UCC) LSC
13 34 R 8258 IGS (trnS-GCU, trnG-UCC) 8261 IGS (trnS-GCU, trnG-UCC) LSC
14 33 F 8261 IGS (trnS-GCU, trnG-UCC) 8283 IGS (trnS-GCU, trnG-UCC) LSC
15 32 R 8263 IGS (trnS-GCU, trnG-UCC) 8280 IGS (trnS-GCU, trnG-UCC) LSC
16 31 F 8261 IGS (trnS-GCU, trnG-UCC) 8264 IGS (trnS-GCU, trnG-UCC) LSC
17 31 R 8261 IGS (trnS-GCU, trnG-UCC) 8280 IGS (trnS-GCU, trnG-UCC) LSC
18 31 R 8267 IGS (trnS-GCU, trnG-UCC) 29,873 IGS (psbM, trnD-GUC) LSC
19 30 P 8267 IGS (trnS-GCU, trnG-UCC) 62,668 IGS (ycf4, cemA) LSC
20 30 P 8280 IGS (trnS-GCU, trnG-UCC) 31,428 IGS (trnE-UUC, trnT-GGU) LSC
21 31 F 9566 trnG-UCC 37,057 trnG-GCC LSC
22 30 P 36,019 IGS (psbC, trnS-UGA) 46,268 trnS-GGA LSC
23 30 F 39,314 psaB 41,538 psaA LSC
24 42 F 44,540 ycf3 (intronII) 123,558 ndhA (intron) LSC, SSC
25 39 F 44,543 ycf3 (intronII) 100,738 IGS (rps12, trnV-GAC) LSC, IRb
26 39 P 44,543 ycf3 (intronII) 143,050 IGS (trnV-GAC, rps12) LSC, IRa
27 30 F 44,555 ycf3 (intronII) 100,750 IGS (rps12, trnV-GAC) LSC, IRb
28 30 P 44,555 ycf3 (intronII) 143,047 IGS (trnV-GAC, rps12) LSC, IRa
29 40 P 76,849 IGS (psbT, psbN) 76,849 IGS (psbT, psbN) LSC
30 30 P 84,344 IGS (trnS-GCU, trnG-UCC) 84,346 IGS (trnS-GCU, trnG-UCC) LSC
31 61 F 93,517 ycf2 93,535 ycf2 IRb
32 61 P 93,517 ycf2 150,231 ycf2 IRb, IRa
33 61 P 93,535 ycf2 150,249 ycf2 IRb, IRa
34 61 F 150,231 ycf2 150,249 ycf2 IRa
35 52 F 93,526 ycf2 93,544 ycf2 IRb
36 52 P 93,526 ycf2 150,231 ycf2 IRb, IRa
37 52 P 93,544 ycf2 150,249 ycf2 IRb, IRa
38 34 F 93,526 ycf2 93,562 ycf2 IRb
39 34 P 93,526 ycf2 150,231 ycf2 IRb, IRa
40 34 P 93,562 ycf2 150,267 ycf2 IRb, IRa
41 43 F 93,517 ycf2 93,533 ycf2 IRb
42 43 P 93,517 ycf2 150,231 ycf2 IRb, IRa
43 43 P 93,553 ycf2 150,267 ycf2 IRb, IRa
44 43 F 150,231 ycf2 150,267 ycf2 IRa
45 40 F 100,738 IGS (rps12, trnV-GAC) 123,561 ndhA (intron) IRb, SSC
46 34 F 109,506 IGS (rrn4.5, rrn5) 109,538 IGS (rrn4.5, rrn5) IRb
47 34 F 109,506 IGS (rrn4.5, rrn5) 134,255 IGS (rrn5, rrn4.5) IRb, IRa
48 34 P 109,538 IGS (rrn4.5, rrn5) 134,287 IGS (rrn5, rrn4.5) IRb, IRa
49 34 P 134,255 IGS (rrn5, rrn4.5) 134,287 IGS (rrn5, rrn4.5) IRa
50 38 P 118,646 IGS (ccsA, ndhD) 118,646 IGS (ccsA, ndhD) SSC
51 40 P 123,561 ndhA (intron) 143,049 IGS(trnV-GAC, rps12) SSC, IRa
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F: forward repeat; P: palindrome (inverted) repeat; R: reverse repeat; C: complement repeat. IGS: intergenic spacer.

