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Javaid et al. BMC Genomics (2024) 25:597
https://doi.org/10.1186/s12864-024-10366-3 BMC Genomics
*Correspondence:
Musarrat Ramzan
musarrat.ramzan@iub.edu.pk
Muhammad Anwar
184370@hainanu.edu.cn
Song Xiqiang
songstrong@hainanu.edu.cn
Full list of author information is available at the end of the article
Abstract
Chrozophora sabulosa Kar. & Kir. is a biennial herbaceous plant that belongs to the Euphorbiaceae family and
has medicinal properties. This research aimed to identify the genetic characteristics and phylogenetic position
of the Chrozophora genus within the Euphorbiaceae family. The evolutionary position of the Chrozophora genus
was previously unknown due to insucient research. Therefore, to determine the evolutionary link between C.
sabulosa and other related species, we conducted a study using the NGS Illumina platform to sequence the C.
sabulosa chloroplast (cp.) genome. The study results showed that the genome was 156,488bp in length. It had a
quadripartite structure consisting of two inverted repeats (IRb and IRa) of 24,649-bp, separated by an 87,696-bp
LSC region and a 19,494-bp SSC region. The CP genome contained 113 unique genes, including four rRNA genes,
30 tRNA genes, and 79 CDS genes. In the second copy of the inverted repeat, there were 18 duplicated genes.
The C. sabulosa lacks the petD, petB, rpl2, and rps16 intron. The analysis of simple sequence repeats (SSRs) revealed
93 SSR loci of 22 types and 78 oligonucleotide repeats of four kinds. The phylogenetic investigation showed that
the Chrozophora genus evolved paraphyletically from other members of the Euphorbiaceae family. To support the
phylogenetic ndings, we selected species from the Euphorbiaceae and Phyllanthaceae families to compare with
C. sabulosa for Ks and Ka substitution rates, InDels investigation, IR contraction and expansion, and SNPs analysis.
The results of these comparative studies align with the phylogenetic ndings. We identied six highly polymorphic
regions shared by both families, which could be used as molecular identiers for the Chrozophora genus (rpl33-
rps18, rps18-rpl20, rps15-ycf1, ndhG-ndhI, psaI-ycf4, petA-psbJ). The cp. genome sequence of C. sabulosa reveals the
evolution of plastid sequences in Chrozophora species. This is the rst time the cp. genome of a Chrozophora genus
has been sequenced, serving as a foundation for future sequencing of other species within the Chrozophoreae
The chloroplast genome of Chrozophora
sabulosa Kar. & Kir. and its exploration in the
evolutionary position uncertainty of genus
Chrozophora
NidaJavaid3, MusarratRamzan3*, ShaguftaJabeen3, YanjunDu1, MuhammadAnwar1,2,4* and SongXiqiang2*
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Page 2 of 18
Javaid et al. BMC Genomics (2024) 25:597
Introduction
e Euphorbiaceae family has approximately 9,000 spe-
cies spread across 340 genera and 52 tribes, primarily
found in various tropical regions [1–7]. e genus Chro-
zophora A. Juss. belongs to the Chrozophoreae tribe of
the Acalyphoideae subfamily of the Euphorbiaceae family
[8]. Chrozophora is found in the Mediterranean, tropical
Africa, and West Asia, as well as in Pakistan’s tropical and
temperate zones [2]. According to “World Flora Online”
http://www.worldoraonline.org/taxon/wfo-4000008162:
Chozophora has 9 accepted species (C. gangetica Gand.,
C. brocchiana Schweinf., C. mujunkumi T. Nasimova, C.
oblongifolia (Delile) A. Juss. ex Spreng., C. plicata (Vahl)
A. Juss. ex Spreng., C. rottleri (Geiseler) Spreng., C.
sabulosa Kar. & Kir., C. senegalensis (Lam.) Spreng., C.
tinctoria (L.) A. Juss.). Chrozophora sabulosa Kar & Kir,
known as Nilakari, is an important medicinal plant from
the Euphorbiaceae family [1].
e family Euphorbiaceae is challenging to understand
due to its wide distribution range, many species, and
poorly dened genera. However, it has been conrmed
that Euphorbiaceae is monophyletic based on molecu-
lar and embryological features [8–10]. Webster [6] has
classied the Euphorbiaceae family into ve subfamilies
based on the number of ovules per ovary locule: Phyl-
lanthoideae, Oldeldioideae, Acalyphoideae, Crotonoi-
deae, and Euphorbioideae. In the Angiosperm Phylogeny
Group (APG II 2003) [11] classication, the family was
divided into four groups: Euphorbiaceae s.s., Phyllan-
thaceae, Picrodendraceae, and Putranjivaceae, all in
the clade Malpighiales. e subfamilies with uniovulate
ovary locules (Euphorbioideae, Acalyphoideae, and Cro-
tonoideae) are considered Euphorbiaceae s.s [11]. e
family was further divided into several subfamilies and
tribes based on molecular data [12], and some genera
were moved to independent families [5]. APG III (2009)
[13] divides it into four subfamilies: Acalyphoideae, Chei-
losoideae, Crotonoideae, and Euphorbioideae. Despite
the extensive research carried out by botanists, who have
conducted studies in taxonomy, anatomy, phytochem-
istry, economic botany, and molecular systematic, the
knowledge of this family still has signicant gaps, even
regarding morphology [5]. Detailed molecular, morpho-
logical, and anatomical studies involving many genera are
required to propose a safer classication for this family.
However, some genera within the family, such as Chro-
zophora, still have confusing taxonomic positions. To
understand the evolutionary relationship between C. sab-
ulosa and related plants, it is necessary to sequence the
chloroplast genomes from the Chrozophora genus and
the Chrozophoreae tribe.
Previous studies have used both molecular and mor-
phological data to perform phylogenetic reconstruc-
tions. However, it has been suggested that molecular
approaches are more reliable in phylogenetics [8–10].
Among molecular approaches, cp. genome sequences
have gained signicant interest in plant phylogenetics,
phylogeography, and molecular evolution investigations
in recent years [14]. e cp. genome has a signicantly
conserved gene content and genome order [15]. More-
over, it is smaller, has fewer nucleotide alterations, and
has fewer genome sequence reorganizations than the
nuclear and mitochondrial genomes. ese characteris-
tics make it an excellent tool for understanding genome
evolution in complicated angiosperm families [14–18].
As a result, cp. genomes provide valuable data that can
be easily combined with source molecular data to vali-
date complicated evolutionary connections and perform
comprehensive phylogenetic analyses [17]. More than
400 species of Euphorbiaceae have had their cp. genomes
sequenced using a high-throughput sequencing tech-
nique [10]. However, data on the chloroplast genomes of
Chrozophora was unavailable, which limits further infor-
mation regarding its phylogenetic position in the family.
Additionally, no members of the Chrozophoreae tribe
have had their cp. genomes sequenced yet, which casts
doubt on the tribe’s exact phylogenetic position. e
present study aimed to investigate the cp. genome of C.
sabulosa to clarify the evolutionary position of the Chro-
zophora genus in the Euphorbiaceae family. By contrib-
uting signicant molecular and phylogenetic data on the
Chrozophora genus, this study may aid in species identi-
cation and determination of its evolutionary position.
e C. sabulosa cp. genome has been marked as the rst
member of the Chrozophora genus and the Chrozopho-
reae tribe to be sequenced. e results of this research
may provide a foundation for phylogenetic investigations
of the Chrozophoreae tribe.
