# Complete Chloroplast Genome Sequence ofJusticia flava: Genome Comparative Analysis and Phylogenetic Relationships among Acanthaceae

Authors: Samaila S. Yaradua; Dhafer A. Alzahrani; Enas J. Albokhary; Abidina Abba; Abubakar Bello
Journal: BioMed Research International
 (2019)

---

## Abstract

The complete chloroplast genome ofJ.flava, an endangered medicinal plant in Saudi Arabia, was sequenced and compared with cp genome of three Acanthaceae species to characterize the cp genome, identify SSRs, and also detect variation among the cp genomes of the sampled Acanthaceae. NOVOPlasty was used to assemble the complete chloroplast genome from the whole genome data. The cp genome ofJ.flavawas 150, 888bp in length with GC content of 38.2%, and has a quadripartite structure; the genome harbors one pair of inverted repeat (IRa and IRb 25, 500bp each) separated by large single copy (LSC, 82, 995 bp) and small single copy (SSC, 16, 893 bp). There are 132 genes in the genome, which includes 80 protein coding genes, 30 tRNA, and 4 rRNA; 113 are unique while the remaining 19 are duplicated in IR regions. The repeat analysis indicates that the genome contained all types of repeats with palindromic occurring more frequently; the analysis also identified total number of 98 simple sequence repeats (SSR) of which majority are mononucleotides A/T and are found in the intergenic spacer. The comparative analysis with other cp genomes sampled indicated that the inverted repeat regions are conserved than the single copy regions and the noncoding regions show high rate of variation than the coding region. All the genomes havendhF andycf1 genes in the border junction of IRb and SSC. Sequence divergence analysis of the protein coding genes showed that seven genes (petB, atpF, psaI, rpl32, rpl16, ycf1, and clpP) are under positive selection. The phylogenetic analysis revealed that Justiceae is sister to Ruellieae. This study reported the first cp genome of the largest genus in Acanthaceae and provided resources for studying genetic diversity ofJ.flavaas well as resolving phylogenetic relationships within the core Acanthaceae.


---

## Body

## 1. Introduction

Justicia L. is one of the largest and the most taxonomically complex genus in Acanthaceae belonging to the tribe Justicieae consisting of ca. 600 species [1–3] typified byJ. hyssopifolia (Sp Pl. 15 1753; Gen. Pl. ed. 5, 10 1754; nonsensu Nees 1832, 1847; nec sunsu Kuntze, 1891) distributed in the tropical and subtropical regions of the world, extending into warm temperate zones in Europe, Asia, and North America [4]. The genus is the type specimen of the tribe Justiceae and is characterized by a number of characters which includes corolla having 2 libbeds (bilobed upper lip and trilobed lower lip) and 2 bithecous stamens, one of the two named thecae above the other and the other one contains stur at the base [1, 3]. Several authors [5–7] include small segregate genera in the genus while author [1] in his own view reported a new classification adding more taxa and defined the genus as having 16 sections based on some floral parts and seeds. Recent molecular studies reported thatJusticia s. l (Graham 1988) is paraphyletic [8, 9]. Lindau classifiedJusticia and several closely related species under a tribe that is characterized by androecium having 2 stamens and Knotchenpollen. There is a problem with this system of classification as reported by some student of Acanthaceae because some species in the tribe do not have this character while several taxa with the characters were classified in other tribes. However, there is still a problem in the status of the tribe Justiceae; [6] divided Ruellioideae into Justicieae, Ruellieae, and Lepidagathideae, whereas authors [10] in their study on phylogenetic relationship among Acanthaceae usingtrnL-trnF reportedRuellia andJusticia as separate lineages. Recently, [11] classified the member of the Justicieae as subtribe under the tribe Ruelloideae.Justicia flava is among the endangered species in Saudi Arabia; the plant is widely used in traditional medicine in treating various ailments such as cough, paralysis, fever epilepsy, convulsion, and spasm and skin infection disorder. The roots are also reported to be used in treating diarrhea and dysentery [12, 13]. As reported by [14] the plant has antimicrobial and antioxidant activity as well as wound healing activity. Despite the endangered nature and uses in traditional medicine of the species, the complete chloroplast genome of the species was not sequenced until this study.Comparison of complete chloroplast genome provides very informative information for reconstruction of phylogeny and resolving evolutionary relationships issues at various taxonomic levels [15–19]. This is as a result of the conservative nature of the chloroplast genome [20]; this conservative nature is because the plastome evolves about half the rate of other genomes like the nuclear [21, 22]. However, rearrangements in the sequence of chloroplast genome were reported by various plant chloroplast genome studies [19, 23–25]. These rearrangements occur as a result of contractions, expansions, and inversions in the single copy regions (large single copy and small single copy) and the inverted repeats [26, 27]. The rearrangements of the genes and inversion in the chloroplast genome are reported to be useful in phylogenetic analyses to solve taxonomic problems at various taxonomic levels because they do not occur often and estimation of their homology and inversion event polarity is simple [22, 28–31]. With the importance of complete chloroplast genome in resolving phylogenetic relationship issues and the large number of genera and species in Acanthaceae only complete chloroplast genome of four genera has been so far reported (Andrographis paniculata (Burm.f.) Nees, NC_022451;Ruellia breedlovei T. F. Daniel, KP300014;Strobilanthes cusia(Nees) O. Kuntze, MG874806, and four speciesEchinocactusMF490441, MH045155, MH045156, and MH045157.)In this study, we reported the characteristics of the complete chloroplast genome ofJusticia flava, the first cp genome in the largest genus of Acanthaceae, and compared the genomes of four Acanthaceae species to understand the variation among the cp genomes, report the simple sequence repeats to provide the tools for genetic diversity and identification of the species, and lastly, resolve the status of Justiceae.