Figure 2.

Figure 2

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Repeat sequences in seven chloroplast genomes of Caryophyllales. REPuter was used to identify repeat sequences with length ≥30 bp and sequence identify ≥90% in the chloroplast genomes. F, P, R and C indicate the repeat types forward, palindrome, reverse and complement, respectively.

There are 92 simple sequence repeats (SSRs) in the T. paniculatum chloroplast genome, the majority of which were mononuclear repeats (68). Twelve dinucleotide repeats, five trinucleotide repeats and seven pentanucleotide repeats were also found in T. paniculatum cp genome (Table 6, Figure 3). Whereas, only seven SSRs located in the CDSs (Table 7), including four mononucleotide, one dinucleotide and two pentanucleotide SSRs. The results indicate that all of the SSRs in CDs are located in the LSC region and most of them are AT repeats, which is consistent with the view that SSRs in cp genomes contribute to AT richness [32].

Table 6.

Frequency of simple sequence repeats in the T. paniculatum chloroplast genome.

Length Unit 10 10 11 12 13 14 15 16 17 18 19 20 Total
A 15 15 3 8 1 1 3 1 1 33
T 14 14 4 7 3 2 1 1 1 33
C 1 1 1
G 1 1 1
AG 1 1 1
AT 4 4 3 7
TA 2 2 1 1 4
AAT 1 1 1
ATA 1 1
TTA 2 2
TAT 1 1
AGGT 1 1
ATGG 1 1
AATT 1 1
CTAC 1 1
TTTC 1 1
TAAT 1 1
GGAA 1 1
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Figure 3.

Figure 3

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Distribution of SSRs present in seven chloroplast genomes of Caryophyllales.

Table 7.

Simple sequence repeats in the CDSs of the T. paniculatum chloroplast genome.

No. Type Motif Size Start End Location Region
1 P1 (A)10 10 47 56 trnH-UGG LSC
2 P1 (A)10 10 637 646 psbA LSC
3 P1 (A)11 11 2104 2114 matK LSC
4 P1 (A)12 12 3942 3953 trnK-UUU (intron) LSC
5 P2 (AT)5 10 755 764 psbA LSC
6 P4 (AATT)3 12 3974 3985 trnK-UUU (intron) LSC
7 P4 (CCAT)3 12 54 65 trnH-UGG LSC
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2.4. matK and rpl23 Diversity Analysis

The chloroplast genes matK and rpl23 of T. paniculatum were compared with those of nine other Caryophyllaceae species. The matK sequence is often used in DNA barcoding, phylogenetic and evolutionary studies [34]. The sequences of matK genes of chloroplast DNA in T. paniculatum, P. oleracea, S. conica, S. chalcedonica, S. europaea, S. bigelovii, A. githago, H. ammodendro and S. oleracea show significant diversity by alignment analysis (Figure 4), while most of the matk (T. paniculatum, P. oleracea, S. conica, S. chalcedonica, S. europaea, S. bigelovii, A. githaggo, H. ammodendro and S. oleracea) were inserted into the introns of trnK-UUU [35]. Oddly, the matK gene is a pseudogene in S. conica, which has the shortest length [36]. The multiple alignment result revealed that the matK gene of T. paniculatum was relatively similar to that of P. oleracea, except for an AATT insert and eight single nucleotide polymorphisms (SNPs) (Figure 4). The matK genes from S. chalcedonica and A. githago displayed relatively high sequence similarity, due to the fact they both belong to the Sileneae in the Caryophyllaceae family [37]. The matK in S. bigelovii and S. europaea had high sequence similarity too, because they belong to the same family of Chenopodiaceae (Figure 4). These results revealed that the closer the relationship between species, the higher the sequence similarity of matK. Hence, consistent with many research results, the matK gene was one of the best choices for DNA barcoding, phylogenetic and evolutionary analysis [38,39,40].