Results
C. sabulosa cp Genome assembly and its characteristics
e Illumina HiSeq2500 generated 10.1 GB of raw data
for C. sabulosa through paired-end sequencing with
150bp reads. e de novo assembled cp. genome had
an average coverage depth of 271 and was 156,488 bp
long, comprising two inverted repeats (IRb and IRa) of
24,649bp, an LSC region of 87,696bp, and an SSC region
of 19,494bp (Fig.1). e total GC content of C. sabulosa
tribe and facilitating in-depth taxonomic research. The results of this research will also aid in identifying new
Chrozophora species.
Keywords Euphorbiaceae, Phyllanthaceae, Chrozophora sabulosa, Chrozophoreae, Phylogenetic ndings
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Javaid et al. BMC Genomics (2024) 25:597
was 36.5%, with the highest GC content (43.3%) found in
the IRs, followed by 34.1% in the LSC region and 30% in
the SSC region. e CP genome contained 113 distinct
genes, including 79 CDS genes, four rRNA genes, and 30
tRNA genes, as shown in Table1. e LSC segment had
84 genes, while the SSC segment had 13. Table S1 listed
the 15 genes (out of 113) with introns, including three
with two introns (clpP, ycf3, rps12), ve tRNA, and ve
CDS genes with one intron. e rps12 gene was repeated
twice, leading to a trans-splitting event. e psbL gene in
C. sabulosa began with a TCG codon, resulting in reo-
nine as the rst amino acid. e cp. genome lacked the
Fig. 1 The cp. genome map of C. sabulosa. Genes outwards the map is demonstrated clockwise, while inside genes are transcribed anticlockwise. Color
coding is used to dierentiate between functional groups of genes. The intensity of the inner circle color indicates the amount of GC (Dark grey) and AT
(Light grey). LSC denotes the large single copy, SSC denotes the small single copy, and IRb and IRa denote inverted repeats
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Page 4 of 18
Javaid et al. BMC Genomics (2024) 25:597
petB, petD, rpl16, and rpl2 introns. Table2 provides a
detailed description of the genes in C. sabulosa based on
their function.
RSCU, and amino acid frequencies in C. sabulosa
All genomes showcase codon usage bias, which aects
translational dynamics, consistency, accuracy, and pro-
tein folding [19]. e Relative synonymous codon usage
(RSCU) ratio is the average usage frequency of a codon
divided by its predicted unbiased usage frequency. Recent
studies have highlighted the signicance of codon usage
in the evolution of the cp. genome [20, 21]. In the coding
sequences in C. sabulosa, there are 52,162 codons within
79,461 bp. e most abundant amino acid in the cp.
genome of C. sabulosa is leucine (11%), followed by iso-
leucine (9%), and the least abundant is cysteine (1%) (for
further details, see Table S2 and Fig.2). We have iden-
tied 31 variant codons with RSCU values greater than
one, indicating that C. sabulosa uses them specically
to encode certain amino acids. e AGA codon, which
codes for arginine, has the highest usage bias (2.05), while
the CGC codon, which also codes for arginine, has the
lowest (0.44). ere was no bias at codons AUG (Methio-
nine), CCC (Proline), and UGG (Tryptophan) in C. sabu-
losa cp. genomes with 1.00 RSCU (Table S3).
Editing sites of RNA
PREP-cp detected 50 RNA editing sites in 21 genes from
C. sabulosa. e ndhB gene has the highest number of
Table 1 The cp. genome of Chrozophora sabulosa is described
in detail
Category Items Descriptions
Construction of
cp. genome
Length of LSC 87,696bp
Length of SSC 19,494bp
Length of IRs (IRA, IRB) 24,649bp
Complete Genome Size 156,488bp
Gene content Total number of genes 131
No. of Protein-coding genes 86
No. of tRNAs 37
No. of rRNAs 8
No. of genes in LSC 84
No. of genes in SSC 13
No. of genes with two copies in IRs 18
Total genes length 104,727bp
The average length of genes 799bp
Gene length/Genome ratio 0.67%
GC content
percentage
GC % in LSC 34.10%
GC % in SSC 30.00%
GC % in IR 43.30%
GC content (%) overall 36.50%
Intron containing
genes
Total intron-containing genes 15
ICGs Protein coding (CDS) 10
ICGs in tRNA 5
ICGs in rRNA 0
1 Intron containing Genes 11
2 Intron-containing Genes clpP, ycf3,
rps12, rps12
Table 2 Chrozophora sabulosa gene functions are summarized in this table
The primary category of genes Functional group of genes
Genes involved in photosynthesis Photosystem subunits (ndhI, ndhJ, ndhK, psaA, psaB, psaC, psaI, psaJ, psbA, psbB, psbC, psbD, psbE, psbF,
psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ)
Cytochrome b/f complex subunits (petA, petB, petD, petG, petL, petN)
Hypothetical chloroplast RF1 (ycf1)
Photosystem I assembly protein Ycf3 (ycf3)
Photosystem I assembly protein Ycf4 (ycf4)
Rubisco large subunit (rbcL)
ATP synthase subunits (atpA, atpB, atpE, atpF, atpH, atpI)
NADH dehydrogenase subunits (ndhA, ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH)
Self-replicating genes rRNA genes (rrn16, rrn23, rrn4.5, rrn5)
tRNA genes (trnH-GUG, trnK-UUU, trnS-CGA, trnC-ACA, trnL-UAA, trnA-UGC, trnS-UGA, trnI-GAU, trnM-CAU,
trnS-GCU, trnS-GGA, trnY-GUA, trnW-CCA, trnL-CAA, trnL-UAG, trnP-UGG, trnD-GUC, trnfM-CAU, trnR-ACG, trnT-
UGU, trnM-CAU, trnE-UUC, trnF-GAA, trnC-GCA, trnT-GGU, trnQ-UUG, trnR-UCU, trnV-GAC, trnN-GUU, trnG-UCC)
Ribosome small subunit (rps11, rps12, rps14, rps15, rps16, rps18, rps19, rps2)
Ribosome large subunit (rps3, rps4, rps7, rps8, rpl2, rpl16, rpl20, rpl22, rpl14, rpl23, rpl32, rpl33)
DNA-dependent RNA polymerase (rpoA, rpoB, rpoC1, rpoC2, rpl36)
Other genes Maturase (matK)
Envelope membrane protein (cemA)
acetyl-CoA subunit (accD)
C-type cytochrome synthesis gene (ccsA)
Translational initiation factor (infA)
Protease (clpP)
Conserved open reading frames (ycf2)
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Javaid et al. BMC Genomics (2024) 25:597
RNA editing sites (9 sites), followed by the ndhD gene (7
sites) (refer to Fig.3). Modifying a nucleotide in the rst
position of a codon resulted in 13 editing sites (26%),
while changes in the second position resulted in 37 edit-
ing sites (74%). Most RNA editing sites (42%) were found
in Serine codons. In C. sabulosa, 90% of the Serine was
converted to Leucine, while the remaining 10% was con-
verted to Phenylalanine. e codon-encoded Proline
had the second-highest conversion rate (18%), while the
codon-encoded reonine had the third-highest con-
version rate (14%). Proline, Serine, reonine, and Ala-
nine showed multiple types of nucleotide conversion,
whereas Leucine, Arginine, and Histidine showed only
one. Hydrophobic amino acid conversions [including
Proline (9), Alanine (2), and Leucine (6)] occurred at 34%
of all RNA editing sites. In contrast, soluble amino acids
resulted in 33 conversions (66%), including Histidine (3),
reonine (7), Serine (21), and Arginine (2). ree non-
polar to polar conversions, 14 non-polar to non-polar
conversions, 30 polar to non-polar conversions, and
three polar-to-polar amino acid conversions have also
been found. For further information, please refer to Table
S4, which details all the RNA editing sites.