## 2. Materials and Methods

### 2.1. Plant Material and DNA Extraction

Plant material (vegetative and floral part) was collected through field survey ofJ.flava in Taif, Saudi Arabia, and identified based on the herbarium specimens and morphological features seen in relevant literatures, the voucher specimen was deposited in the herbarium of King Abdulaziz University, Jeddah, Saudi Arabia. Leaves were collected from the specimen for genomic DNA extraction. The genomic DNA was extracted using Plant Genomic DNA Kit according to manufacturer’s protocol.


### 2.2. Library Construction, Sequencing, and Assembly

A total amount of 1.0μg DNA was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® DNA Library Prep Kit following manufacturer’s recommendations and indices were added to each sample. The genomic DNA is randomly fragmented to a size of 350bp by shearing, then DNA fragments were end polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing and further PCR enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system) and resulting libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR. The qualified libraries are fed into Illumina sequencers after pooling according to its effective concentration and expected data volume. The raw reads were filtered to get the clean reads (5 Gb) using PRINSEQ lite v0.20.4 [32] and were subjected to de novo assembly using NOVOPlasty2.7.2 [33] with kmer (K-mer= 31) to assemble the complete chloroplast genome from the whole genome sequence. ndhAx1-ndhAx2 intergenic spacer fromJusticia flava (KY632456) was used as seed and the plastome sequence ofRuellia breedlovei (KP300014.1) was used as the reference. Finally, one contig containing the complete chloroplast genome sequence was generated.


### 2.3. Gene Annotation

The programme DOGMA (Dual Organellar GenoMe Annotator, University of Texas at Austin, Austin, TX, USA) [34] was used to annotate the genes in the assembled chloroplast genome. The positions of start and stop codon were adjusted manually. trnAscan-SE server (http://lowelab.ucsc.edu/tRNAscan-SE/) [35] was used to verified the tRNA genes and finally, the plastome genome circular map was drawn using OGDRAW (Organellar Genome DRAW) [36]. The sequence of the chloroplast genome ofJ. Flava was deposited in the GenBank database with accession number (MK548577,).


### 2.4. Sequence Analysis

MEGA 6.0 was used to analyze the relative synonymous codon usage values (RSCU), base composition, and codon usage. Possible RNA editing sites present in the protein coding genes ofJ. flava cp genome were determined using PREP suite [37] with 0.8 as the cutoff value.


### 2.5. Repeat Analysis inJ. flava Chloroplast Genome

Simple sequence repeats (SSRs) were identified in theJ.flava chloroplast genome and genome of other three species of Acanthaceae using the online software MIcroSAtellite (MISA) [38] with the following parameters: eight, five, four, and three repeats units for mononucleotides, dinucleotides, trinucleotides, and tetra-, penta-, hexanucleotides SSR motifs, respectively. For analysis of long repeats (palindromic, forward, reverse, and complement) the program REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) [39] with default parameters was used to identify the size and location of the repeats inJ.flava chloroplast genome and genome of other 3 species of Acanthaceae.


### 2.6. Genome Comparison

The program mVISTA [40] was used to compare the chloroplast genome ofJ. flava with the cp genomes ofE.longzhouensis, R.breedlovei, andS.cusia using the annotation ofJ. flava as reference in the Shuffle-LAGAN mode [41]. The border region between the large single copy (LSC) and inverted repeat (IR) and small single copy SSC and inverted repeat (IR) junction were compared among the four species of Acanthaceae.


