molecules

Article
Complete Chloroplast Genome Sequences of
Kaempferia Galanga and Kaempferia Elegans:
Molecular Structures and Comparative Analysis
Dong-Mei Li *, Chao-Yi Zhao and Xiao-Fei Liu
Guangdong Key Lab of Ornamental Plant Germplasm Innovation and Utilization, Environmental Horticulture
Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
zhaoc2009@126.com (C.-Y.Z.); 13580525912@139.com (X.-F.L.)
* Correspondence: biology.li2008@163.com; Tel.: +86-20-875-93429




Received: 27 December 2018; Accepted: 25 January 2019; Published: 29 January 2019


Abstract: Kaempferia galanga and Kaempferia elegans, which belong to the genus Kaempferia
family Zingiberaceae, are used as valuable herbal medicine and ornamental plants, respectively.
The chloroplast genomes have been used for molecular markers, species identification and
phylogenetic studies. In this study, the complete chloroplast genome sequences of K. galanga and K.
elegans are reported. Results show that the complete chloroplast genome of K. galanga is 163,811 bp
long, having a quadripartite structure with large single copy (LSC) of 88,405 bp and a small single
copy (SSC) of 15,812 bp separated by inverted repeats (IRs) of 29,797 bp. Similarly, the complete
chloroplast genome of K. elegans is 163,555 bp long, having a quadripartite structure in which IRs
of 29,773 bp length separates 88,020 bp of LSC and 15,989 bp of SSC. A total of 111 genes in K.
galanga and 113 genes in K. elegans comprised 79 protein-coding genes and 4 ribosomal RNA (rRNA)
genes, as well as 28 and 30 transfer RNA (tRNA) genes in K. galanga and K. elegans, respectively.
The gene order, GC content and orientation of the two Kaempferia chloroplast genomes exhibited
high similarity. The location and distribution of simple sequence repeats (SSRs) and long repeat
sequences were determined. Eight highly variable regions between the two Kaempferia species
were identified and 643 mutation events, including 536 single-nucleotide polymorphisms (SNPs)
and 107 insertion/deletions (indels), were accurately located. Sequence divergences of the whole
chloroplast genomes were calculated among related Zingiberaceae species. The phylogenetic analysis
based on SNPs among eleven species strongly supported that K. galanga and K. elegans formed a
cluster within Zingiberaceae. This study identified the unique characteristics of the entire K. galanga
and K. elegans chloroplast genomes that contribute to our understanding of the chloroplast DNA
evolution within Zingiberaceae species. It provides valuable information for phylogenetic analysis
and species identification within genus Kaempferia.

Keywords: Kaempferia galanga; Kaempferia elegans; chloroplast genome; Illumina sequencing; PacBio
sequencing; genomic structure; comparative analysis

1. Introduction
The genus Kaempferia belongs to the family Zingiberaceae, which consists of approximately
50 species in the world [1–3]. Kaempferia species are distributed in tropical Asia regions [1,2]. Kaempferia
species are grown primarily for their ornamental foliage rather than for their flowers [3]. In addition,
several species have long been cultivated for their medicinal properties [3]. Kaempferia galanga and
Kaempferia elegans are valuable herbal medicine and ornamental plants in this genus, respectively.
K. galanga is mainly distributed in the regions of Southern and Northwestern China (Guangdong,

Molecules 2019, 24, 474; doi:10.3390/molecules24030474 www.mdpi.com/journal/molecules

Molecules 2019, 24, 474 2 of 18

Guangxi, Guizhou, Yunnan and Sichuan provinces) and is widely cultivated in Southeast Asia; whereas
K. elegans is only produced in Sichuan province of China and is commonly cultivated in tropical Asia
regions [1,2]. Morphology data had been used to determine the differences between K. galanga and
K. elegans [1–3]. From these studies, the two Kaempferia species had been characterized differently
by leaf shape, petioles, flower color and rhizomes [1–3]. The leaves of K. galanga spread flat on
ground, subsessile, green, orbicular, 7–20 × 3–17 cm, glabrous on both surfaces or villous abaxially,
margin usually white, apex mucronate or acute; the leaves of K. elegans have petioles, to 10 cm,
leaf adaxially green, abaxially pale green, oblong or elliptic, 13–15 × 5–8 cm, margin usually red,
base rounded, apex acute. The petals are white in K. galanga, whereas the petals are purple in K.
elegans. The rhizomes of K. galanga are pale green or greenish white inside, tuberous and fragrant;
the rhizomes of K. elegans are bearing globose tubers with fibrous roots. K. galanga can be used as
aromatic medicinal plant and also as ornamental plant with important economic value, whereas K.
elegans is mainly used as potted plant with ornamental value. In detail, the rhizomes of K. galanga
have been used as aromatic stomachic, with effects of dissipating cold, dampness, warm the spleen
and stomach, also used as flavoring spices, for example, a famous Guangdong delicacy of sand ginger
salted chicken [2]. With increased demand for rhizomes of K. galanga, researches related to the high
rhizomes yield for its cultivation has been lacking, leading to having a high price in the market.
However, sometimes morphological identification of Kaempferia species was difficult owing to the
morphological similarity of vegetative parts among species and other genera in Zingiberaceae, such
as Boesenbergia, Cornukaempferia, Curcuma and Scaphochlamys [3,4]. In addition, intraspecific variation
caused more complicated problems in the morphological taxonomy of the genus Kaempferia [3,4].
Based only on morphological characteristics, we could not conclusively distinguish and identify the
Kaempferia species and hybrids and other genera species in Zingiberaceae. Therefore, the morphological
classification and relationships within Kaempferia species need further investigation together with more
molecular analyses. Combined evidences from morphology characteristics and chloroplast DNA have
proven useful and powerful in species identification and phylogenetic relationships analysis [4–8].
In angiosperm plants, chloroplasts play an important role in photosynthesis and the metabolism
of starch, fatty acids, nitrogen, amino acids and internal redox signals [9–11]. In general, the chloroplast
genomes of angiosperms encode 110–130 genes with a size range of 120-180 kb, which have a
typical quadripartite structure consisting of a large single copy (LSC) region, a small single copy
(SSC) region and two copies of inverted repeats (IRs) [12–15]. As the chloroplast is the center of
photosynthesis, the research of the chloroplast genome is important to discover the mechanisms of
plant photosynthesis. The third-generation sequencing platform PacBio utilizes a single-molecule
real-time sequencing technology, which has been successfully used to determine many chloroplast
genome sequences [16–18]. The main advantage of this method is the long read length of over 10 kb
on average, which provides many benefits in genome assembly, including longer contigs and fewer
unresolved gaps [16,17]. However, PacBio sequencing has high rates of random error in its single-pass
reads; therefore, in combination with Illumina sequencing can reduce these random errors [17,18].
In this study, we sequenced and analyzed the complete chloroplast genomes from K. galanga
and K. elegans, respectively, using Illumina and PacBio sequencing. We also characterized the long
repeats and SSRs detected in the genome, including repeat types, distribution patterns and so on.
Comparative sequence analysis and phylogenetic relationships were also analyzed among other
members in the family Zingiberaceae. These results will improve the genetic information of the genus
Kaempferia we already have and will be beneficial for DNA molecular studies in Kaempferia.