Detecting simple sequence repeats (SSRs) in C. sabulosa
In this study, MISA obtained a set of 93 SSRs, which
had 22 types that were at least 10bp in size (Table S5).
Among these, there were two types of mononucleotides
(A/T), six types of dinucleotides (AG/CT, AT/AT, and
AT/AT) (Table S6 & S7), two types of trinucleotides
(AAT/ATT), 12 types of tetranucleotides (ACCT/AGGT,
AAGG/CCTT, AAAT/ATTT, AATT/AATT, AATG/
ATTC, AAAG/CTTT), and four types of pentaucleo-
tides (AAAAG/CTTTT, AAAAT/ATTTT). e mono-
nucleotides were the most common type of SSR detected
(68%), the pentanucleotides were the most extended
form of SSR, and hexanucleotides were not found in C.
sabulosa (Fig.4a). e number of SSRs identied in inter-
genic spacer regions was higher than in other locations
Fig. 3 RNA editing sites of C. sabulosa cp. genome
Fig. 2 Amino acids frequency (%) of C. sabulosa
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Page 6 of 18
Javaid et al. BMC Genomics (2024) 25:597
(Fig.4b). e LSC had the most SSRs, followed by the
SSC, while the inverted repeats had the fewest (Fig.4c).
Oligonucleotide repeats analysis of C. sabulosa
We used the REPuter tool to identify 79 oligonucleotide
repetitions, which had the following values: P = 27, F = 27,
C = 7, and R = 18, as illustrated in Fig.5. ese repeats had
a size ranging from 20 to 51bp (as shown in Fig.5b). We
found that the LSC contained 57 oligonucleotide repeats,
the SSC had 11, and the IRs had four. Furthermore, we
discovered that IR and LSC shared three repeat layouts,
with two shared by LSC/SSC and one shared by SSC/
Fig. 5 Oligonucleotide repeats analysis in C. sabulosa
Fig. 4 SSR analysis of C. sabulosa. (a) Types of SSRs. (b) Distribution of SSRs in active cp. genome regions. (c) Location of SSRs
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Page 7 of 18
Javaid et al. BMC Genomics (2024) 25:597
IR (as depicted in Fig. 5c). e number of oligonucle-
otide repetitions in intergenic spacer areas was highest
(55), followed by CDS (9), transfer RNA (3), and intronic
region (2). We also detected mutual repeats in the IGS/
CDS (2), IGS/Intron (4), and IGS/trn (3) regions (as
shown in Fig. 5d). Palindromic repeats were more fre-
quent than other repetitions (as seen in Fig. 5a). e
locations, positions, and areas of oligonucleotide repeat
sequences are provided in Table S8.
Phylogenetic analyses
We created a maximum likelihood tree using CDS data
from Euphorbiaceae and Phyllanthaceae cp. genomes
(Fig. 6, Table S12) to investigate the evolutionary rela-
tionship of Chrozophora. We constructed the tree by
using C. sabulosa, 18 other genera from Euphorbiaceae,
and 10 genera from Phyllanthaceae. e alignment of 31
species using MAFFT produced an 80,283 bp consen-
sus sequence with 22,018 (22.5%) identical positions and
84.8% pair-wise identity. e tree comprised 28 nodes
with bootstrap values ranging from 48 to 100. e best-
t model for this tree was GTR + F + R6. Its log-likelihood
was − 471155.9363, AIC score 942465.8726, AICc score
942465.9954, and BIC score 943196.6990. e over-
all length of the tree was 0.9525, with internal branch
lengths of 0.2132 (22% of tree length). Our analysis indi-
cated that the Chrozophora genus evolved from other
Euphorbiaceae members in a paraphyletic manner. e
Chrozophora genus was closely related to the Bischoa
genus (Bischoa polycarpa) of the Phyllanthaceae family.
is nding suggests that the Chrozophora genus is more
closely associated with Phyllanthaceae members than
other Euphorbiaceae members. e tree also conrmed
the common ancestor of both families and showed that
the members of the Phyllanthaceae family share a molec-
ular basis with members of the Euphorbiaceae family.
Additionally, our analysis revealed Chrozophora’s unique
position within the Euphorbiaceae family.
C. sabulosa’s comparison with other Euphorbiaceae and
phyllanthaceae species
We have selected eight plant species from two families,
Euphorbiaceae and Phyllanthaceae, to compare their
chloroplast genome with C. sabulosa. e four selected
species of Euphorbiaceae are Ricinus communis, Manihot
esculenta, Jatropha curcas, and Euphorbia helioscopia,
while four Phyllanthaceae species are Antidesma bunius,
Breynia fruticosa, Glochidion chodoense, and Phyllan-
thus urinaria. A detailed basic comparison is shown in
Table3. We have compared the length of the chloroplast
sequence and the quadripartite structure of each spe-
cies. e length of the chloroplast sequence varied from
155,630bp (B. fruticosa) to 163,856bp (J. curcas), and
each segment of the quadripartite structure was compa-
rable across the analyzed plastomes. M. esculenta and
E. helioscopia had the highest number of genes (132),
while B. fruticosa and P. urinaria had the fewest (129).
e overall GC content of these cp. genomes varied from
35.4 to 36.7%, and the gene component was comparable,
except for a few missing or added genes. e infA gene
was present in C. sabulosa and A. bunius but not in the
other seven species. e rps16 gene was absent in J. cur-
cas and E. helioscopia, while the petD intron was absent
in C. sabulosa, R. communis, E. helioscopia, and G. cho-
doense. e intron of petB and rpl16 was absent in C. sab-
ulosa, R. communis, and E. helioscopia. e rpl2 intron
Fig. 6 CDS-based ML tree of Euphorbiaceae and Phyllanthaceae species
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Page 8 of 18
Javaid et al. BMC Genomics (2024) 25:597
was only absent in the plastome of C. sabulosa. We used
Geneious Prime 2021.1.1 and the MAFFT alignment of
cp. genomes from the nine species to compare the rela-
tive placements of genes across the species. e 192,904-
bp consensus sequence had 103,626 (54%) identical sites
and 80% pair-wise identity. is analysis demonstrates
that both family members have a close association, and
C. sabulosa demonstrated a close link with both fam-
ily members. ese comparisons conrm the phyloge-
netic results and show that the chloroplast genome helps
understand the evolutionary relationships within the
Euphorbiaceae and Phyllanthaceae families.