### 2.7. Characterization of Substitution Rate

DNAsp v5.10.01 [42] was used to analyze synonymous (dS) and nonsynonymous (dN) substitution rate and dN/dS ratio to detect the genes that are under selection pressure; the chloroplast genome ofJ. flava was compared with the cp genome ofE.longzhouensis, R.breedlovei, andS.cusia. Individual protein coding genes were aligned separately in Genious V 8.1.3; the aligned sequences were then translated into protein sequence.


### 2.8. Phylogenetic Analysis

The complete chloroplast genome of three Acanthaceae and five species from the order Lamiales were downloaded from Genbank. The downloaded sequences were aligned with sequenced cp genome ofJ.flava using MAFFT v.7 [43]. The data were analyzed using Maximum Parsimony (PAUP version 4.0b10) [44] using heuristic searches with 1000 replicates of random taxon addition, tree bisection-reconnection branch swapping, MulTrees on, saving a maximum of 100 trees each replicate. Missing characters were treated as gaps. Support was assessed using 1000 replicates of nonparametric bootstrap analysis. Bayesian analysis was carried out using MrBayes version 3.2.6 [45]. jModelTest version 3.7 [46] was used to select the suitable model.


## 2.1. Plant Material and DNA Extraction

Plant material (vegetative and floral part) was collected through field survey ofJ.flava in Taif, Saudi Arabia, and identified based on the herbarium specimens and morphological features seen in relevant literatures, the voucher specimen was deposited in the herbarium of King Abdulaziz University, Jeddah, Saudi Arabia. Leaves were collected from the specimen for genomic DNA extraction. The genomic DNA was extracted using Plant Genomic DNA Kit according to manufacturer’s protocol.


## 2.2. Library Construction, Sequencing, and Assembly

A total amount of 1.0μg DNA was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® DNA Library Prep Kit following manufacturer’s recommendations and indices were added to each sample. The genomic DNA is randomly fragmented to a size of 350bp by shearing, then DNA fragments were end polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing and further PCR enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system) and resulting libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR. The qualified libraries are fed into Illumina sequencers after pooling according to its effective concentration and expected data volume. The raw reads were filtered to get the clean reads (5 Gb) using PRINSEQ lite v0.20.4 [32] and were subjected to de novo assembly using NOVOPlasty2.7.2 [33] with kmer (K-mer= 31) to assemble the complete chloroplast genome from the whole genome sequence. ndhAx1-ndhAx2 intergenic spacer fromJusticia flava (KY632456) was used as seed and the plastome sequence ofRuellia breedlovei (KP300014.1) was used as the reference. Finally, one contig containing the complete chloroplast genome sequence was generated.


## 2.3. Gene Annotation

The programme DOGMA (Dual Organellar GenoMe Annotator, University of Texas at Austin, Austin, TX, USA) [34] was used to annotate the genes in the assembled chloroplast genome. The positions of start and stop codon were adjusted manually. trnAscan-SE server (http://lowelab.ucsc.edu/tRNAscan-SE/) [35] was used to verified the tRNA genes and finally, the plastome genome circular map was drawn using OGDRAW (Organellar Genome DRAW) [36]. The sequence of the chloroplast genome ofJ. Flava was deposited in the GenBank database with accession number (MK548577,).


## 2.4. Sequence Analysis

MEGA 6.0 was used to analyze the relative synonymous codon usage values (RSCU), base composition, and codon usage. Possible RNA editing sites present in the protein coding genes ofJ. flava cp genome were determined using PREP suite [37] with 0.8 as the cutoff value.


## 2.5. Repeat Analysis inJ. flava Chloroplast Genome

Simple sequence repeats (SSRs) were identified in theJ.flava chloroplast genome and genome of other three species of Acanthaceae using the online software MIcroSAtellite (MISA) [38] with the following parameters: eight, five, four, and three repeats units for mononucleotides, dinucleotides, trinucleotides, and tetra-, penta-, hexanucleotides SSR motifs, respectively. For analysis of long repeats (palindromic, forward, reverse, and complement) the program REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) [39] with default parameters was used to identify the size and location of the repeats inJ.flava chloroplast genome and genome of other 3 species of Acanthaceae.


## 2.6. Genome Comparison

The program mVISTA [40] was used to compare the chloroplast genome ofJ. flava with the cp genomes ofE.longzhouensis, R.breedlovei, andS.cusia using the annotation ofJ. flava as reference in the Shuffle-LAGAN mode [41]. The border region between the large single copy (LSC) and inverted repeat (IR) and small single copy SSC and inverted repeat (IR) junction were compared among the four species of Acanthaceae.