Figure 4.

Figure 4

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Sequence alignment of matK among nine species cp genome in Caryophyllales. As the matK gene are too long, only the sequences with greater variation were shown here.

Ribosomal protein L23 (rpl23) is a protein component of the 60S large ribosomal subunit and is also a negative regulator of cellular apoptosis in animals [41]. The alignment of rpl23 was carried out in T. paniculatum, P. oleracea, C. longiscapa, S. conica, H. ammodendron, S. bigelovii, S. europaea (Figure 5). The results revealed that rpl23 gene of T. paniculatum was a pseudogene as in H. ammodendron, S. bigelovii and S. europaea. The rpl23 gene lacked the CTTGACACCAAAGA sequence in H. ammodendron, S. bigelovii and S. europaea. However, the rpl23 of T. paniculatum had AATT inserted. Interestingly, the rpl23 was not presented in A. githago, S. chalcedonica and A. hypochondriacus [37]. The rpl23 were normal genec in P. oleracea and C. longiscapa, which had some SNPs between them [42].

Figure 5.

Figure 5

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The gene of rpl23 sequence alignment among six Caryophyllales species.

2.5. Comparative Analysis of the Chloroplast Genomes of the Caryophyllales

The genome sequence of T. paniculatum was compared with eight species of Caryophyllales using the mVISTA software (Figure 6). T. paniculatum had the biggest cp genome with the biggest IR region (25,751 bp), while C. gigantea had the smallest cp genome with one IR region loss [24]. The length differences among them were mainly caused by deletions in the non-coding regions. Meanwhile, the results showed that the non-coding region variation was significantly higher than that of the coding regions, and the IR region was more conserved than the LSC and SSC regions [43,44]. The matK, accD, ndhF, infA, trnS-GGA, trnT-UGU, trnL-UAA, trnG-UCC, ycf2, ycf1, rpl23, ccsA, ndhG and rps19 genes were the most divergent coding regions. The introns of ycf3, ndhA, rpl16, clpP, petB, trnK-UUU, rpoC, pet D were relatively highly divergent, too.

Figure 6.

Figure 6

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Complete chloroplast genome sequence comparison of eight species using mVISTA, with T. paniculatum as a reference. The horizontal axis corresponds to the coordinates within the chloroplast genome. The vertical scale represents the identity percentage. The grey lines and the arrows show the genes with their orientation and position. CNS: conserved noncoding sequences.

2.6. IR Expansion and Contraction

IR contraction and expansion of T. paniculatum was analyzed by comparing the LSC/IRb/SSC/IRa boundary regions with seven families in the Caryophyllales (Figure 7). Among them, the chloroplast genome of T. paniculatum was the longest. The ndhF gene of T. paniculatum did not cross the IRb region and the SSC region, compared to P. oleracea, A. githago, A. hypochndriacus and S. oleracea, however it was the largest IR region among them (Figure 7). The rps19 gene in the T. paniculatum cp genome was shifted by 117 bp from LSC to IRb at the LSC/IRb border, which was the smallest shift length compared to P. oleracea, A. githago, H. ammodendron, S. oleracea and S. bigelovii (Figure 7). Differently from other LSC/IRb border, the rps19 gene of S. chalcedonica was entirely located in the LSC region. Unlike most LSC/IRa borders, the trnH gene of S. bigelovii was located in the LSC region, and the trnH of T. paniculatum was located in the LSC region as most of them. Oddly, a significant bigger IR size but the smallest rps19 and ycf1 length in the IR region were found in T. paniculatum. This phenomenon may be for two reasons: on one hand, pseudogenes exist, and on the other hand, there is a low occurrence of contraction by the fragment deletions in the intergenic regions in T. paniculatum [32].

Figure 7.