The expansion and contraction of IRs
A study was conducted on the margins of four key areas
(LSC, IRB, SSC, IRA) and their surrounding genes in
C. sabulosa and selected cp. genomes (Fig. 7). e ycf1
gene on the JSA (SSC/IRA) junction was found to be
functional in all species. However, in R. communis, M.
esculenta, J. curcas, E. helioscopia, (A) bunius, and G.
chodoense, a pseudo copy of ycf1 was detected at the JSB
(IRB/SSC) border, while it was absent in C. sabulosa, (B)
fruticosa, and P. urinaria. e size and position of the
ndhF gene varied at the JSB border. e rps19 and rpl2
genes were entirely in the IRs in (C) sabulosa, E. heliosco-
pia, A. bunius, and P. urinaria, but they were found in
varied locations in the other ve species. e trnH gene
was detected at the JLA (IRA/LSC) boundary in all spe-
cies except A. bunius, which had two copies of this gene
found in IR regions. e study revealed that identical
genes varied in locations and sizes at every junction of
the cp. genomes, indicating a variety of gene content.
A thorough analysis of IR contraction and expansion is
shown in Fig.7 (IRSCOPE analysis). ese ndings sug-
gest that these nine cp. genomes were slightly dierent
due to dierences in size and gene placement in these
species.
The Ka, Ks substitutions, and Ka/Ks rate
Pair-wise alignments of C. sabulosa genes were per-
formed with eight selected cp. genomes to determine the
Ka/Ks ratio (Fig. 8). All comparisons of selected genes
with all species had a Ka/Ks ratio that was typically less
than one. Genes for which Ka/Ks ratios were unavailable
(N/A) were set to zero (See Table S10). After excluding
the genes with a Ka or Ks value of zero, the average Ka/
Ks ratio was 0.20, demonstrating that the genes in the
cp. genome of C. sabulosa were subjected to signicant
purifying selection forces. e average Ka/Ks ratio for
Euphorbiaceae species was 0.212, while it was 0.184 for
the Phyllanthaceae species. e psbI and petN have zero
Ka/Ks ratio in all species, making them the most stable
genes among both families. Most of the genes exhib-
ited a Ka/Ks ratio of below one in all comparisons, and
their proportions were consistent, except for petD, ndhK,
cemA, rpl23, and rpl20 genes, which had challenging
ratios. For instance, in R. communis, E. helioscopia, and
G. chodoense, the Ka/Ks rate of petD was above one, but
in the other ve comparisons, it was less than one. Com-
pared to R. communis, the Ka/Ks ratio of petD was 9.1,
whereas it was only 0.03 when compared to J. curcas.
Similarly, the Ka/Ks value of ndhK was 1.12 compared
to R. communis. e Ka/Ks values of the ve challeng-
ing genes are displayed in Table4. e comprehensive Ka
and Ks values and their ratios are available in Supplemen-
tary Table 9.
Investigating SNPs and InDel mutations analysis in C.
sabulosa
We conducted pair-wise alignments between C. sabulosa
and selected species of Euphorbiaceae and Phyllantha-
ceae families. As a result, we discovered single nucleotide
polymorphisms (SNPs) and InDels mutation events in
the IR, SSC, and LSC regions of C. sabulosa. e highest
number of SNPs (18,625) was found when comparing C.
sabulosa to E. helioscopia, while the lowest number was
found when compared to M. esculenta (17,028 SNPs). All
Table 3 Described the comparison of C. sabulosa cp. genome with four Euphorbiaceae and four Phyllanthaceae species
Genome features C.
sabulosa
EUPHORBIACEAE PHYLLANTHACEAE
Ricinus
communis
Manihot
esculenta
Jatro-
pha
curcas
Euphorbia
helioscopia
Antidesma
bunius
Breynia
fruticosa
Glochidion
chodoense
Phyl-
lanthus
urinaria
Genome Size (bp) 156,488 163,161 161,453 163,856 160,041 162,160 155,630 157,085 157,673
Length of LSC (bp) 87,696 89,651 89,295 91,731 88,832 89,499 85,065 85,304 85,189
Length of SSC (bp) 19,494 18,816 18,250 17,849 17,145 19,051 19,441 17,635 17,134
Length of IR (bp) 24,649 27,347 26,954 27,138 27,032 26,805 25,562 27,073 27,675
GC content % 36.5 35.7 35.9 35.4 35.9 36.4 36.7 36.7 36.5
Total No. of genes 131 131 132 130 132 131 129 130 129
Protein Coding Genes 86 86 86 85 85 87 84 85 85
No. of tRNA genes 37 37 38 37 39 38 37 37 36
No. of rRNA genes 8 8 8 8 8 8 8 8 8
Accession Number MW541931 JF937588 NC010433 FJ695500 MN199031 ON022043 MT863745 MK056235 OL693862
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Page 9 of 18
Javaid et al. BMC Genomics (2024) 25:597
species in Table5 had a transition-to-transversion ratio
greater than one due to more transitions than transver-
sions. e LSC and SSC regions showed a higher rate of
substitutions than the IR regions. In terms of inDels, the
inverted repeat regions had the fewest, while the SSC
and LSC regions had the most. Pair-wise alignment of J.
curcas resulted in the most signicant number of inDels
(24,369), followed by P. urinaria with 23,217 inDels, and
E. helioscopia with the least (21,134). Table6 provides an
in-depth description of inDels and its relevant parame-
ters. e similar values of InDels and SNPs observed in
all the Euphorbiaceae and Phyllanthaceae species indi-
cate their closer relationship.
Fig. 8 Ka/Ks values for Euphorbiaceae and Phyllanthaceae species
Fig. 7 The IRSCOPE analysis of Euphorbiaceae and Phyllanthaceae species
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Page 10 of 18
Javaid et al. BMC Genomics (2024) 25:597
The nucleotide diversity and highly polymorphic loci of
Euphorbiaceae and Phyllanthaceae species, with particular
reference to C. sabulosa
We conducted an independent analysis by comparing C.
sabulosa with representatives of both Euphorbiaceae and
Phyllanthaceae families. We aimed to examine the nucle-
otide diversity and highly polymorphic loci for C. sabu-
losa and other chosen species of these two families. We
found that the average nucleotide diversity in Euphor-
biaceae species was 0.0958, whereas in Phyllanthaceae
species, it was 0.0981. e nucleotide diversity values var-
ied across species and regions. In Euphorbiaceae species
(Table S10), it ranged from 0.0076(rps7) to 0.2549(rpl33-
rps18), whereas in Phyllanthaceae species (Table S11),
it ranged from 0.0068(rps7) to 0.2467(rps15-ycf1). We
observed that the average nucleotide diversity in cod-
ing areas was the lowest (Euphorbiaceae species 0.0624,
Phyllanthaceae species 0.0652), followed by IGS regions
(Euphorbiaceae species 0.1934, Phyllanthaceae species
0.1953), and Intronic regions (Euphorbiaceae species
0.6178, Phyllanthaceae species 0.6193). Additionally, we
identied six highly polymorphic sites shared by both
families (Table7), which can be used as mutational mark-
ers to identify and classify Chrozophora species. Figure9
shows the nucleotide diversity values for the 93 locations
selected from both families. We found that C. sabulosa
had identical nucleotide diversity values with both family
members, indicating that it shares characteristics of both
families.