## 2.7. Characterization of Substitution Rate

DNAsp v5.10.01 [42] was used to analyze synonymous (dS) and nonsynonymous (dN) substitution rate and dN/dS ratio to detect the genes that are under selection pressure; the chloroplast genome ofJ. flava was compared with the cp genome ofE.longzhouensis, R.breedlovei, andS.cusia. Individual protein coding genes were aligned separately in Genious V 8.1.3; the aligned sequences were then translated into protein sequence.


## 2.8. Phylogenetic Analysis

The complete chloroplast genome of three Acanthaceae and five species from the order Lamiales were downloaded from Genbank. The downloaded sequences were aligned with sequenced cp genome ofJ.flava using MAFFT v.7 [43]. The data were analyzed using Maximum Parsimony (PAUP version 4.0b10) [44] using heuristic searches with 1000 replicates of random taxon addition, tree bisection-reconnection branch swapping, MulTrees on, saving a maximum of 100 trees each replicate. Missing characters were treated as gaps. Support was assessed using 1000 replicates of nonparametric bootstrap analysis. Bayesian analysis was carried out using MrBayes version 3.2.6 [45]. jModelTest version 3.7 [46] was used to select the suitable model.


## 3. Results and Discussion

### 3.1. Characteristics ofJ. flava Chloroplast Genome

The complete chloroplast genome ofJ.flava is a circular molecule and has quadripartite structure; the genome was found to have 150, 888bp in length. The genome is divided into four regions, namely, large single copy (LSC), small single copy (SSC), and two inverted repeats (IRa and IRb). The coding region is 98, 671bp in length and constitutes 65.39% of the genome; the rest of 52, 217 bp is the intergenic spacer region including intron (34.60%). The LSC and SSC regions possessed 82, 995bp and 16, 893bp, respectively, the inverted repeats IRa and IRb have 25, 500bp and are separated by SSC region (Figure 1 and Table 1). The organization and structure of theJ.flava cp genome are similar to other sequenced Acanthaceae cp genomes [47, 48]. The percentage of occurrence of AT and GC content in the genome showed that the LSC, SSC, IRA, and IRB regions possessed 63.8% and 36.2%, 67.8% and 32.2%, 56.7% and 43.4%, and 56.7% and 3.4, respectively. The whole chloroplast genome has AT content of 61.8% and GC content of 38.2%; this is similar to cp genome ofStrobilanthes cusia [49]. The GC content in the inverted repeat is found to be higher than the single copy regions both the LSC and SSC.Table 1
Base composition in theJ. flavachloroplast genome.
Region  T(U) (%) C (%) A (%) G (%) Total (bp) cp genome  31 19 31 19 150888 LSC  32 19 31 18 82995 SSC  34 17 34 15 16893 IRA  28 23 28 21 25500 IRB  28 21 28 23 25500 1st Position 31 20 30 19 50296 2nd Position 31 19 31 19 50296 3rd Position 31 20 30 19 50296Figure 1
Gene map of theJ. flava chloroplast genome. Genes outside the circles are transcribed in counterclockwise direction and those inside in clockwise direction. Known functional genes are indicated in the coloured bar. The GC and AT content are denoted by the dark grey and light grey colour in the inner circle, respectively. LSC indicates large single copy; SSC, indicates small single copy, and IR indicates inverted repeat.The result of the genes annotation in the chloroplast genome ofJ. flava revealed a total of 132 genes among which 113 are unique; the remaining 19 are duplicated in the inverted region. The genome harbored 80 protein coding genes, 30 tRNA genes, and 4 rRNA genes (Figure 1 and Table 2). The numbers and orientation of the genes in the cp genome are the same as other cp genomes of Acanthaceae [47, 48]. The inverted repeat region contained eight protein coding genes, seven tRNA, and four rRNA while in the single copy region, the LSC contained 61 protein coding genes and 22 tRNA genes; the rest of 12 protein coding genes and 1 tRNA are located within the SSC region. Almost all the protein coding genes start with the ATG codon that code for methionine whereas some of the genes start with codon like ATC, GTG and ACG; this is common in most flowering plant (angiosperms) chloroplast genome [49–51].Table 2