Figure 7

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Comparison of the borders of the LSC, SSC and IR regions in nine Caryophyllales species. #indicates that the gene is a pseudogene.

2.7. Phylogenetic Analysis

The cp genome sequence is a useful resource for studying the taxonomy in the Angiosperm clade, and for analyzing evolutionary relationships within families. Here, to obtain a reasonable phylogenetic status of T. paniculatum, we performed multiple sequence alignments of cp genome protein coding genes. A total of 35 complete cp genomes of Caryophyllales and two outgroup species were subjected to phylogenetic analysis based on a 48-gene data matrix, which used the MP and ML methods. MP analysis resulted in a single tree with a length of 24,669, a consistency index (CI) of 0.6111, and a retention index (RI) of 0.7786 (Figure 8). Bootstrap analysis showed that 28 out of the 33 nodes had bootstrap values >95%.

Figure 8.

Figure 8

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Phylogenetic tree of the 35 species in Caryophyllales using maximum parsimony (MP) and tree bisection-reconnection (TBR) analysis based on 48 protein-coding genes using a non-partitioning scheme. The phylogenetic tree was drawn using Cistanche deserticola and Rehmannia chingiis as outgroup.

As we all know, the specific relationships within the Poltulaca and Talinum remain obscure due to their complex past evolutionary histories. Recently, according to molecular and morphological evidence, the Anacampserotaceae, Basellaceae, Cactaceae, Didiereaceae, Portulacaceae, and Talinaceae were classified into four single independent families [17,18,19,20]. The Portulacaeae are now comprised only a single Portulace genus [21]. However, few reports have been published on the genetic diversity of chloroplast DNA from the Anacampserotaceae, Basellaceae, Cactaceae, Didiereaceae and Portulacaceae. From Figure 8, the cp genome protein coding genes of the phylogenetic trees show that P. oleracea and T. paniculatum do not belong to the same family, although the above data shows that there are many similarities between P. oleracea and T. paniculatum, which is consistent with the recent classification studies [20,45,46]. Hence, whole cp genome or the key segments of evolutionary variation should be used for phylogenetic study.

3. Discussion

We report a genome sequence of T. paniculatum, which provides an important resource for studying the evolution of the Caryophyllales and the molecular breeding of T. paniculatum with high pharmaceutical efficacy. Despite the fact that the chloroplast genomes of Angiosperms are well-conserved in the genomic structure in terms of gene order and number, length variations of the whole chloroplast genome sequences and LSC, SSC and IR regions, the IR expansion and contraction occur frequently. The results reported here are congruent with the recent studies which showed that the trnH-GUG gene was situated in the LSC region in some species of Caryophyllales, while the SSC/IRA border extends into the ycf1 with subsequent formation of a ycf1 pseudogene [47,48]. Boundary expansion and contraction between the single copy and IR boundary regions lead to sequence variation, which might be a base of plant lineages [49].

In this study, we analyzed codon usage frequency and RSCU in the T. paniculatum. As previously reported, leucine and isoleucine are the more commonly seen amino acids in the cp genomes of Angiosperms [33,50,51,52,53]. Likewise, like in earlier studies about repeats and SSRs, mononucleotide repeats are more abundant with A/T repeats, which is consistent with AT richness in Angiosperm chloroplast genomes [54,55,56]. Complement repeats were rarely found in T. paniculatum and C. gigantea [24]. Meanwhile, all of the SSRs in CDs locate in the LSC region [32]. These cp SSR markers could be a resource for molecular-marker-assisted selection breeding for T. paniculatum for production of high levels of biologically active compounds.

Here, we compared the matK and rpl23 sequences of several species of Caryophyllales. The results revealed that the pseudogene rpl23 of T. paniculatum had inserted AATT, which is different from other Caryophyllales. The gene of matK is often used to identity the relationship between species as a barcoding marker [57]. Most reports have revealed that the closer the relationship between species, the higher the sequence similarity of matK. matK represents one of the best choices for DNA barcoding, phylogenetic and evolutionary analysis [38,39,40]. However, though T. paniculatum has a high matK gene similarity to P. oleracea, they belong to different families, hence, multiple marks should be used for barcoding.