Discussion
We are excited to present our novel ndings on the chlo-
roplast genome of C. sabulosa, a rst-time report in the
scientic community. To determine the phylogenetic
position of this genus, we conducted a comprehensive
comparative analysis with members of the Euphorbiaceae
and Phyllanthaceae families. Specically, we compared C.
sabulosa to four Euphorbiaceae species (Ricinus commu-
nis, Manihot esculenta, Jatropha curcas, and Euphorbia
helioscopia) and four Phyllanthaceae species (Antidesma
bunius, Breynia fruticosa, Glochidion chodoense, and
Phyllanthus urinaria). Our investigation covered various
aspects such as cp. genome structure, gene details and
their functions, GC content, intron presence or absence,
amino acid frequencies, relative codon use values, RNA
editing sites, SSRs, and oligonucleotide repeats. Notably,
the cp. genome of C. sabulosa exhibits a typical quad-
ripartite architecture and comparable structure and
genomic data to other Euphorbiaceae and Phyllanthaceae
species [9, 10, 22–26].
Our research on the chloroplast genome of C. sabu-
losa unveiled unique and intriguing ndings. e TCG
codon in the psbL gene of C. sabulosa leads to reo-
nine as the rst amino acid, a behavior similar to that
observed in the cp. genomes of other plant species such
as Indigofera genus [27], Spinacia oleracea (NC_002202),
Nicotiana tabacum (NC_001879), Ampelopsis glandulosa
(KT831767), Lycium barbarum (MH032560), and Lycium
chinense (MK040922). is underscores the conserved
structure of the chloroplast genome, a feature observed
in various other angiosperm lineages [17, 28–31]. Our
ndings also revealed that the DNA GC percentage is
not uniform within the chloroplast genomic domains,
with the GC concentration in the IR area being more
Table 4 Genes showing elusive Ka/Ks ratio for eight selected
species
Sr. no GENES Species which pair-wise
aligned with C. sabulosa
Ka/Ks
1petD Ricinus communis 9.085106383
Jatropha curcas 0.029435163
Euphorbia helioscopia 5.663157895
Manihot esculenta 0.037310924
Antidesma bunius 0.056277056
Breynia fruticosa 0.059561966
Glochidion chodoense 5.253968254
Phyllanthus urinaria 0.056277056
2cemA Ricinus communis 0.164969982
Jatropha curcas 0.188277087
Euphorbia helioscopia 0.27484472
Manihot esculenta 0.249648876
Antidesma bunius 1.910591472
Breynia fruticosa 0.208214193
Glochidion chodoense 0.182541788
Phyllanthus urinaria 0.195567867
3ndhK Ricinus communis 1.122105263
Jatropha curcas 0.103290415
Euphorbia helioscopia 0.109882353
Manihot esculenta 0.102958937
Antidesma bunius 0.122624778
Breynia fruticosa 0.158464035
Glochidion chodoense 0.129590208
Phyllanthus urinaria 0.105896157
4rpl20 Ricinus communis 0.606166783
Jatropha curcas 1.008688097
Euphorbia helioscopia 0.760762174
Manihot esculenta 0.457788945
Antidesma bunius 0.528846154
Breynia fruticosa 0.514962037
Glochidion chodoense 0.445371143
Phyllanthus urinaria 0.479802513
5rpl23 Ricinus communis 0.486597938
Jatropha curcas 0.486597938
Euphorbia helioscopia 0.486597938
Manihot esculenta 0.7375
Antidesma bunius 0.392116183
Breynia fruticosa 1
Glochidion chodoense 1.103950104
Phyllanthus urinaria 1
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Page 11 of 18
Javaid et al. BMC Genomics (2024) 25:597
signicant than that of the other regions, likely due to the
high GC concentration found in the four rRNAs in the
inverted repeats [31].
Furthermore, our comparison of the cp. genome of
C. sabulosa with other species in the Euphorbiaceae
and Phyllanthaceae families yielded signicant ndings.
e cp. genomes and gene content were similar across
selected species. However, the infA gene was only pres-
ent in C. sabulosa and (A) bunius and was missing in
other Euphorbiaceae and Phyllanthaceae cp. genomes.
e pseudo copy of ycf1 was absent in C. sabulosa, (B)
fruticosa, and P. urinaria. ese ndings highlight the
unique gene content and organization in (C) sabulosa
and its evolutionary implications [32–37]. We also dis-
covered that the petD, petB, rpl16, and rpl2 genes of C.
sabulosa and some other species lacked introns, a phe-
nomenon documented in various other angiosperms [38,
39]. e genes in which intron loss was reported earlier
in other angiosperms are rpoC2, atpF, rpl2, rps12, atpF,
rps16, and clpP [38–42]. is underscores the critical role
of introns in gene expression control and their potential
to boost exogenous gene expression at plant genome
regions to achieve desirable agronomic features [37]. e
lack of specic introns may cause changes in gene expres-
sion [37].
e codon usage bias in the cp. genome of plants is
an essential evolutionary characteristic that aects the
translation of mRNA, gene identication, and molecu-
lar biological investigations [32]. Some genes in plastoms
have shown a bias towards specic codons, likely due to
external pressure [43]. C. sabulosa’s cp. genome uses leu-
cine most commonly and cysteine uncommonly. Similar
ndings have been reported in other cp. genomes, such
as Eruca sativa [43], Farsetia hamiltonii [17], and Nas-
turtium ocinale [44]. AGA codon in C. sabulosa’s cp.
genome had the highest usage bias for Arginine. ese
ndings suggest that codon usage signicantly impacts
the reshaping and translation of the cp. genome [17, 19–
21, 43–47]. Our results also support earlier studies on the
adaptational evolution of the large A/T concentration in
chloroplast genomes, which have also shown a prefer-
ence for specic codons [17, 19, 22, 23].