2. Results

2.1. Chloroplast Genome Organization of Two Kaempferia Species

The complete chloroplast genome of K. galanga and K. elegans consisted of a single circular
molecule with quadripartite structure (Figure 1). The sizes of K. galanga and K. elegans chloroplast
Molecules 2019, 24, 474 3 of 18

genomes were 163,811 and 163,555 bp, respectively. They consisted of a pair of inverted repeats (IRs)
of 29,797 bp in K. galanga and 29,773 bp in K. elegans, a large single copy (LSC) region of 88,405
Molecules 2019, 24, x FOR PEER REVIEW
bp in
3 of 18
K. galanga and 88,020 bp in K. elegans and a small single copy (SSC) region of 15,812 bp in K. galanga
of 29,797
and 15,989 bpbpininK.K.elegans
galanga(Figure
and 29,773
1 and bp Table
in K. elegans,
1). Thea GC
largecontent
single copy (LSC)
of the region was
genomes of 88,405
36.1%bpboth
in in
K. galanga
K. galanga andand 88,020 bp
K. elegans butin the
K. elegans and a had
IR regions smallhigher
single copy (SSC) region
GC contents of 15,812
(41.2% bp in K.
and 41.1% ingalanga
K. galanga
andelegans,
and K. 15,989 respectively)
bp in K. elegansthan (Figure
that1 of
and Table
the LSC1).regions
The GC(33.9%
contentboth
of the
ingenomes
K. galangawas 36.1%
and both in and
K. elegans)
K. galanga and K. elegans but the IR regions had higher GC contents (41.2%
SSC regions (29.5% and 29.4% in K. galanga and K. elegans, respectively). Approximately 48.3–50.7% and 41.1% in K. galanga
and K. elegans, respectively) than that of the LSC regions (33.9% both in K. galanga and K. elegans) and
of the two Kaempferia species chloroplast genomes consisted of protein-coding genes (83,172 bp in K.
SSC regions (29.5% and 29.4% in K. galanga and K. elegans, respectively). Approximately 48.3–50.7%
galanga and 79,117 bp in K. elegans), 1.7% of tRNAs (2870 bp K. galanga and 2852 bp in K. elegans) and
of the two Kaempferia species chloroplast genomes consisted of protein-coding genes (83,172 bp in K.
5.5%galanga
of rRNAs (9046 bp
and 79,117 bp in
inK.K.elegans),
galanga1.7% andof9046
tRNAs in K. elegans).
bp (2,870 Forand
bp K. galanga protein-coding
2,852 bp in K. regions, the AT
elegans) and
content
5.5%for
of the first,(9,046
rRNAs secondbp inandK. third
galanga codons were
and 9,046 bp55.4%, 62.6% For
in K. elegans). 71.1% in K. galanga,
andprotein-coding regions,respectively
the AT
and 66.9%, in K. elegans,
content for the first, second and third codons were 55.4%, 62.6% and 71.1% in K. galanga, respectively of
56.7% and 64.7% respectively (Table 1). The non-coding regions consisting
introns,
and pseudogenes
66.9%, 56.7% and and64.7%
intergenic spacersrespectively
in K. elegans, accounted(Table
for 49.3% and
1). The the K. galanga
51.7% forregions
non-coding and K.
consisting
of introns,
elegans pseudogenes
chloroplast genomes, and intergenic spacers
respectively (Table accounted
1). for 49.3% and 51.7% for the K. galanga and
K. elegans chloroplast genomes, respectively (Table 1).

Figure 1. Circular
Figure gene
1. Circular map
gene map ofof
chloroplast
chloroplastgenomes
genomes ofof two Kaempferiaspecies.
twoKaempferia species.
TheThe
graygray arrowheads
arrowheads
indicate
indicate the direction
the direction of the
of the genes.Genes
genes. Genesshown
shown inside
inside the
thecircle
circleare
aretranscribed clockwise
transcribed clockwiseand and
thosethose
outside
outside are transcribed
are transcribed counterclockwise.Different
counterclockwise. Different genes
genes are
arecolor
colorcoded. The
coded. innermost
The innermostdarker gray gray
darker
corresponds
corresponds to GC
to GC content,
content, whereasthe
whereas thelighter
lighter gray
gray corresponds
correspondstoto ATATcontent. IR, IR,
content. inverted repeat;
inverted repeat;
LSC, large single copy region; SSC, small single copy
LSC, large single copy region; SSC, small single copy region. region.
Molecules 2019, 24, 474 4 of 18

Table 1. Features of the chloroplast genomes of K. galanga and K. elegans.

Species Regions Positions Length (bp) T/U (%) C (%) A (%) G (%) AT/U (%)
Genome 163,811 32.2 18.3 31.7 17.7 63.9
LSC 88,405 33.7 17.3 32.4 16.4 66.1
IRa 29,797 28.8 19.8 30.0 21.2 58.8
SSC 15,812 34.5 15.5 35.9 13.9 70.5
IRb 29,797 28.8 19.8 30.0 21.2 58.8
K. galanga Protein coding genes 83,172 31.5 17.2 31.4 19.7 63.0
1st position 27,724 23.9 18.2 31.4 26.3 55.4
2nd position 27,724 32.4 20.0 30.1 17.3 62.6
3rd position 27,724 38.3 13.3 32.8 15.5 71.1
tRNA 2,870 24.9 23.6 22.0 29.3 47.0
rRNA 9,046 18.6 23.6 26.1 31.5 44.8
Genome 163,555 32.2 18.3 31.7 17.7 63.9
LSC 88,020 33.7 17.4 32.4 16.5 66.1
IRa 29,773 28.8 19.8 30.1 21.3 58.9
SSC 15,989 34.6 15.5 36.1 13.8 70.6
IRb 29,773 28.8 19.8 30.1 21.3 58.9
K. elegans Protein coding genes 79,117 31.6 17.3 31.2 19.9 62.8
1st position 26,372 35.0 14.3 31.9 18.8 66.9
2nd position 26,372 26.6 19.4 30.1 23.9 56.7
3rd position 26,372 33.3 18.2 31.4 17.1 64.7
tRNA 2,852 24.9 23.7 22.0 29.4 46.9
rRNA 9,046 18.7 23.6 26.1 31.5 44.8

There were 111 predicted genes in the K. galanga chloroplast genome including 79 protein-coding
genes, 28 tRNA genes and 4 rRNA genes, while 113 genes predicted in the K. elegans chloroplast
genome consisted of 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes (Table 2). Among the
protein-coding genes in K. galanga chloroplast genome, 61 genes were located in the LSC region,
12 genes were in the SSC region and 8 genes were duplicated in the IR regions (Supplementary file
1). In total, there were 18 intron-containing genes in the K. galanga chloroplast genome, 16 of which
contained one intron and two of which (ycf3 and clpP) contained two introns (Table 3). Among the
protein-coding genes in the K. elegans chloroplast genome, 63 genes were located in the LSC region,
12 genes were in the SSC region and 6 genes were duplicated in the IR regions (Supplementary file
2). In total, there were 17 intron-containing genes in the K. elegans chloroplast genome, 15 of which
contained one intron and two of which (ycf3 and clpP) contained two introns (Table 3).

Table 2. Genes present in the chloroplast genomes of K. galanga and K. elegans.

Category Gene Name

Photosystem I psaA, psaB, psaC, psaI, psaJ
Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, lhbA
Cytochrome b/f petA, petB *, petD *, petG, petL, petN
ATP synthase atpA, atpB, atpE, atpF *, atpH, atpI
NADH dehydrogenase ndhA *, ndhB(×2) *, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Rubisco rbcL
RNA polymerase rpoA, rpoB, rpoC1 *, rpoC2
Large subunit ribosomal proteins rpl2(×2) *, rpl14, rpl16 *, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36
Small subunit ribosomal proteins rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×2) *, rps14, rps15, rps16 *, rps18, rps19(×2)
Other proteins accD, ccsA, cemA, clpP *, infA, matK
Proteins of unknown function ycf1(×2), ycf2(×2), ycf3*, ycf4
Ribosomal RNAs rrn4.5(×2), rrn5(×2), rrn16(×2), rrn23(×2)
trnA-UGC (×2) *, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC (Kg×2,
Ke) **, trnG-UCC, trnH-GUG (×2), trnI-CAU (×2), trnI-GAU (×2) *, trnK-UUU (×2) *,
Transfer RNAs trnL-CAA (×2), trnL-UAA (×2) *, trnL-UAG, trnM-CAU, trnN-GUU (×2), trnP-UGG,
trnQ-UUG, trnR-ACG (×2), trnR-UCU, trnS-GCU (Kg×2, Ke), trnS-UGA, trnT-GGU (Kg×2,
Ke), trnV-GAC (×2), trnV-UAC (×2) *, trnW-CCA, trnY-GUA, trnS-GGA (Ke), trnT-UGU (Ke)
Kg: K. galanga; Ke: K. elegans; ×2: Gene with two copies; *: Genes containing introns both in K. galanga and K. elegans;
**: Genes containing introns only in K. galanga.
Molecules 2019, 24, 474 5 of 18

Table 3. Genes with introns in the chloroplast genomes of K. galanga and K. elegans, including the exon
and intron lengths.