4. Conclusions

In this study, the complete cp genome of T. paniculatum was reported and analyzed for the first time. T. paniculatum is one of the key traditional Chinese medicines used against cancer, diabetes, hepatic disorders, leishmaniasis and reproductive disorders and is also an edible vegetable. Comparing the cp genomes of T. paniculatum with other Caryophyllale species, the cp genome of T. paniculatum is the largest IRs, but has the smallest rps19 and ycf1 length in the IR border, most likely due to low occurrence of contraction by the fragment deletions in the intergenic regions. The pseudogene of rpl23 was inserted by AATT, and trnK-UUU and rpl16 have larger introns than other Caryophyllale species. The matK genes show rich divergence. All these results provide good opportunities for future barcoding molecular marker development. Our phylogenetic analysis showed that T. paniculatum and P. oleracea don’t belong to the same family. This information will be useful for the phylogenetic study of T. paniculatum, and might also contribute to the genetics and breeding of T. paniculatum.

5. Materials and Methods

5.1. DNA Sequencing and Genome Assembly

Total DNA of T. paniculatum was obtained from approximately 100 g of fresh leaves using the CTAB method [58]. Quality of the DNA was evaluated by measuring A260 using a Nanodrop2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, the DNA was sheared to fragments of 300~500 bp. Paired-end libraries were prepared with the TruSeqTM DNA sample Prep Kit and the TruSeq PE Cluster Kit. The genome was then sequenced using the HiSeq4000 platform (Illumina Inc., San Diego, CA, USA). The assembly of the cp genome of T. paniculatum was first carried out through the error correction and production of initial contigs using the GS FLX De Novo Assembler Software (Newbler V2.6). PCR amplification and Sanger sequencing were performed to verify the four junction regions between the IRs and the LSC/SSC. The final cp genome of T. paniculatum was submitted to GenBank with the accession number MG710385.

5.2. Gene Annotation and Codon Usage Analysis

The cp genome was annotated by manual corrections using BLAST and DOGMA [59]. The tRNAscan-SE [60] was used to identify the tRNA genes. OGDRAW [61] was used to draw the circular genome map. MEGA5 were used for revealing the characteristics of the variations in synonymous codon usage [62]. The relative synonymous codon usage values (RSCU), codon usage and GC content were also determined by MEGA5.

5.3. 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 identified by REPuter [63] and MISA respectively, with the same parameters as described in Ni et al. [43].

5.4. Comparative Genome Analysis of the T. paniculatum with Eight cp Genomes of Caryophyllales

Comparison of the overall cp genome of T. paniculatum with eight cp genomes of Caryophyllales were performed by mVISTA [64,65], using the annotation of T. paniculatum as a reference.

5.5. Phylogenetic Analysis

A total of 37 complete cp genome sequences were downloaded from the NCBI Organelle Genome Resources database (http://www.ncbi.nlm.nih.gov/genomes/). For the phylogenetic analysis, a set of 48 protein-coding genes that were common in the 37 analyzed genomes, was used. Maximum parsimony (MP) analysis was performed with PAUP*4.0b10 [66], using a heuristic search combined with the random addition of 1000 replicates and tree bisection-reconnection (TBR) branch swapping, in the Multrees option. Bootstrap analysis was also performed with 1000 replicates and TBR branch swapping. Rehmannia chingii and Lindenbergia philippensis were set as outgroups.

Acknowledgments

This work was supported by grants from the Natural Science Foundation of Tianjin (17JCZDJC34300) and Key Projects in the National Science and Technology Pillar Program during the Twelve Five-year Plan Period of China (2015BAD16B02).

Author Contributions

X.L. conceived and designed the experiments; Y.L. and H.Y. performed the experiments; X.L., Y.L. and B.Z. analysed the data; X.L. and Y.L. wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the T. paniculatum are available from the authors.

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