RNA editing is a modication after transcription, sig-
nicantly impacting the sequencing and performance
of related proteins and genetic material [45]. Analyz-
ing the RNA editing sites in C. sabulosa could provide
evolutionary insights into how RNA editing systems
Table 5 Ts, Tv substitutions, and Ts/Tv ratio (in IRs, SSC, and LSC) of selected Euphorbiaceae and Phyllanthaceae species
Region Species
(C. sabulosa as
reference)
Transition substi-
tuations (Ts)
Total Ts
substituations
Transversion substituaions (Tv) Total Tv
substituaions
Ts/
Tv
A/G C/T A/T A/C C/G G/T
Substitution
Type
RY W M S K
Large Single
Copy
R. communis 3505 3806 7311 2000 1303 569 1395 5267 1.39
J. curcas 3683 3921 7604 2350 1399 551 1424 5724 1.33
E. helioscopia 3832 4061 7893 2512 1553 706 1624 6395 1.23
M. esculenta 3546 3845 7391 2145 1260 560 1419 5384 1.37
A. bunius 3723 3970 7693 2130 1371 647 1473 5621 1.37
B. fruticosa 3697 3876 7573 2214 1344 653 1446 5657 1.34
G. chodoense 3682 3908 7590 2234 1391 634 1470 5729 1.32
P. urinaria 3661 3924 7585 2167 1358 647 1488 5660 1.34
Inverted
Repeat
R. communis 273 330 603 54 113 65 112 344 1.75
J. curcas 257 303 560 46 96 65 102 309 1.81
E. helioscopia 295 337 632 61 118 79 113 371 1.70
M. esculenta 288 340 628 57 106 65 115 343 1.83
A. bunius 305 328 633 71 120 72 133 396 1.60
B. fruticosa 308 329 637 66 110 67 124 367 1.74
G. chodoense 298 344 642 65 107 68 138 378 1.70
P. urinaria 306 337 643 75 119 69 139 402 1.60
Small Single
Copy
R. communis 960 959 1919 658 367 146 433 1604 1.20
J. curcas 913 880 1793 617 326 128 379 1450 1.24
E. helioscopia 870 894 1764 566 374 189 441 1570 1.12
M. esculenta 914 918 1832 577 349 123 401 1450 1.26
A. bunius 970 927 1897 641 386 153 477 1657 1.14
B. fruticosa 967 1004 1971 658 381 189 457 1685 1.17
G. chodoense 861 942 1803 615 361 168 410 1554 1.16
P. urinaria 846 891 1737 581 330 172 403 1486 1.17
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Page 12 of 18
Javaid et al. BMC Genomics (2024) 25:597
evolved during the evolution of plant life on Earth and
which editing sites may have been maintained to carry
out essential functions. Most RNA editing sites were
found in the ndhB gene, which encodes for NADH
dehydrogenase subunits. is demonstrated that a single
gene could translate a wide range of protein products
using RNA editing [41]. Changes at the second position
of the nucleotide were more prevalent than changes at
Table 6 The detailed analysis of InDels, Average InDel Length, InDel Diversity K(i), InDel Diversity per site Pi(i), and alignment length in
LSC, IR, and SSC regions of C. sabulosa by making pairwise alignment with eight selected species of Euphorbiaceae and Phyllanthaceae
families
Region Species (C. sabulosa as
reference)
Alignment length No. of InDels InDel average
Length
InDel diversity k(i) InDel
diver-
sity per
site Pi(i)
Large single copy R. communis 96,729 16,111 15.857 1016.000 0.01050
J. curcas 97,699 15,971 14.159 1128.000 0.01155
E. helioscopia 95,542 14,556 13.693 1063.000 0.01113
M. esculenta 95,837 14,703 14.030 1048.000 0.01094
A. bunius 96,398 15,601 14.621 1067.000 0.01107
B. fruticosa 93,881 15,001 14.867 1009.000 0.01075
G. chodoense 93,909 14,818 14.527 1020.000 0.01086
P. urinaria 94,125 15,365 15.427 996.000 0.01058
Inverted Repeat R. communis 27,634 3272 30.868 106.000 0.00384
J. curcas 28,159 4531 43.990 103.000 0.00366
E. helioscopia 27,302 2923 27.838 105.000 0.00385
M. esculenta 27,397 3191 28.491 112.000 0.00409
A. bunius 27,284 3114 31.140 100.000 0.00367
B. fruticosa 26,187 2163 19.313 112.000 0.00428
G. chodoense 27,836 3950 34.052 116.000 0.00417
P. urinaria 28,112 3900 34.821 112.000 0.00398
Small Single Copy R. communis 20,641 2972 16.697 178.000 0.00862
J. curcas 20,605 3867 22.224 174.000 0.00844
E. helioscopia 20,147 3655 24.205 151.000 0.00749
M. esculenta 20,614 3484 20.862 167.000 0.00810
A. bunius 20,697 2849 16.096 177.000 0.00855
B. fruticosa 20,749 2563 14.901 172.000 0.00829
G. chodoense 20,473 3817 25.447 150.000 0.00733
P. urinaria 20,290 3952 26.523 149.000 0.00734
Table 7 Six mutual highly polymorphic regions of selected Euphorbiaceae and Phyllanthaceae species
Sr. No Region Location Nucleotide Diversity with Euphorbiaceae Species Nucleotide Diversity with Phyllanthaceae Species
1rpl33-rps18 IGS 0.255 0.208
2rps18-rpl20 IGS 0.218 0.208
3rps15-ycf1 IGS 0.214 0.247
4ndhG-ndhI IGS 0.214 0.24
5psaI-ycf4 IGS 0.209 0.222
6petA-psbJ IGS 0.193 0.192
Fig. 9 Nucleotide diversity (π) in 93 regions common in Euphorbiaceae and Phyllanthaceae family members. The π values of Euphorbiaceae members
are displayed in sky blue, whereas the π values of Phyllanthaceae members are depicted in red
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Page 13 of 18
Javaid et al. BMC Genomics (2024) 25:597
other positions among the RNA editing sites examined.
RNA editing, particularly at the second codon position,
can alter the encoding amino acid and the primary, sec-
ondary, and tertiary protein organization, which may be
essential for their function [41, 45]. Most of the RNA
editing sites were discovered in Serine codons, with the
most signicant transformation of Serine into Leucine,
possibly increasing the hydrophobicity of the associated
peptide. Our ndings also supported that RNA edit-
ing sites can restore amino acid conservation, improve
hydrophobicity, and impact protein architecture [41].
ese ndings were consistent with the fundamental
properties of chloroplast gene RNA editing in higher
plants [17, 41, 45].
Our study on the C. sabulosa cp. genome revealed that
mono-nucleotide repeats were the most common and
pentanucleotide was the most extended SSR type. Our
results were consistent with similar studies on angio-
sperm species, indicating that polyadenine and polythy-
mine repeats are more abundant in cp. genomes [17, 26,
29, 44, 51, 52]. We did not observe any hexanucleotide
SSRs in the C. sabulosa cp. genome, which is a shared
trait with Brassica napa, Nasturtium ocinale, Raphanus
sativus, and Fritillaria cp. genomes [44, 48, 49]. Single-
copy regions had a higher percentage of oligonucleotide
repeats than inverted repeats, conrming the reverse
nature of inverted repeats [17]. e IGS had more repeats
than other cp. genome regions, indicating higher suscep-
tibility to mutations and recombination [17, 29]. Palin-
dromic repeats were more frequent than other types of
repetitions, suggesting the existence of various identical
or comparable sequences, either continuous or separated
by a spacer region [17, 28, 29]. Our ndings were consis-
tent with several studies conducted on angiosperms [17,
28, 29, 37, 53–55].
High-dimensional sequencing methods have made it
easier to access CP genomes with vast amounts of genetic
material [17, 43]. For phylogenetic research, cp. genome
sequences are an excellent resource [17, 43, 53, 56, 57].
e Euphorbiaceae family is one of the most diverse
angiosperm families [4, 5, 58]. However, there is conict
in sub-famil classication within the Euphorbiaceae fam-
ily. Previously, based on pollen morphology, the Euphor-
biaceae was classied into ve sub-families, including
Phyllanthoideae, Oldeldioideae, Acalyphoideae, Croto-
noideae, and Euphorbioideae [6]. Later, the Angiosperm
Phylogeny Group [13, 59] separated the Phyllanthaceae
from the Euphorbiaceae, giving it a separate family status.