Genes present in the chloroplast genome ofJ. flava.
Category Group of genes Name of genes RNA genes ribosomal RNA genes (rRNA) rrn5, rrn4.5, rrn16, rrn23  Transfer RNA genes (tRNA) trnH-GUG, trnK-UUU +, trnQ-UUG, trnS-GCU, trnS-CGA+, trnR-UCU,trnC-GCA, trnD-GUC, trnY-GUA, trnE-UUC, trnT-GGU, trnS-UGA, trnfM-CAU, trnG-GCC, trnS-GGA, trnL-UAA+, trnT-UGU, trnF-GAA, trnV-UAC+, trnM-CAU, trnW-CCA, trnP-UGG, trnI-CAUa, trnL-CAAa,trnV-GACa, trnI-GAU+,a,trnA-UGC+,a, trnR-ACGa, trnN-GUUa, trnL-UAG,  Ribosomal proteins Small subunit of ribosome rps2, rps3, rps4, r p s 7 a, rps8, rps11,rps12a, rps14, rps15,rps,16+, rps18,rps19  Transcription Large subunit of ribosome r p l 2 + , a, rpl14, rpl16, rpl20, rpl22,rpl23a, rpl32, rpl33, rpl36  DNA dependent RNA polymerase rpoA, rpoB, rpoC1 +, rpoC2  Protein genes Photosystem I psaA, psaB, psaC, psaI,psaJ,ycf3 ++  Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ  Subunit of cytochrome petA, petB, petD, petG, petL, petN  Subunit of synthase atpA, atpB, atpE, atpF +, atpH, atpI  Large subunit of rubisco rbcL  NADH dehydrogenase ndhA +,ndhB+a, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK  ATP dependent protease subunit P clpP ++  Chloroplast envelope membrane protein cemA  Other genes Maturase matK  Subunit acetyl-coA carboxylase accD  C-type cytochrome synthesis ccsA  Hypothetical proteins y c f 2 a, ycf4, ycf15a  Component of TIC complex y c f 1 a G e n e   + with one intron. Gene++ with two intron. a Gene with copies.TheJ. flava chloroplast genome is found to contain intron in some of the protein coding and tRNA genes, like other chloroplast genomes of angiosperms [49, 50]. There are 14 genes that contain intron out of the 113 different genes (Table 3); among the 14 genes 8 are protein coding genes while the remaining six are tRNA genes (Table 3). Four genes that have the intron, namely,rpl2,ndhB,trnI-GAU,andtrnA-UGCare located in the IR region while the remaining 12 are located in the LSC region. Only two genesycf3 andclpP have two introns, the other 12 have only one intron, and this is also seen inS. cusia [49]. The tRNA,trnK-UUUhas the longest intron of 2460 bp (Table 3); this is as a result of position of thematK gene in the intron.Table 3
Genes with intron theJ. flava chloroplast genome and length of introns and exons.
Gene Location Exon I (bp) Intron I (bp) Exon II (bp) Intron II (bp) Exon III (bp) atpF LSC 143 659 467 rpoC1 LSC 443 769 1628 ycf3 LSC 128 682 227 721 152 clpP LSC 68 763 293 635 227 rpl2 IR 392 664 434 ndhB IR 776 680 755 ndhA SSC 551 954 539 trnK-UU LSC 36 2460 37 trnS-CGA LSC 31 667 59 trnL-UAA LSC 36 501 49 trnV-UAC LSC 37 593 36 trnI-GAU IR 41 938 34 trnA-UGC IR 37 818 34The codon usage bias in the plastome was computed using the protein coding genes and tRNA genes nucleotide sequences 89, 377bp. The relative synonymous codon usage of each codon in the genome is presented in (Table4); the result revealed that all the genes are encoded by 29, 790 codons. Codons, coding for the amino acids Leucine, are the most frequent codons 3,329 (11.7%) (Figure 2), similar to that ofAilanthus altissima [52], whereas codons coding for Trp are the least 570 (1.91%) in the genome. G- and C-ending are found to be more frequent than their counterpart A and T; this is not the case in other plastomes sequences [53–55]. The result of the analysis (Table 4) showed that codon usage bias is low in the chloroplast genome ofJ.flava. The RSCU values of 29 codons were >1 and all of them have A/T ending while for 30 codons were <1 and are all of G/C ending. Only two amino acids Tryptophan and Methionine have RSCU value of 1; therefore they are the only amino acids with no codon bias.Table 4