Species Gene Location Exon I (bp) Intron I (bp) Exon II (bp) Intron II (bp) Exon III (bp)
trnA-UGC IR 38 801 35
trnG-GCC LSC 14 711 48
trnI-GAU IR 42 935 35
trnK-UUU LSC 35 2646 37
trnL-UAA LSC 35 536 50
trnV-UAC LSC 37 598 38
rps12 * LSC/IR 114 - 231 540 27
rps16 LSC 212 749 40
rpl2 IR 443 650 315
K. galanga
rpl16 LSC 402 1058 9
petB LSC 6 783 642
petD LSC 8 740 481
atpF LSC 425 816 145
ndhA SSC 518 1083 562
ndhB IR 778 673 782
rpoC1 LSC 1632 726 432
clpP LSC 252 636 306 856 60
ycf3 LSC 153 794 228 714 132
trnA-UGC IR 38 801 35
trnI-GAU IR 42 935 35
trnK-UUU LSC 35 2663 37
trnL-UAA LSC 35 535 50
trnV-UAC LSC 37 598 38
rps12 * LSC/IR 114 - 231 540 27
rps16 LSC 212 729 40
rpl2 IR 432 659 387
K. elegans rpl16 LSC 402 1056 9
petB LSC 6 784 642
petD LSC 8 741 481
atpF LSC 411 816 144
ndhA SSC 540 1079 552
ndhB IR 756 700 777
rpoC1 LSC 1632 728 432
clpP LSC 255 636 291 854 69
ycf3 LSC 153 794 228 723 132
* The rps12 gene is divided into 50 -rps12 in the LSC region and 30 -rps12 in the IR region.

Relative synonymous codon usage (RSCU) is the ratio between frequency of use and expected
frequency of a particular codon. RSCU values <1.00 indicate use of a codon less frequent than expected,
while codons used more frequently than expected have a score of >1.00 [19,20]. The codon usage of
the K. galanga and K. elegans chloroplast genomes are summarized in Table S1. Protein-coding genes
comprised 27,724 and 27,675 codons in both the K. galanga and K. elegans, respectively. Among these
codons, those for leucine and isoleucine were the most common in both K. galanga and K. elegans
chloroplast genomes (Figure 2 and Table S1). The use of the codons ATG and TGG, encoding Met and
Trp respectively, exhibited no bias (RSCU = 1.00) in these two Kaempferia species chloroplast genomes
(Table S1). Codons ending in A and/or U accounted for 71.1% and 64.7% of all protein-coding gene
codons of the chloroplast genomes of K. galanga and K. elegans, respectively (Table 1 and Table S1).
The findings also revealed that all of the types of preferred synonymous codons (RSCU>1.00) ended
with A or U except for trL-CAA in these two Kaempferia species (Table S1).
Molecules 2019, 24, x FOR PEER REVIEW 6 of 18
Molecules 2019, 24, 474 6 of 18
Molecules 2019, 24, x FOR PEER REVIEW 6 of 18

Figure 2. Amino acid frequencies in K. galanga and K. elegans protein-coding sequences.

Figure
Figure2.2.Amino
Aminoacid frequencies
acid in K.ingalanga
frequencies and K.
K. galanga elegans
and protein-coding
K. elegans sequences.
protein-coding sequences.
2.2. Analysis of SSRs and Long Repeats
2.2. Analysis
2.2. Analysis ofofSSRs
SSRsandandLong
LongRepeats
Repeats
SSRs or microsatellites, are tandem repeat sequences consisting of 1-6 nucleotide repeat units
SSRs or microsatellites, are tandem repeat sequences consisting of 1-6 nucleotide repeat units
and areSSRs or microsatellites,
widely distributed inare tandem repeat
chloroplast genomes sequences
[5,7,12]. consisting
SSRs wereof 1-6 nucleotide
detected using MISA repeat in units
both
and are widely distributed in chloroplast genomes [5,7,12]. SSRs were detected using MISA in both
and are widely
Kaempferia species distributed
chloroplast in chloroplast
genomes. genomes
We detected [5,7,12].
240 andSSRs 248were
SSRs detected
in K. using and
galanga MISA K. in both
elegans
Kaempferia species chloroplast genomes. We detected 240 and 248 SSRs in K. galanga and K. elegans
Kaempferia
chloroplast species
chloroplast genomes,
genomes, chloroplast genomes.
respectively.
respectively. We detected
Mononucleotide
Mononucleotide motifs 240
motifs
were and the248
were the
most SSRsmostin abundant
abundant K. galanga and K.
type of type
repeat of elegans
repeat
chloroplast
and dinucleotides were the second most abundant in both Kaempferia species chloroplast genomes repeat
and genomes,
dinucleotides were respectively.
the second Mononucleotide
most abundant motifs
in both were the
Kaempferia most abundant
species type
chloroplast of
genomes
and dinucleotides
(Figure
(Figure 33and
andTable were
TableS2). the second
S2).There
There were
were177 most
177 abundant
momo-,
momo-, 3232di-,in
di-, both
tri-,21Kaempferia
6 6tri-, tetra-,
21 tetra-, 3 species
3 penta-
penta- and chloroplast
andone hexa-genomes
hexa-nucleotide
one
(Figure
SSRs in 3K.and
nucleotide SSRs Table S2). There
in K.chloroplast
galanga galanga weregenome;
chloroplast
genome; 177bymomo-, 32there
by contrast,
contrast, di-,
there6were
tri-,
were 21188tetra-,
188 momo-,
momo-, 3 penta-
di-,7and
33 di-, 7tri-, one
19
tri-, 19 hexa-
tetra-
tetra-
and 1 and
nucleotide 1 SSRs
penta-nucleotide
penta-nucleotide SSRsSSRs
in K. galanga in elegans
in K. K. elegans
chloroplast chloroplast
genome;
chloroplast genome there
by contrast,
genome (Figure
(Figure 3).
were3).The
Themajority
188 momo-,
majority of SSRs
33ofdi-, 7 tri-,
SSRs were19
were located
tetra- and
located in the LSC
1 penta-nucleotide
in the regions
LSC regions rather rather
SSRsthan than in
in K.inelegans IR and
IR andchloroplastSSC
SSC regions regions
genome of both
of both(Figure Kaempferia
3). The
Kaempferia species
majority
species of SSRs
chloroplast
chloroplast
were
genomes located genomes
(Figurein the (Figure
3 andLSC 3 and
Table S2).Table
regions SSRs S2).
ratherwereSSRs
than were
in IR
more more
andabundant
abundant SSC in non-coding
in regions
non-coding of both regions than
Kaempferia
regions than species
in coding
in coding regions of both genomes (Figure 3). Furthermore, almost all SSR loci were composed of A
chloroplast
regions genomes
of both genomes (Figure 3 and
(Figure 3). Table S2). SSRs
Furthermore, were all
almost more SSRabundant
loci were in non-coding
composed of Aregions than
or T, which
or T, which contributed to the bias in base composition (A/T; both 63.9%) in the chloroplast genomes
in coding regions
contributed to the of both
bias genomes
in base (Figure 3).
composition Furthermore,
(A/T; both 63.9%) almost
in theall SSR loci were
chloroplast composed
genomes of the of A
two
of the two Kaempferia species.
or T, whichspecies.
Kaempferia contributed to the bias in base composition (A/T; both 63.9%) in the chloroplast genomes
of the two Kaempferia species.

(A) (B)

(A) (B)

Figure 3. Cont.
Molecules 2019,24,
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of 18
18
Molecules 2019, 24, x FOR PEER REVIEW 7 of 18