Recently, Euphorbiaceae has been divided into four sub-
families based on molecular data [59, 60]. However, this
classication is also unclear due to a lack of available data
on its species. Chloroplast genomes have been used to
determine the phylogenetic relationships in the Euphor-
biaceae family [18, 23–26]. e systematic position of
Chrozophora was unclear until this study. e ndings
suggest that Chrozophora is closely related to the Phyl-
lanthaceae family, which supports the historical record
of the Euphorbiaceae family being the ancestor of the
Phyllanthaceae family [6]. e Chrozophora genus is con-
sidered to be distinct from the other Euphorbiaceae fam-
ily genera, indicating its paraphyletic origin. Our results
also conrmed that the Euphorbiaceae family’s immense
diversity, morphological divergences, variable ecologi-
cal range distribution, and the scarcity of literature on
numerous species make phylogenetic interpretations
challenging [60]. Additionally, there is a critical need to
sequence more chloroplast genomes of the Chrozopho-
reae tribe to clarify its position among these two families.
e cp. genome is known to be stable across dierent
plant lineages. However, the expansion and contraction
of IRs can alter the size of the cp. genome and its seg-
ments [17, 28, 53, 61, 62]. e expansion and contraction
of IRs aect genes, substitutions, and genome length,
ultimately determining a species’ phylogenetic position
[17, 34, 63]. Previous studies have found that changes in
the boundaries of the cp. genome are caused by dier-
ences in the number and location of genes at the interface
of inverted repeats [17, 34]. We studied the IR regions of
C. sabulosa, four Euphorbiaceae, and four Phyllantha-
ceae species. Our ndings revealed both similarities and
genetic variations in these plastomes. e conservation
of the IR region was higher in all plastomes, while most
substitutions occurred in the LSC and SSC regions. ese
results are consistent with similar studies on other plastid
genomes [17, 28, 62–64]. Our research also showed that
gene migration between single copies and inverted repeat
regions causes mutation rate variations, making chloro-
plast genomes either conserved [17, 28, 31, 34] or widely
polymorphic in gene content and structure [64–67].
e Ka/Ks ratio conrms the selective forces that have
acted upon the genes during evolution. ese forces can
be impartial, pure, or positive depending on the Ka/Ks
proportion [17, 68, 69]. We compared the cp. genome
of C. sabulosa to eight selected species to validate our
phylogenetic ndings. Our analysis showed that most C.
sabulosa genes underwent purifying selection to main-
tain their preserved function. Purifying selection on most
chloroplast genes in the Euphorbiaceae and Phyllantha-
ceae family species contributed to corroborating phylo-
genetic results. However, a few genes showed abnormal
behavior regarding Ka/Ks values, indicating petD, ndhK,
cemA, rpl23, and rpl20 in both family representatives.
Our research ndings are in collaboration with the pre-
viously published similar results [17, 43, 46, 70, 71].
InDels and SNPs were more frequent in the LSC sec-
tion than in the inverted repeats region. e IRs had the
lowest number of mutations, indicating their conserved
nature over the single-copy regions [17, 28, 62–64]. e
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 14 of 18
Javaid et al. BMC Genomics (2024) 25:597
transition-to-transversion ratio was more signicant
than one in all selected species, indicating a higher tran-
sition rate than the transversion rate. is suggests that
the species are distant and have more SNPs [17, 22]. e
high Ts/Tv ratio may be due to the GC-rich composi-
tion of the chloroplast genome [61]; similarly, the nuclear
genomes have already been reported to have a Ts/Tv ratio
due to their GC-rich makeup [72]. e high number of
InDels and SNPs (signicant mutations) indicated Chro-
zophora’s unique phylogenetic position and paraphyletic
evolution.
We analyzed nucleotide diversity in C. sabulosa and
selected species from the Euphorbiaceae and Phyllan-
thaceae families. Results showed that IGS regions have
higher rates of genetic recombination and polymor-
phisms than protein-coding regions. ese ndings con-
rmed that IGS regions are more susceptible to genetic
recombination and polymorphisms than protein-coding
regions. ese outcomes also supported the conserved
status of the protein-coding genes reported earlier in
other plastid genomes [17, 28, 31, 34]. Nucleotide diver-
sity is low in Euphobiaceae and Phyllanthaceae spe-
cies, ranging from 0.007 to 0.24. is suggests that the
plastome architecture is conserved in both families, con-
sistent with previous studies [17, 72–76]. We identied
six highly polymorphic regions shared by both families
that could be used as molecular identiers for the Chro-
zophora genus (rpl33-rps18, rps18-rpl20, rps15-ycf1,
ndhG-ndhI, psaI-ycf4, petA-psbJ) all with π > 0.5. For fur-
ther validation of the results of this study, more species
of the Chrozophora genus and the Chrozophoreae tribe
must be sequenced.
Conclusion
is is the rst cp. genome of C. sabulosa, which is also
the rst member of the genus and tribe reported. It has
a typical quadripartite structure and gene content that
is quite similar to other chloroplast genomes. Our com-
parative analysis with other Euphorbiaceae and Phyllan-
thaceae species has highlighted the conserved structure
of the chloroplast genome, the non-uniform distribu-
tion of GC percentage, and the unique gene content and
organization of C. sabulosa. Our investigation into codon
usage bias and RNA editing sites has provided insights
into the evolutionary characteristics of the cp. genome
and the potential impact on protein organization and
function. e phylogenetic analysis in this study revealed
Chrozophora’s unique position in the Euphorbiaceae
family, supporting the idea that this genus is paraphy-
letic. is chloroplast genome from C. sabulosa will be
useful for the molecular characterization of related Chro-
zophoreae tribe species in the future. e phylogenetic
data presented by this study will also aid in determining
the genus’ location in Euphorbiaceae family. e highly
polymorphic loci identied in this study could be used
as markers for future Chrozophora species identication.
Furthermore, it is essential to sequence its sister Chro-
zophora species to fully comprehend its phylogenetic
position and evolutionary dynamics. Overall, our study
provides valuable information on the chloroplast genome
of C. sabulosa and its evolutionary implications for the
scientic community.
Materials and methods
Collection of plant material and its sequencing
Nida Javaid and Shagufta Jabeen conducted the formal
plant material identication for this study. Fresh leaves
of Chrozophora sabulosa plants were collected from
the Lesser Cholistan desert in Pakistan (28.7719699,
71.3346211) and the verication process was carried out
at the Cholistan Institute of Desert Studies (CIDS) of the
Islamia University Bahawalpur. e herbarium of plants
was submitted to the CIDS for identication. A voucher
number was issued for Chrozophora sabulosa Kar. & Kir.
Nilkari of Euphorbiaceae which is CIDS/IUB-1601/59.
For DNA extraction, the phenol-chloroform (Organic)
[77] procedure was used with a few modications. ese
modications included using 1 µL 2-Mercaptoethanol
and precipitating DNA with absolute ethanol following
a wash with 70% ethanol. e extracted DNA was then
analyzed for its quantity and quality using Nanodrop
and 1% agarose gel electrophoresis. A full genome shot-
gun was generated by a Paired-end library of 150bp with
350bp insert size using Illumina Hiseq2500 at the Beijing
Institute of Genomics (BIG), Beijing, China.
Chloroplast genome assembly and genes annotation
We used FastQC software [78] to verify raw reads, and
NOVOPlasty [79] to assemble the cp. genome. e LSC,
SSC, and IR regions were dened manually by inspect-
ing the sequence scaolding. We employed GeSeq [80]
(https://chlorobox.mpimp-golm.mpg.de/geseq.html) and
CpGAVAS (http://www.herbalgenomics.org/cpgavas) to
annotate the assembled cp. genome [81]. e annotation
was manually veried using MAFFT alignment (Multiple
Alignment with Fast Fourier Transform) [82] in Geneious
Prime 2023.2.1 software [83]. To conrm the tRNA
genes, tRNAscan-SE 1.23 program was used [84]. We
determined the average sequencing coverage depth for
the assembled C. sabulosa genome by mapping sequenc-
ing short reads to their respective de novo assembled cp.
genomes using Tablet [85] and BWA [86]. We created
the circular map of the cp. genome using OGDraw v1.2
(https://chlorobox.mpimp-golm.mpg.de/OGDraw.html)
[87]. e C. sabulosa cp. genome was submitted to Gen-
Bank and assigned the accession number MW541931.