Codon-anticodon recognition patterns and codon usage of theJ. flava chloroplast genome.
Codon Amino Acid RSCU tRNA Codon Amino Acid RSCU tRNA UUU Phe 1.18 trnF-GAA UAU Tyr 1.38 trnY-GUA UUC Phe 0.82  UAC Tyr 0.62 UUA Leu 1.31 trnL-UAA UAA Stop 1.01 UUG Leu 1.31 trnL-CAA UAG Stop 1.03 CUU Leu 1.23 trnL-UAG CAU His 1.26 trnH-GUG CUC Leu 0.65  CAC His 0.74 CUA Leu 0.92  CAA Gln 1.37 trnQ-UUG CUG Leu 0.59  CAG Gln 0.63 AUU Ile 1.22 trnI-GAU AAU Asn 1.34 trnG-GUU AUC Ile 0.82  AAC Asn 0.66 AUA Ile 0.95 trnI-CAU AAA Lys 1.29 trnK-UUU AUG Met 1 trnM-CAU AAG Lys 0.71 GUU Val 1.45 trnV-GAC GAU Asp 1.44 trnD-GUC GUC Val 0.65  GAC Asp 0.56 GUG Val 0.74  GAA Glu 1.38 trnE-UUC GUA Val 1.16 trnV-UAC GAG Glu 0.62 UCU Ser 1.46 trnS-GGA UGU Cys 1.14 trnC-GCA UCC Ser 0.95  UGC Cys 0.86 UCG Ser 0.76  UGA Stop 0.96 UCA Ser 1.33 trnS-UGA UGG Trp 1 trnW-CCA CCU Pro 1.21 trnP-UGG CGU Arg 0.84 trnR-ACG CCC Pro 0.87  CGC Arg 0.35 trnR-UCU CCA Pro 1.13  CGA Arg 1.16 CCG Pro 0.79  CGG Arg 0.77 ACU Thr 1.22  AGA Arg 1.8 ACC Thr 0.87  AGG Arg 1.07 ACG Thr 0.72 trnT-GGU AGU Ser 0.92 trnS-GCU ACA Thr 1.2 trnT-UGU AGC Ser 0.58 GCU Ala 1.33 trnA-UGC GGU Gly 1.18 trnG-GCC GCC Ala 0.76  GGC Gly 0.54 GCA Ala 1.18  GGA Gly 1.28 GCG Ala 0.72  GGG Gly 0.99 trnG-UCCFigure 2
Amino acids frequencies inJ. flava chloroplast genome protein coding sequences.The RNA editing site in theJ. flavachloroplast genomes was predicted using the program PREP suite; all the analysis was done using the first codon position of the first nucleotide. The result (Table 5) shows that the majority of the conversion in the codon positions is from the amino acid Serine to Leucine (Table 5). In all, the programme revealed 61 editing sites in the genome; the editing sites are distributed among 18 protein coding genes. As reported in previous researches [56–58] thendhB gene has the highest number of editing sites (9 sites) followed byrpoB (7 site) andndhG,atpF,rpl2,rpl20, andrps2 have the least 1 site each. Among all the conversion in the RNA editing site, one site changed the amino acid from apolar group to polar group (Proline to Serine). The following genes do not have RNA predicting site in their first codon of the first nucleotidesatpB, ccsA, clpP, ndhC, ndhE psaB, petD, petG, rpoA, and petL, among others.Table 5
Predicted RNA editing site in theJ. flava chloroplast genome.
gene Nucleotide Position Amino Acid Position Codon Conversion Amino Acid Conversion Score accD 800 267 TCG => TTG S => L 0.8 844 282 CCC => TCC P => S 0.8 atpA 776 259 ACC => ATC T => I 1 914 305 TCA => TTA S => L 1 1270 424 CCC => TCC P => S 1 atpF 92 31 CCA => CTA P => L 0.86 atpI 404 135 GCT => GTT A => V 1 620 207 TCA => TTA S => L 1 matK 640 214 CAT => TAT H => Y 1 1249 417 CAT => TAT H => Y 1 ndhA 326 109 ACT => ATT T => I 1 566 189 TCA => TTA S => L 1 922 308 CTT => TTT L => F 1 ndhB 149 50 TCA => TTA S => L 1 467 156 CCA => CTA P => L 1 586 196 CAT => TAT H => Y 1 737 246 CCA => CTA P => L 1 746 249 TCT => TTT S => F 1 830 277 TCA => TTA S => L 1 836 279 TCA => TTA S => L 1 1292 431 TCC => TTC S => F 1 1481 494 CCA => CTA P => L 1 ndhD 2 1 ACG => ATG T => M 1 32 11 GCA => GTA A => V 1 878 293 TCA => TTA S => L 1 1445 482 GCT => GTT A => V 1 ndhF 124 42 CTT => TTT L => F 1 671 224 CCA => CTA P => L 1 713 238 GCT => GTT A => V 0.8 1505 502 TCT => TTT S => F 1 1667 556 CCC => CTC P => L 1 2173 725 CTC => TTC L => F 1 ndhG 314 105 ACA => ATA T => I 0.8 petB 617 206 CCA=> CTA P => L 1 psaI 22 8 CCT => TCT P => S 1 28 10 CTT => TTT L => F 0.86 rpl2 596 199 GCG => GTG A => V 0.86 rpl20 308 103 TCA => TTA S => L 0.86 rpoB 338 113 TCT => TTT S => F 1 473 158 TCA => TTA S => L 0.86 551 184 TCA => TTA S => L 1 566 189 TCG => TTG S => L 1 593 198 GCT => GTT A => V 0.86 2000 667 TCT => TTT S => F 1 2426 809 TCA => TTA S => L 0.86 rpoC2 2290 764 CGG => TGG R => W 1 3202 1068 CTT => TTT L => F 0.86 3719 1240 TCA => TTA S => L 0.86 rps2 248 83 TCA => TTA S => L 1 266 89 ACA => ATA T => I 0.86 rps14 80 27 TCA => TTA S => L 1