(C) (D)
(C) (D)
Figure3.3. Distribution
Figure Distribution of
of SSRs in the chloroplast genomes
genomes ofof K.
K. galanga and K.
galanga and K. elegans.
elegans.(A)
(A)Number
Number
Figure
of 3. Distribution
ofdifferent
different SSR
SSRtypes of SSRs inin
types detected
detected inthe
thechloroplast
the two genomes
two Kaempferia
Kaempferia of K.
species
species galanga and
chloroplast
chloroplast K. elegans.
genomes;
genomes; (B)(A)
(B) Number of
Frequency
Frequency of
of
different
identified SSR
identifiedSSR types
SSRmotifs detected
motifsin in
indifferent the
differentrepeattwo Kaempferia
repeatclass
classtypes; species
types;(C)
(C)SSR chloroplast
SSRdistribution
distributioningenomes;
indifferent (B)
differentgenomicFrequency
genomicregions of
regions
identified
of SSR motifs
twoKaempferia
oftwo Kaempferia in different
species
species repeat
chloroplast
chloroplast class types;
genomes;
genomes; (D) (C)
(D)SSR SSR distribution
SSRdistribution
distribution in different
between
between codinggenomic
coding and regions
andnon-coding
non-coding
of two
regions Kaempferia
regionsof species
twoKaempferia
oftwo Kaempferia chloroplast
species
species genomes;
chloroplast
chloroplast (D) SSR
genomes.
genomes. distribution between coding and non-coding
regions of two Kaempferia species chloroplast genomes.
Long
Long repeat sequences in
repeat sequences theK.K.galanga
inthe galangaand andK.K.elegans
eleganschloroplast
chloroplastgenomes
genomeswere were analyzed
analyzed by
by Long
REPuter
REPuter repeat
andand sequences
results
results shownshownin the K. galanga
in Figure
in Figure 4 and and
4 andTableK.
Tableelegans
S3.S3. chloroplast
In the
In the K. galangagenomes
K. galanga were analyzed
chloroplast
chloroplast genome,
genome, by
21
REPuter
21 forward and results
repeats, 20shown in
palindrome Figure 4 and
repeats, Table
5 reverseS3. In the
repeats K. galanga
and 4
forward repeats, 20 palindrome repeats, 5 reverse repeats and 4 complement repeats were detected chloroplast
complement genome,
repeats were21 forward
detected
repeats, 20 palindrome
(identity>90%)
(identity>90%) (Figure
(Figure4 and4 repeats,
andTable 5 S3).
S3).
Table reverse repeatsinand
In comparison,
In comparison, theinK.4thecomplement
elegans repeats
chloroplast
K. elegans were
genome,
chloroplast 26 detected
forward
genome, 26
(identity>90%)
repeats, 17 (Figure
palindrome 4 and
repeats, Table
4 S3).
reverse In comparison,
repeats and 2 in the
complement
forward repeats, 17 palindrome repeats, 4 reverse repeats and 2 complement repeats were detectedK. elegans
repeats chloroplast
were genome,
detected (Figure 26
4
forward
and Table repeats,
S3). Out 17
of palindrome
the 50 repeats repeats,
in K. 4 reverse
galanga repeats
chloroplast and
genome,2 complement
(Figure 4 and Table S3). Out of the 50 repeats in K. galanga chloroplast genome, 38 repeats (76.0%)38 repeats repeats
(76.0%) were
were detected
30–39 bp
(Figure
long, 4 and
were 930–39
repeatsbpTable
long,S3).
(18.0%) Out 40–49
were
9 repeats of (18.0%)
the bp
50 repeats
long
wereand in 3K.repeats
40–49 galanga
bp chloroplast
long(6.0%)
and 3wererepeatsgenome,
≥ 50(6.0%) 38
bp long repeats
(Figure
were ≥50 bp(76.0%)
4 and
long
were
Table 30–39
S3). By bp long,
contrast, 9 repeats
of the 49(18.0%)
repeats were
in K.40–49
elegans bp long and
chloroplast 3 repeats
genome,
(Figure 4 and Table S3). By contrast, of the 49 repeats in K. elegans chloroplast genome, 37 repeats (6.0%)
37 were
repeats ≥50
(75.5%) bp long
were
(Figure
30–39
(75.5%)bp4were
and30–39
long, 6Table
repeats
bpS3). By contrast,
(12.2%)
long, of
were 40–49
6 repeats thebp49long
(12.2%) repeats
were and
40–49 6inrepeats
K. elegans
bp chloroplast
long(12.2%)
and were ≥50
6 repeats genome,
bp long
(12.2%) 37 repeats
(Figure
were ≥50 bp4
(75.5%)
and
longTable were 30–39
S3). 4The
(Figure bp
andmajoritylong,
Table S3). 6 repeats
of these (12.2%)
repeats were
The majority were 40–49
mainly
of these bp
forward
repeats long
were and
and 6 repeats
palindromic
mainly forward (12.2%)
types were
and with ≥50 bp
lengths
palindromic
long
mainly (Figure
in the 4 and
range Table
of 30–50 S3).
bp The
in majority
both of
Kaempferia these repeats
species.
types with lengths mainly in the range of 30–50 bp in both Kaempferia species. were mainly forward and palindromic
types with lengths mainly in the range of 30–50 bp in both Kaempferia species.

(A) (B)
(A) (B)
Figure 4. Analysis of long repeat sequences in the chloroplast genomes of K. galanga and K. elegans.
Figure
Figure 4.4. Analysis
Analysis
(A) Frequency of long
of
of long long repeat
repeat
repeats sequences
sequences
types; in the
in the chloroplast
(B) Frequency chloroplast genomes
of long repeats of K. galanga and K. elegans.
by length.
(A)Frequency
(A) Frequencyof oflong
longrepeats
repeatstypes;
types;(B)
(B)Frequency
Frequencyofoflong
longrepeats
repeatsby
bylength.
length.
2.3. IR Contraction and Expansion
2.3.
2.3. IR
IR Contraction
Contraction and
and Expansion
Expansion
A detailed comparison was performed for four junctions, LSC/IRa, LSC/IRb, SSC/IRa and
A
A detailed
detailed comparison
comparison was performed for
forfour junctions, LSC/IRa, LSC/IRb, SSC/IRa and
SSC/IRb, between the two IRswas (IRaperformed
and IRb) and four
the twojunctions, LSC/IRa,
single-copy regionsLSC/IRb,
(LSC andSSC/IRa
SSC) among and
SSC/IRb,
SSC/IRb, between
between the
the two
two IRs
IRs (IRa
(IRaand
and IRb)
IRb)and
andthe
the two
twosingle-copy
single-copyregions (LSC
regions (LSCand
andSSC)
SSC)among
amongA.
A. zerumbet, C. flaviflora and Z. spectabile in comparison to K. galanga and K. elegans (Figure 5).
zerumbet,
A. C.
zerumbet,flaviflora and Z. spectabile in comparison to K. galanga and K. elegans (Figure 5). Although the
Although theC.
IR flaviflora
region ofand Z. spectabile
the five in comparison
Zingiberaceae to K. galanga
species chloroplast and was
genomes K. elegans
highly (Figure
conserved,5).
IR region of
Although the the
IR five Zingiberaceae
region of the five species chloroplast
Zingiberaceae species genomes was
chloroplast highlywas
genomes conserved,
highly structure
conserved,
structure variation was still found in the IR/SC boundary regions. As shown in Figure 5, the rpl22-
structure
rps19 genesvariation was stillinfound
were located in the IR/SC
the junctions boundary
of the LSC/IRbregions. Asin
regions shown in Figure
K. galanga, K. 5,elegans,
the rpl22-
A.
rps19 genes were located in the junctions of the LSC/IRb regions in K. galanga, K. elegans, A. zerumbet
Molecules 2019, 24, 474 8 of 18

variation was still found in the IR/SC boundary regions. As shown in Figure 5, the rpl22-rps19 genes
were located in the junctions of the LSC/IRb regions in K. galanga, K. elegans, A. zerumbet and C.
Molecules 2019, 24, x FOR PEER REVIEW 8 of 18
flaviflora, though the trnM-ycf2 sequence in Z. spectabile, one of which was missing the rpl22/-rps19
gene in the junctions of the LSC/IRb regions. The ycf1-ndhF genes were located
and C. flaviflora, though the trnM-ycf2 sequence in Z. spectabile, one of which was missing the rpl22/- at the junctions of the
IRb/SSC regions in the five Zingiberaceae species chloroplast genomes. The
rps19 gene in the junctions of the LSC/IRb regions. The ycf1-ndhF genes were located at the junctions ndhF gene was 23, 98,
251,
of the133 and 33
IRb/SSC bp from
regions in thethe IRb/SSC
five in K. galanga,
borderspecies
Zingiberaceae K. genomes.
chloroplast elegans, A. zerumbet,
The ndhF gene C.was
flaviflora
23, and Z.
spectabile,
98, 251, 133respectively
and 33 bp from (Figure 5). The border
the IRb/SSC SSC/IRa junctions
in K. in elegans,
galanga, K. the fiveA.Zingiberaceae speciesand
zerumbet, C. flaviflora chloroplast
genomes were crossed by the ycf1 gene, with 665–3888 bp in the IRa region. Like the IRb/SSC boundary
Z. spectabile, respectively (Figure 5). The SSC/IRa junctions in the five Zingiberaceae species
chloroplast
regions, thegenomes
IRa/LSC were crossed
regions wereby also
the ycf1 gene, with
variable. 665–3888 bpgenes
The rps19-psbA in thewere
IRa region.
locatedLike thejunctions
in the
IRb/SSC
of boundary
the IRa/LSC regions,
regions in the IRa/LSC K.
K. galanga, regions
elegans,were also variable.
A. zerumbet and The rps19-psbA
C. flaviflora, genes the
though weretrnH-psbA
located in the junctions of the IRa/LSC regions in K. galanga, K. elegans, A. zerumbet and
genes in Z. spectabile, one of which was missing the rps19 gene in the junctions of the IRa/LSC region. C. flaviflora,
though the trnH-psbA genes in Z. spectabile, one of which was missing the rps19 gene in the junctions
The rps19-psbA genes of K. elegans were located at the junctions of IRa/LSC regions with 136 and
of the IRa/LSC region. The rps19-psbA genes of K. elegans were located at the junctions of IRa/LSC
123 bp, respectively, separating the spacer from the end of the IRa region. However, in Z. spectabile,
regions with 136 and 123 bp, respectively, separating the spacer from the end of the IRa region.
the trnH gene
However, in Z. was the last
spectabile, the gene at one
trnH gene wasend
theoflast
thegene
IRaatregion,
one end 256
of bp
the away from 256
IRa region, the bpIRa/LSC
away border.
Overall,
from the IRa/LSC border. Overall, contraction and expansion of the IR regions was detected across species
contraction and expansion of the IR regions was detected across the five Zingiberaceae
chloroplast genomes.species chloroplast genomes.
the five Zingiberaceae