We also submitted the raw data obtained in this work
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Page 15 of 18
Javaid et al. BMC Genomics (2024) 25:597
to Sequence Read Archive (SRA) under project number
PRJNA660981.
RNA editing site, codon usage, and amino acid frequency
We used Geneious Prime 2023.2.1 to analyze the amino
acid frequency, and MEGA-X to examine Relative Syn-
onymous Codon Usage (RSCU) in protein-coding
sequences of C. sabulosa [88]. Additionally, we used Pre-
dictive RNA Editors for Plants Chloroplast (PREP-cp:
http://prep.unl.edu/) to nd RNA editing sites in 21 pro-
tein-coding genes [89].
Detecting simple sequence repeats (SSRs) and
oligonucleotide repeats
e Perl script MIcroSAtellite Identication Tools
(MISA) [90] software (https://webblast.ipk-gatersleben.
de/misa/) was used to identify simple sequence repeats
(SSRs), with minimal repeat count of ten for mono-, ve
for di-, four for tri-, three tetra-, three Penta-, and three
for hexanucleotides. Additionally, the REPuter program
(https://bibiserv.cebitec.uni-bielefeld.de/reputer) was
utilized to detect reverse (R), complementary (C), pal-
indromic (P), and forward (F) oligonucleotide repeats
with an edit distance of two, and minimum repeat size
of 10bp. e maximum calculated repeat was set to 100
[91].
Phylogenetic analysis
e National Center for Biotechnology Information
(NCBI) was used to select 18 species plastomes from the
Euphorbiaceae family for phylogenetic analysis (Table
S12). ese species represent 11 tribes and three sub-
families of the Euphorbiaceae, including Acalyphoideae,
Crotonoideae, and Euphorbioideae. Additionally, 10 spe-
cies of the Phyllanthaceae family and two out-groups
(Mangifera indica and Lannea coromandelica) from the
Anacardiaceae family were chosen for analysis. In total,
31 species were included in the phylogenetic tree. e
protein-coding sequences for each species were extracted
and concatenated using Geneious Prime 2021.1.1. e
sequences were then aligned using MAFFT in Geneious
Prime 2023.2.1. e maximum likelihood tree was con-
structed online in Galaxy (https://usegalaxy.org) using
IQ-TREE [92] and 1000 bootstrap replications with
Ultrafast bootstrap settings [93]. e best-t model was
selected according to the Akaike information criterion
(AIC) [94]. We completed the tree display using iTOL
(https://itol.embl.de/#) [95].
Comparative analyses with C. Sabulosa
e cp. genomes of four species from the Euphorbiaceae
family (Ricinus communis, Manihot esculenta, Jatro-
pha curcas, and Euphorbia helioscopia) and four species
from the Phyllanthaceae familys (Antidesma bunius,
Breynia fruticosa, Glochidion chodoense, and Phyllan-
thus urinaria) were compared to that of C. sabulosa.
is was done using phylogenetic analysis outcomes. e
Geneious Prime 2023.2.1 was used to perform a basic
comparison of the plastomes. IRscope (https://irscope.
shinyapps.io/irapp/) was used to observe IR contrac-
tion and expansion in the LSC/IRB/SSC/IRA junctions
among these selected species [96]. Pairwise comparisons
of the 78 protein-coding genes common in C. sabulosa
and the eight selected species were performed to esti-
mate synonymous (Ks) and non-synonymous (Ka) sub-
stitution rates. C. sabulosa was used as the reference
member to make pairwise alignments with every gene of
the selected species. Firstly, MAFFT in Geneious Prime
2023.2.1 software was used to perform 624 pairwise
alignments of the identied genes among species [82, 83],
and then DnaSP [97] was employed to examine Ka and
Ks substitutions. Geneious Prime 2023.2.1 was used to
calculate the number, coordinate placements, and types
of substitutions (transition and transversion). DnaSP
[97] was used to nd InDels mutations for each part of
the pairwise aligned cp. genomes. e alignment length,
inDel average length, k(i) inDel diversity, and Pi(i) inDel
diversity per site were also calculated.
Nucleotide diversity to determine highly polymorphic loci
We analyzed to compare Nucleotide diversity (π) values
among representatives of both families along with C. sab-
ulosa. We extracted a total of 837 regions, which included
59 CDS genes, 27 IGS regions, and seven intronic regions
that were common in all species. To create multiple
alignments of 93 locations of C. sabulosa with members
of both families separately, we used MAFFT [82]. We
only selected sequences that were longer than 200 base
pairs [98]. To calculate the Nucleotide diversity (π), we
used DnaSP [97]. We identied six highly polymorphic
loci that had greater nucleotide diversity to compare
among the selected species [98].
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12864-024-10366-3.
Supplementary Material 1
Acknowledgements
The authors acknowledge the contribution of Beijing Institute of Genomics
(BIG), China in sequencing the chloroplast genome of C. sabulosa.
Author contributions
Conceptualization: Nida Javaid, Musarrat Ramzan, Muhammad Anwar
Methodology = Nida Javaid, Musarrat Ramzan, Shagufta Jabeen Writing
original draft preparation = Nida Javaid, Musarrat Ramzan, Shagufta Jabeen,
Muhammad Anwar Data collection and analysis= Nida Javaid and Musarrat
Ramzanundertook the formal identication of the plant material used in this
study and analysis. Funding acquisition = Muhammad Anwar, Yanjun Du, Song
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 16 of 18
Javaid et al. BMC Genomics (2024) 25:597
Xiqiang Supervision = Musarrat Ramzan, Muhammad Anwar, SONG Xiqiang.
All authors have read and agreed to the published version of the manuscript.
Funding
This research is supported by Hainan University Research Initiation Project
Fund (XJ2400005264), Nature National Science Foundation NSFC(N0.
32371959).
Data availability
The datasets generated and/or analyzed during the current study are available
in the NCBI repository, https://www.ncbi.nlm.nih.gov/nuccore/2419468502
ACCESSION MW541931. Raw data submitted Sequence Read Archive (SRA)
under the project number PRJNA660981. Further data is present in the
manuscript. For more in-depth details, there is a supplementary le.
Declarations
Ethics approval and consent to participate
We all declare that manuscript reporting studies do not involve any human
participants, human data, or human tissue. So, it is not applicable. Nida
Javaid and Musarrat Ramzan undertook the formal identication of the plant
material used in this study. We conrmed that we have taken permission to
collect the plant material used in this study. This study complies with relevant
institutional, national, and international guidelines. Plant voucher number for
Euphorbiaceae Chrozophora sabulosa Kar. & Kir. Nilkari is CIDS/ IUB-1601/59
Consent for publication
Not Applicable (NA).
Competing interests
The authors declare no competing interests.
Author details
1School of Tropical Agriculture and Forestry (School of Agriculture and
Rural Aairs,School of Rural Revitalization), Hainan University, Haikou, P.R.
China
2Key Laboratory of Genetic and Germplasm Innovation of Tropical
Special Forest Trees and Ornamental Plants, Ministry of Education, Hainan
University, Haikou, P.R. China
3The Islamia University, Bahawalpur, Pakistan
4Haikou, P.R. China
Received: 2 June 2023 / Accepted: 29 April 2024
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