### 3.2. Repeat Analysis

#### 3.2.1. Long Repeats

REPuter programme was used to identify the repeat sequence in the chloroplast genome ofJ. flava using default settings; the result obtained showed that all the four types of repeats (palindromic, forward, reverse, and complement) were present in the genome (Table 6). The analysis showed 16 palindromic repeats, 23 forward repeats, 9 reverse repeats, and only one complement repeat (Table 6). Majority of the repeats size is between 20 and 29bp (46.93%), followed by 10-19bp (40.81%), whereas 30-39bp and 40-49bp are the least with 8.16% and 4.08%, respectively. In total, there are 49 repeats in the chloroplast genome ofJ.flava. In the first location the intergenic spacer harbored 65.30% of the repeats; this has also been reported in cp genome ofFagopyrum dibotrys [59]. tRNA contained 8 repeats (16.32%); the remaining 9 repeats (18.36%) are located in the protein coding genes specificallyatpB,psaB,ndhC,ycf1, andycf2. Within the protein coding genesycf2 contained 1 reverse and 2 palindromic and forward repeats.Table 6
Repeat sequences present in theJ. flava chloroplast genome.
S/N Repeat Size Repeat Position 1 Repeat Type Repeat Location 1 Repeat Position 2 Repeat Location 2 E-Value 1 41 97201 F IGS 117423 IGS 1.32E-15 2 41 117423 P IGS 136591 IGS 1.32E-15 3 39 42922 F ycf3 Intron 97203 IGS 2.12E-14 4 39 42922 F ycf3 Intron 117425 IGS 2.12E-14 5 39 42922 P ycf3 Intron 136591 IGS 2.12E-14 6 30 7623 P IGS-trnS-GCU 44420 IGS-trnSGCA 5.55E-09 7 26 86664 P ycf2 86664 ycf2 1.42E-06 8 26 86664 F ycf2 147143 ycf2 1.42E-06 9 26 120785 F IGS 120808 IGS 1.42E-06 10 26 147143 P ycf2 147143 ycf2 1.42E-06 11 25 118049 F ndhA Intron 118074 ndhA Intron 5.69E-06 12 24 58638 F IGS 58661 IGS 2.27E-05 13 23 31057 R IGS 31057 IGS 9.10E-05 14 22 9120 F trnG-GCC 35746 trnG-UCC 3.64E-04 15 21 7629 F trnS-GCU-IGS 34812 trnS-UGA 1.46E-03 16 21 11762 R IGS 11762 IGS 1.46E-03 17 21 34812 P trnS-UGA 44423 trnS-GGA 1.46E-03 18 21 45956 R trnT-UGU 45956 trnT-UGU 1.46E-03 19 21 106777 F IGS 123747 ycf1 1.46E-03 20 21 123747 P ycf1 127035 IGS 1.46E-03 21 20 29704 R IGS 29704 IGS 5.82E-03 22 20 30622 P IGS 30622 IGS 5.82E-03 23 20 49490 R ndhC 49490 ndhC 5.82E-03 24 20 51290 P trnV-UAC 101924 trnA-UGC 5.82E-03 25 20 51290 F trnV-UAC 131889 trnA-UGC 5.82E-03 26 20 92988 F IGS 93005 IGS 5.82E-03 27 20 92988 P IGS 140808 IGS 5.82E-03 28 20 93005 P IGS 140825 IGS 5.82E-03 29 20 140808 F IGS 140825 IGS 5.82E-03 30 19 11760 P IGS 80056 IGS 2.33E-02 31 19 43979 F IGS 43997 IGS 2.33E-02 32 19 58511 R IGS 58511 IGS 2.33E-02 33 19 108471 R ycf2 108471 ycf2 2.33E-02 34 19 120905 R IGS 120905 IGS 2.33E-02 35 19 123419 F ycf1 123443 ycf1 2.33E-02 36 18 235 P IGS 269 IGS 9.32E-02 37 18 5052 F IGS 5069 IGS 9.32E-02 38 18 7694 F trnS-GCU 34882 trnS-UGAIGS 9.32E-02 39 18 7758 P IGS 67000 IGS 9.32E-02 40 18 11761 F atpF Intron 54061 IGS 9.32E-02 41 18 21857 F rpoC1 Intron 27884 IGS 9.32E-02 42 18 30254 P IGS 30254 IGS 9.32E-02 43 18 30895 R IGS 30895 IGS 9.32E-02 44 18 37376 F psaB 39591 psaA 9.32E-02 45 18 37925 F psaB 40149 psaA 9.32E-02 46 18 46008 P IGS 46008 IGS 9.32E-02 47 18 50038 C IGS 80055 IGS 9.32E-02 48 18 53019 F atpB 125148 ycf1 9.32E-02 49 18 54060 F IGS 106779 IGS 9.32E-02We compared the frequency of repeats among four Acanthaceae cp genomes and found that all the types of repeats (palindromic, forward, reverse, and complement) are present in all the genomes (Figure3).S.cusiahas the highest frequency of palindromic repeats (23) whileJ. flava has the lowest with (16).R. breedlovei andS. cusia have the same number of forward repeats 15 each, and number of reverse repeats is the same in the genome ofJ. flava andS. cusia(Figure 3). Complement repeats are found to be the less type of repeat in the genome withE. longzhouensis andJ. flava having 1 and the other two species having 3 each.Figure 3
Number of different repeats in four chloroplast genome of Acanthaceae. P= palindromic, F = forward, R=reverse, and C= complement.