8 bp 1586 bp 147 bp
107 bp 148 bp 123 bp
3879 bp 23 bp 3880 bp
rpl22 rps19 Ψycf1 ndhF ycf1 rps19 psbA
Kaempferia galanga LSC IRb SSC IRa LSC
88,405 bp 29,797 bp 15,812 bp 29,797 bp
114 bp 137 bp 3888bp 98 bp 1583 bp 3888 bp136 bp 123 bp
rpl22 rps19 Ψycf1 ndhF ycf1 rps19 psbA
Kaempferia elegans LSC IRb SSC IRa LSC
88,020 bp 29,773 bp 15,989 bp 25,773 bp
47 bp 130 bp 1047 bp 251 bp
4340 bp1048 bp 130 bp 108 bp
rpl22 rps19 Ψycf1 ndhF ycf1 rps19 psbA
Alpinia zerumbet LSC IRb SSC IRa LSC
87,644 bp 26,917 bp 18,295 bp 26,917 bp
108 bp 131 bp 4 bp 131 bp 132 bp
133 bp 4376 bp 1072 bp
rpl22 rps19 Ψycf1 ndhF ycf1 rps19 psbA
Curcuma flaviflora LSC IRb SSC IRa LSC
88,008 bp 26,950 bp 18,570 bp 26,950 bp
125 bp 33 bp 4789 bp 665 bp 256 bp 205 bp
924 bp
trnM ycf2 Ψycf1 ndhF ycf1 trnH psbA
Zingiber spectabile LSC IRb SSC IRa LSC
88,460 bp 25,451 bp 16,528 bp 25,451 bp

Figure 5. Comparison of the borders of the LSC, SSC and IR regions among five Zingiberaceae
chloroplast
Figure genomes.ofΨ,the
5. Comparison pseudogenes.
borders of theBoxes
LSC,above the main
SSC and line indicate
IR regions among fivethe Zingiberaceae
adjacent border genes.
The figure genomes.
chloroplast is not to scale with respectBoxes
Ψ, pseudogenes. to sequence length
above the main and only shows
line indicate relativeborder
the adjacent changes at or near the
genes.
The figure is
IR/SC borders.not to scale with respect to sequence length and only shows relative changes at or near
the IR/SC borders.
2.4. Comparative Chloroplast Genomic Analysis
To characterize genome divergence, we performed multiple sequence alignments between the
five Zingiberaceae species chloroplast genomes using the program mVISTA, with K. galanga being
used as a reference (Figure 6). The comparison demonstrated that the two IR regions were less
Molecules 2019, 24, x FOR PEER REVIEW 9 of 18

2.4. Comparative Chloroplast Genomic Analysis

Molecules 2019, 24, 474 9 of 18
To characterize genome divergence, we performed multiple sequence alignments between the
five Zingiberaceae species chloroplast genomes using the program mVISTA, with K. galanga being
used as a reference
divergent (Figure
than the LSC 6). The
and SSC. comparison
Moreover, demonstrated
the coding regions arethat
moretheconserved
two IR regions
than the were less
non-coding
divergent
regions. than the LSC
The most anddivergent
highly SSC. Moreover,
regions the coding
among the regions are moregenomes
five chloroplast conserved than
were the among
found non-
coding regions. The most highly divergent regions among the five chloroplast genomes were
the intergenic spacers, including trnH-psbA, rps16-psbK, atpH-atpI, petN-psbM, trnE-psbD psbC-rps14, found
among the intergenic
rps4-ycf3, rps4-ndhJ,spacers, including
ndhC-atpE, trnH-psbA,
ycf4-cemA rps16-psbK,
and petA-psbJ in LSCatpH-atpI,
as well petN-psbM, trnE-psbD
as rpl32-ccsA, psbC-
psaC-ndhG and
rps14, rps4-ycf3,
ndhG-ndhI rps4-ndhJ,
in SSC. HigherndhC-atpE,
divergenceycf4-cemA and petA-psbJ
in the coding in LSC
regions was as well
found as matK,
in the rpl32-ccsA,
rpoA,psaC-ndhG
rps16, rps19,
and ndhG-ndhI
ndhF, in SSC.
ccsA, psaC, Higher
ndhD, ndhE,divergence
ndhG, ndhI,in the and
ndhA coding
ycf1regions was found in the matK, rpoA, rps16,
sequences.
rps19, ndhF, ccsA, psaC, ndhD, ndhE, ndhG, ndhI, ndhA and ycf1 sequences.

Figure 6. Comparison of five chloroplast genomes, with K. galanga as a reference using mVISTA
alignment
Figure program. Gray
6. Comparison arrows
of five and thick
chloroplast black lines
genomes, withabove the alignment
K. galanga indicateusing
as a reference gene orientation.
mVISTA
Purple bars represent exons, sky-blue bars represent transfer RNA (tRNA) and ribosomal RNA (rRNA),
alignment program. Gray arrows and thick black lines above the alignment indicate gene orientation.
red bars
Purple barsrepresent non-coding
represent sequences
exons, sky-blue bars(CNS) and white
represent peaks
transfer RNArepresent
(tRNA)differences of chloroplast
and ribosomal RNA
genomes. The y-axis represents the identity percentage ranging from 50 to 100%.
(rRNA), red bars represent non-coding sequences (CNS) and white peaks represent differences of
chloroplast genomes. The y-axis represents the identity percentage ranging from 50 to 100%.
Furthermore, sliding window analysis using DnaSP detected highly variable regions in the
chloroplast genomes between K. galanga and K. elegans (Figure 7A). The average value of nucleotide
Furthermore, sliding window analysis using DnaSP detected highly variable regions in the
diversity (Pi) was 0.01075. The IR regions showed lower variability than the LSC and SSC regions.
chloroplast genomes between K. galanga and K. elegans (Figure 7A). The average value of nucleotide
There were 7 mutational hotspots that exhibited remarkably higher Pi values (>0.03) and were located
diversity (Pi) was 0.01075. The IR regions showed lower variability than the LSC and SSC regions.
at the LSC and SSC regions, which included trnS-trnG, rps12-clpP, psbT-psbN, ycf1-ndhF, ndhF-rpl32,
There were 7 mutational hotspots that exhibited remarkably higher Pi values (>0.03) and were located
psaC-ndhE and ccsA-ndhD regions from the chloroplast genomes (Figure 7A). By contrast, there was
at the LSC and SSC regions, which included trnS-trnG, rps12-clpP, psbT-psbN, ycf1-ndhF, ndhF-rpl32,
only 1 mutational hotspot (rpl2-trnH) that exhibited remarkably higher Pi values (>0.03) located at the
psaC-ndhE and ccsA-ndhD regions from the chloroplast genomes (Figure 7A). By contrast, there was
IR regions (Figure 7A).
only 1 mutational hotspot (rpl2-trnH) that exhibited remarkably higher Pi values (>0.03) located at
the IR regions (Figure 7A).
Molecules 2019, 24, 474 10 of 18
Molecules 2019, 24, x FOR PEER REVIEW 10 of 18

psbT-psbN ycf1-ndhF ndhF-rpl32

(A)
trnH-rpl2 rpl2-trnH
rps12-clpP
psaC-ndhE
trnS-trnG
ccsA-ndhD

LSC IRa SSC IRb

(B) psbT-psbN trnH-rpl2 rpl2-trnH

trnS-trnG psaC-ndhE
trnI-ycf2 ycf2-trnI

ccsA-ndhD

LSC IRa SSC IRb

Figure 7.
Figure 7. Sliding
Slidingwindow
window analysis of the
analysis whole
of the chloroplast
whole genomes.
chloroplast Window
genomes. length: 800
Window bp; step
length: 800 bp; step
size: 200 bp. X-axis:position of the midpoint of a window. Y-axis: nucleotide diversity of each
size: 200 bp. X-axis:position of the midpoint of a window. Y-axis: nucleotide diversity of each window.
window. (A) Pi between K. galanga and K. elegans. (B) Pi among two Kaempferia species, Alpinia
(A) Pi between K. galanga and K. elegans. (B) Pi among two Kaempferia species, Alpinia zerumbet, Curcuma
zerumbet, Curcuma flaviflora and Zingiber spectabile.
flaviflora and Zingiber spectabile.
Figure 7B showed that the average value of Pi was 0.01591 among two Kaempferia species, A.
Figure 7B showed that the average value of Pi was 0.01591 among two Kaempferia species, A.
zerumbet, C. flaviflora and Z. spectabile. The Pi values of these five species were commonly higher than
zerumbet, C. two
those of the flaviflora and Z.
Kaempferia spectabile.
species. The Piseven
Particularly, values of these
highly five species
divergent wereremarkably
loci showed commonly higher
than those of the two Kaempferia species. Particularly, seven highly divergent loci showed
higher Pi values (>0.045), including trnS-trnG, psbT-psbN, trnH-rpl2, trnI-ycf2, ccsA-ndhD, psaC-ndhE remarkably
higher Pi values
and ycf2-trnI (>0.045),
regions including
from the trnS-trnG,
chloroplast genomespsbT-psbN,
(Figure 7B).trnH-rpl2, trnI-ycf2,
These regions ccsA-ndhD,
may be undergoingpsaC-ndhE
rapid
and nucleotide
ycf2-trnI substitution
regions from theat chloroplast
the species level,
genomes indicating
(Figurepotential application
7B). These regionsofmay
molecular
be undergoing
markers
rapid for plantsubstitution
nucleotide identificationat
and
thephylogenetic analysis.
species level, indicating potential application of molecular markers
The chloroplast genomes of K. galanga and
for plant identification and phylogenetic analysis. K. elegans were found to show a 256 bp difference in
length (Table 1). In addition to the total length difference, we assessed SNP and Indel variations
The chloroplast genomes of K. galanga and K. elegans were found to show a 256 bp difference
between the two Kaempferia species chloroplast genomes in their entirety. There were 536 SNPs
in length (Table 1). In addition to the total length difference, we assessed SNP and Indel variations
identified in the two chloroplast genomes (Table S4). The most frequently occurring mutations were
between
located inthe two Kaempferia
intergenic species
region, which chloroplast
included 357 SNPs. genomes
The codingin their contained
regions entirety. 91There were 536 SNPs
synonymous
identified in the two chloroplast genomes (Table S4). The most frequently
SNPs, 87 nonsynonymous SNPs and 1 stop mutation. There were 107 indels in the chloroplast occurring mutations were
located
genomeinidentified
intergenic region,K.which
between included
galanga and K. 357 SNPs.
elegans TheS5),
(Table coding regions
including 47contained
deletions 91
andsynonymous
60
SNPs, 87 nonsynonymous SNPs and 1 stop mutation. There were 107 indels in the chloroplast genome
identified between K. galanga and K. elegans (Table S5), including 47 deletions and 60 insertions.
Of the 107 indel markers between K. galanga and K. elegans genomes, the longest indels (10 bp) were
Molecules 2019,24,
Molecules2019, 24,474
x FOR PEER REVIEW 11 of
11 of 18
18

insertions. Of the 107 indel markers between K. galanga and K. elegans genomes, the longest indels (10
located within the two intergenic sequences (petA-psbJ and atpH-atpI) and two coding sequences (atpF
bp) were located within the two intergenic sequences (petA-psbJ and atpH-atpI) and two coding
and rps12).
sequences (atpF and rps12).
2.5. Phylogenetic Analysis
2.5. Phylogenetic Analysis
In this study, phylogenetic trees were constructed with SNPs from eleven species using ML and
In this study, phylogenetic trees were constructed with SNPs from eleven species using ML and
MP methods, respectively, including nine Zingiberaceae plants and using C. pulverulentus and C. indica
MP methods, respectively, including nine Zingiberaceae plants and using C. pulverulentus and C.
as outgroups (Figure 8). Both the ML and MP phylogenetic trees strongly indicated that K. galanga and
indica as outgroups (Figure 8). Both the ML and MP phylogenetic trees strongly indicated that K.
K. elegans formed a cluster within Zingiberaceae and the C. pulverulentus and C. indica species were
galanga and K. elegans formed a cluster within Zingiberaceae and the C. pulverulentus and C. indica
clearly separated from Zingiberaceae species (Figure 8). Among nine Zingiberaceae species, they were
species were clearly separated from Zingiberaceae species (Figure 8). Among nine Zingiberaceae
clustered into four clusters. The first cluster comprised the genus Kaempferia (K. galanga and K. elegans).
species, they were clustered into four clusters. The first cluster comprised the genus Kaempferia (K.
The second cluster comprised the two genera—Zingiber and Curcuma (Z. spectabile, C. flaviflora and C.
galanga and K. elegans). The second cluster comprised the two genera—Zingiber and Curcuma (Z.
roscoeana). The third cluster comprised the genus Amomum (A. kravanh and A. compactum). The fourth
spectabile, C. flaviflora and C. roscoeana). The third cluster comprised the genus Amomum (A. kravanh
cluster comprised the genus Alpinia (A. zerumbet and A. oxyphylla).
and A. compactum). The fourth cluster comprised the genus Alpinia (A. zerumbet and A. oxyphylla).

Figure 8. Phylogenetic trees constructed with SNPs from 11 species using maximum likelihood (ML,
Figure 8. Phylogenetic trees constructed with SNPs from 11 species using maximum likelihood (ML,
left) and maximum parsimony (MP, right) methods. Numbers at nodes on the tree indicate bootstrap
left) and maximum parsimony (MP, right) methods. Numbers at nodes on the tree indicate bootstrap
values (>50%).
values (>50%).
2.6. Potential RNA Editing Sites
2.6. Potential RNA Editing Sites
In the present study, potential RNA editing sites were predicted for 34 genes; as a result, a total
of 54 andIn the80present study, sites
RNA editing potential
wereRNA editing
identified in sites
the K.were predicted
galanga and K.for 34 genes;
elegans as a result,
chloroplast a total
genomes,
of 54 and 80 RNA editing sites were identified in the K. galanga and K. elegans
respectively (Table S6). No potential editing sites were identified in seven genes (petG, petL, psaB, psaI, chloroplast genomes,
respectively
psbL, (Table
rpl2, rpl23) S6). No
in both potential editing
chloroplast genomes. sites
Ofwere
the identified
54 editingin seven
sites, genesoccurred
which (petG, petL, psaB,
in 21 psaI,
genes,
psbL,
15 rpl2, and
(27.8%) rpl23)39in both chloroplast
(72.2%) were located genomes. Of the
at the first and54 editing
the second sites,
codon which occurred
position, in 21 genes,
respectively, 15
in K.
(27.8%) and 39 (72.2%) were located at the first and the second codon position,
galanga. Of the 80 editing sites, which occurred in 26 genes, 21 (26.2%) and 59 (73.8%) were located at respectively, in K.
the first codon and the second codon position, respectively, in K. elegans. No editing sites were foundat
galanga. Of the 80 editing sites, which occurred in 26 genes, 21 (26.2%) and 59 (73.8%) were located
atthe
thefirst codon
third codonand the second
position codon
in both position,
Kaempferia respectively, in K. elegans. No editing sites were found
species.
at the third codon position in both Kaempferia species.
We also observed that RNA editing sites were all C to U conversion both in K. galanga and K.
elegans. WeInalso observed
K. galanga, thethat
ndhBRNAgeneediting sites were
was predicted all C ten
to have to U conversion
editing both and
sites; accD in K.matK,
galanga
five;and
rpoBK.
elegans. In K. galanga, the ndhB gene was predicted to have ten editing sites;
and rpoC2, four; rpl20, rpoA and rps14, three; atpB, petB, psbE and rpoC1, two; and one each in atpA, accD and matK, five; rpoB
and atpI,
atpF, rpoC2, four;
ccsA, rpl20,
clpP, psbB,rpoA and
psbF, rps14,
rps2 three;Meanwhile
and rps8. atpB, petB,in psbE and rpoC1,
K. elegans, two;of
the gene and onewas
ndhB each in atpA,
predicted
atpF,
to have atpI,
tenccsA, clpP,
editing psbB,
sites; psbF,and
ndhA rps2ndhD,
and rps8.
seven;Meanwhile
accD, matK, in ndhF
K. elegans, the gene
and rpoC2, of rpoB
five; ndhBand
wasycf3,
predicted
four;
to have ten editing sites; ndhA and ndhD, seven; accD, matK, ndhF and rpoC2, five; rpoB and ycf3, four;
Molecules 2019, 24, 474 12 of 18

rpl20, rpoA and rps14, three; atpB, ndhG, petB, psbB and rpoC1, two; and one each in atpA, atpF, atpI, ccsA,
clpP, psbF, rps2, rps8 and rps16.

3. Materials and Methods

3.1. Plant Material and DNA Isolation

Fresh leaves were collected from potted K. galanga and K. elegans plants, respectively, from
greenhouse in environmental horticulture research institute, Guangdong academy of agricultural
sciences, Guangzhou, China. Total chloroplast DNA was extracted from about 100 g of leaves using
the sucrose gradient centrifugation method as improved by Li et al. [21]. The chloroplast DNA
concentration for each sample was estimated using an ND-2000 spectrometer (Nanodrop technologies,
Wilmington, DE, USA), whereas visual examination was performed using gel electrophoresis.

3.2. Chloroplast Genome Sequencing and Genome Assembly

The chloroplast DNA was first fragmented into 300–500 bp using a Covaris
M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA) and used to construct short-insert
libraries (insert size about 430 bp) according to the manufacturer’s instructions (Illumina, San Diego,
CA, USA). The short fragments were sequenced using an Illumina Hiseq X Ten platform (Novogene,
Beijing, China). The Illumina raw reads were cleaned by removing the adapter sequences and low
quality sequences, which included the reads with ambiguous nucleotides and ones containing more
than 10% nucleotides in read with Q-value ≤ 20 and short reads (length < 50 bp).
The chloroplast DNA was also fragmented into 8-10 kb fragments, which were subjected to DNA
sequencing following the standard protocol provided by PacBio platform (Novogene, Beijing, China).
The PacBio raw reads were pre-processed by trimming the adapter sequences, low quality (Q < 0.80)
reads, short reads (length < 100 bp) and short subreads (length <500 bp).
Initially, the Illumina clean reads were assembled using SOAPdenovo (version 2.04, Hongkong,
China) with default parameters into principal contigs [22] and all contigs were sorted and joined into
a single draft sequence using the software Geneious version 11.0.4 (Auckland, New Zealand) [23].
Next, BLASR software (San Diego, CA, USA) was used to compare the PacBio clean data with the
single draft sequence and to extract the correction and error correction [24]. Next, the corrected
PacBio clean data were assembled using Celera Assembler (version 8.0, Rockville, MD, USA) with
default parameters, thus generating scaffolds [25]. Next, the assembled scaffolds were mapped back
to the Illumina clean reads using GapCloser (version 1.12, Hongkong, China) for gap closing [22].
Finally, the redundant fragments sequences were removed, thus generating the final assembled
chloroplast genomic sequence.

3.3. Chloroplast Genome Annotation and Codon Usage

The initial gene annotation of the chloroplast genome was carried out with BLAST homology
searches and DOGMA (Dual Organellar Genome Annotator) [26]. tRNA genes were identified using
tRNAscanSE with default settings [27]. The gene homologies were confirmed by comparing them
with National Center for Biotechnology Information (NCBI)’s non-redundant (Nr) protein database,
Clusters of orthologous groups (COG) for eukaryotic complete genomes database (http://www.ncbi.
nlm.nih.gov/COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/),
Gene Ontology (GO) (http://www.geneontology.org) and SWISS-PROT (http://web.expasy.org/
docs/swissprotguideline.html) databases. The structural features of chloroplast genome maps were
drawn using OGDRAWv1.2 (Potsdam-Golm, Germany) [28]. Codon usage was determined for all
protein-coding genes. To examine the deviation in synonymous codon usage, the relative synonymous
codon usage (RSCU) was calculated using MEGA6 software (Version 6.0, Jeddah, Saudi Arabia) [29].
Amino acid (AA) frequency was also calculated and expressed by the percentage of the codons
encoding the same amino acid divided by the total number of codons. The final chloroplast genomic
Molecules 2019, 24, 474 13 of 18

sequences have been submitted to GenBank under accession numbers MK209001 and MK209002 for K.
galanga and K. elegans, respectively.

3.4. SSRs and Long Repeat Structure

SSRs were identified using MIcroSAtellite (MISA) [30]. The parameters for SSRs were adjusted
for identification of perfect mono-, di-, tri-, tetra-, pena- and hexanucleotide motifs with a minimum
of 8, 5, 4, 3, 3 and 3 repeats, respectively. The online REPuter software was used to identify and
locate forward, palindrome, reverse and complement repeat sequences with repeat sizes ≥30 bp and
sequences identity ≥90% [31].

3.5. Comparative and Divergence Analysis of Chloroplast Genomes of K. galanga and K. elegans
The complete chloroplast genome of K. galanga was employed as a reference and was compared
with the chloroplast genomes of K. elegans, Alpinia zerumbet (JX088668), Curcuma flaviflora (KR967361)
and Zingiber spectabile (JX088661), the last three of which were obtained from GenBank, using mVISTA
program (http://genome.lbl.gov/vista/mvista/about.shtml) in the Shuffle-LAGAN mode [32].
To calculate nucleotide variability (Pi) between K. galanga and K. elegans chloroplast genomes, sliding
window analysis was performed using DnaSP version 5.1 software [33] with window length of 600 bp
and the step size of 200 bp.
The complete chloroplast sequences of K. galanga and K. elegans were also aligned using MUMmer
software (Maryland, USA) [34] and adjusted manually where necessary using Se-Al 2.0 [35]. The single
nucleotide polymorphisms (SNPs) and insertion/deletions (indels) were recorded separately as well
as their locations in the chloroplast genome.

3.6. Phylogenetic Analysis

A molecular phylogenetic tree was constructed using SNP arrays from 11 species including K.
galanga and K. elegans. Among these 11 species, nine complete chloroplast genome sequences were
downloaded from NCBI: A. zerumbet (JX088668), C. flaviflora (KR967361), Z. spectabile (JX088661), C.
roscoeana (NC_022928.1), Alpinia oxyphylla (NC_035895.1), Amomum kravanh (NC_036935.1), Amomum
compactum (NC_036992.1), Costus pulverulentus (KF601573) and Canna indica (KF601570). K. galanga
chloroplast genome was used as reference. Costus pulverulentus and Canna indica were set as outgroups
of the family Zingiberaceae. Firstly, using MUMmer software [34], each chloroplast genome above was
compared globally with the reference genome and the difference between each chloroplast genome
and the reference genome found and preliminary filtering performed to detect the potential SNP sites.
Secondly, the sequence of 100 bp on each side of the reference sequence SNP site was extracted and the
extracted sequence and assembly results were compared using the BLAT software [36,37] to verify the
SNP site. If the length of the alignment is less than 101 bp, it is considered to be a non-trusted SNP and
is removed; if compared several times, the SNP that is considered to be a duplicate region and will also
be removed; and finally a reliable SNP will be obtained. Thirdly, for each chloroplast genome, all SNPs
are connected in the same order to obtain a sequence in FASTA format. Multiple FASTA format
sequences alignments were carried out using ClustalX version 1.81 [38]. To examine the phylogenetic
applications of rapidly evolving SNP markers, the maximum likelihood (ML) and maximum parsimony
(MP) methods with 1000 bootstrap replicates were employed to construct phylogenetic trees using
MEGA6 software, respectively [29].

3.7. RNA Editing Analysis

Thirty-four protein-coding genes of K. galanga and K. elegans chloroplast genomes were used to
predict potential RNA editing sites using the online program Predictive RNA Editor for Plants (PREP)
suite (http://prep.unl.edu/) with a cutoff value of 0.8 (Bielefeld, Germany) [39].
Molecules 2019, 24, 474 14 of 18

4. Discussion
In this study, we obtained the complete chloroplast genomes of K. galanga and K. elegans by
using Illumina and PacBio sequencing, ranging from 163.5-163.8 kb in length. Both chloroplast
genomes exhibit a typical quadripartite structure, as reported for other Zingiberaceae species, such
as A. oxyphylla, A. zerumbet, C. flaviflora, Z. spectabile, C. roscoeana, A. compactum and A. kravanh [12].
Both genomes encode about 111-113 genes, including 79 protein-coding genes, 4 rRNA genes as well
as 28 and 30 tRNA genes distributed throughout their genomes, respectively. This conformed with the
protein-coding genes found in other Zingiberaceae members [12].
The molecular markers obtained from chloroplast genome sequences such as highly variable
sequences, SSRs, SNPs and indels are useful tools in research. In Camellia species, 1.5% high divergent
sequences were used for phylogenetics, taxonomy and species identification [40]. In this study, eight
highly variable regions had been detected between K. galanga and K. elegans chloroplast genomes,
including trnS-trnG, rps12-clpP, psbT-psbN, ycf1-ndhF, ndhF-rpl32, psaC-ndhE, ccsA-ndhD and rpl2-trnH
(Figure 7A). As our results displayed, most of them occurred in the LSC and SSC regions but only one
in the IR regions. Among these highly variable regions, ndhF-rpl32, ccsA-ndhD, trnS-trnG, psbT-psbN
and psaC-ndhE had been reported as highly variable regions in many species, such as Papaver [5],
Machilus [41], Citrullus [42], Fagopyrum [43], Citrus [44] and Oryza [45]. In addition, the ndhF-rpl32,
ccsA-ndhD and trnS-trnG regions had been used as molecular markers for phylogenetic analysis,
to resolve origin problems and phylogeographic studies [13,41–43,45]. Therefore, these eight highly
variable regions could serve to enrich the molecular marker resources of genus Kaempferia in studies of
phylogeny, evolution and species identification.
Besides the highly variable regions, we were able to retrieve SSRs and long repeats. Of the
240 SSRs identified in K. galanga, 64.58% (155 SSRs) were located in the LSC region, 16.66% (49 SSRs) in
the SSC region and 18.75% (45 SSRs) in the IR regions. In contrast, out of the 248 SSRs identified in K.
elegans, 64.11% (159 SSRs) were present in the LSC region, 16.93% (42 SSRs) in the SSC region and the
remaining 19.75% (49 SSRs) located in the IR regions, as reported in other plants like A. kravanh [12],
Talinum paniculatum [20] and Oryza minuta [45]. Among the SSRs types, the most abundant was found
to be mononucleotides in both K. galanga and K. elegans (Figure 3). These findings were in agreement
with results from previous studies in A. kravanh [12], T. paniculatum [20] and buckwheat species [43]
but were different from O. minuta which possessed a majority of dinucleotide repeat motif SSRs [45].
AT/AT (12.5%) was the most frequent dinucleotide motifs followed by AAAT/ATTT in both K. galanga
and K. elegans, respectively (Figure 3). These SSRs and long repeats identified in our study could be
useful in molecular studies, such as genetic diversity and phylogenetic relationship analysis, species
identification and evolution studies [7,12,21,40,43].
In the present study, we also identified 536 SNPs and 107 indels between the two Kaempferia
species (Tables S4 and S5). From the SNPs results, 536 nucleotide substitutions were detected between
K. galanga and K. elegans chloroplast genomes. It indicated that the nucleotide substitution events
in the chloroplast genomes of Kaempferia species were more than that between species of Oryza,
Machilus, cultivated Fagopyrum, Citrus and Panax but less than species of Solanum and wild Fagopyrum.
Comparative analysis of chloroplast genomes found 159 SNPs between Oryza nivara and O. sativa [46],
231 SNPs between Machilus yunnanensis and M. balansae [41], 317 SNPs between cultivated species
Fagopyrum dibotrys and F. tataricum [43], 330 SNPs between Citrus sinensis and C. aurantiifolia [44],
464 SNPs between Panax notoginseng and P. ginseng [47], 591 SNPs between Solanum tuberosum and
S. bulbocastanum [48], 6260 SNPs between wild species F. luojishanense and F. esculentum [43]. Out of
the 107 indels found between K. galanga and K. elegans chloroplast genomes, two longest intergenic
sequences petA-psbJ and atpH-atpI were detected. The petA-psbJ and partial psbA-trnH spacer sequences
can be used for species identification of most Kaempferia and outgroup species [4]. Similarly, a single
large 241-bp deletion in S. tuberosum clearly discriminated a cultivated potato from the wild potato
species S. bulbocastanum [48]. The indels and SNPs of 12 Triticeae species chloroplasts were used to
estimate wheat, barley, rye and their relatives evolution [49]. These indels and SNPs found in our study
Molecules 2019, 24, 474 15 of 18

could be useful in phylogenetic analysis, species identification and evolutionary studies as well as the
65 indels detected between M. yunnanensis and M. balansae [41], 156 indels between P. notoginseng and
P. ginseng [47] and 53 indels between Aconitum pseudolaeve and A. longecassidatum [50].
The chloroplast genome sequences provide a useful genomic resource for phylogenetic studies
and many studies have successfully used protein-coding sequences or whole chloroplast genome
sequences in these analyses [7,12,20,43]. Specifically, the chloroplast psbA-trnH and partial petA-psbJ
sequences and matK gene had been utilized in Zingiberaceae species phylogenetic studies before [4,51].
In this study, we constructed phylogenetic trees using ML and MP methods based on SNPs commonly
present in the chloroplast genomes of eleven species, including two Kaempferia species from the current
study. Our phylogenetic analysis clearly revealed that the two Kaempferia species clustered together,
with bootstrap values of 100%, as well as Amomum and Alpinia species, which segregated in two sister
clades (Figure 8). In a previous study, a phylogenetic tree constructed by using whole chloroplast
genome sequences strongly supported the position in the Zingiberaceae of A. kravanh as a sister of the
closely related species A. zerumbet [12]. Our phylogenetic trees using SNPs were in broad agreement
with the previous study [12]. Therefore, phylogenetic analysis using SNPs among chloroplast genome
sequences could provide useful information for revealing relationships among Zingiberaceae species.
In conclusion, we assembled and analyzed the complete chloroplast genomes of K. galanga and K.
elegans and compared them with other Zingiberaceae species for the first time. The chloroplast
genomes organization, gene order, GC content and codon usage of the two Kaempferia species
showed high similarity. The location and distribution of SSRs and long repeat sequences were
determined. Eight highly variable regions between the two Kaempferia species were identified and
643 mutation events, including 536 SNPs and 107 indels, were accurately located. Sequence divergences
of chloroplast genomes were also calculated for the two Kaempferia species and related Zingiberaceae
species. The phylogenetic analysis based on SNPs among eleven species strongly supported that
K. galanga and K. elegans formed a cluster within Zingiberaceae. Our results provided insights into
the characteristics of the entire K. galanga and K. elegans chloroplast genomes and the phylogenetic
relationships within Zingiberaceae species.

Supplementary Materials: The Supplementary Materials are available online. Table S1: Codon Usage for K.
galanga (Kg) and K. elegans (Ke), Table S2: SSRs, Table S3: Long repeats, Table S4: SNPs detected between K.
galanga and K. elegans chloroplast genomes, Table S5: InDels detected between K. galanga and K. elegans chloroplast
genomes, Table S6: RNA editing results, Supplementary file1: Genes distribution in the K. galanga chloroplast
genome, Supplementary file2: Genes distribution in the K. elegans chloroplast genome.
Author Contributions: D.-M.L. designed the experiments, performed the experiments, collected plant materials,
carried out sequence analysis and draft the manuscript. C.-Y.Z., X.-F.L. and D.-M.L. identified and collected plant
materials. All authors contributed to the experiments and approved the final manuscript.
Funding: This research was funded by Guangzhou Municipal Science and Technology Project (No. 201607010101),
Guangdong Science and Technology Project (No.2015A020209078, 2015B070701016) and the National Natural
Science Foundation of China (31501788).
Conflicts of Interest: The authors declare that they have no conflict of interests.

Abbreviations
LSC Large single copy
SSC Small single copy
IR Inverted repeat
tRNA Transfer RNA
rRNA Ribosomal RNA
SSRs Simple Sequence Repeats
SNPs Single-Nucleotide Polymorphisms
indels insertion/deletions
Molecules 2019, 24, 474 16 of 18

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Sample Availability: Samples of the compounds are available from the authors.

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