#### 3.2.2. Simple Sequence Repeats (SSRs)

There are short repeats of nucleotide series (1-6 bp) that are dispensed all through genome called microsatellites (SSRs). This short repeat in plastid genome is passed from a single parent. As a result, they are used as molecular indicators in developmental studies such as genetic heterogeneity and also contribute in recognition of species [60–62]. The sums of 98 microsatellites were found in plastid genome ofJ. flava in this study (Table 7). Majority of SSRs in the cp genome are mononucleotide (83.67%) of which most are poly T and A (Figure 4). Poly T (polythymine) constituted 58.53% whereas poly A (polyadenine) 40.24%; this is consistent in previous studies [63, 64]. Only a single poly C (polycytosine) 1.21% is present in the genome whereas 2 poly G (polyguanine) is found in the genome. Among the dinucleotide only AT/AT is found in the genome. Reflecting series complementary, three trinucleotide AAG/CTT, ATC/ATG, and AAT/ATT, five tetra AAAC/GTTT, AAAG/CTTT, AAAT/ATTT, AATC/ATTG, and AATT/AATT, and two penta AATAC/ATTGT and AATTC/AATTG were discovered in the genome while no hexanucleotide repeat is present (Figure 4). The intergenic spacer region harbored most of the microsatellite (62.24%) than the coding region (33.67%) (Figure 5). Most but not all the repeats (70.40%) were detected in the LSC region and the SSC region incorporates the least number of repeats in the genome.Table 7
Simple sequence repeats in the chloroplast genome ofJ. flava.
Repeat Length (bp) Number Start position A 8 17 1, 941; 4,077; 7, 866; 11, 688; 13, 037; 17, 946; 41, 684; 43, 752; 51, 259; 54, 200; 68, 846; 66, 730; 95, 631; 110, 063; 111, 010; 113, 860; 118, 261; 150, 742; 150, 809 11 3 7, 592; 14, 787; 15, 599 10 3 14, 665; 21, 867; 28, 269; 9 8 27, 894; 43, 551; 45, 670; 63, 290; 88, 481; 114, 354; 132, 510; 150, 784 14 1 80, 057; 13 1 127, 037;  C 12  4, 487  G 8 2 57, 723; 74, 644  T 8 25 7, 383; 25, 533; 32, 688; 59, 519; 60, 372; 65, 495; 66, 271; 66, 377; 68, 096; 68, 556; 74, 497; 81, 050; 82, 410; 82, 710; 83, 018; 83, 085; 109, 106; 109, 136; 112, 173; 112, 563; 113, 364; 123, 499; 123, 541; 125, 326; 138, 196 10 4 9, 482; 30, 515; 53, 621; 123, 624; 15 1 11, 766; 9 15 12, 488; 15, 906; 17, 805; 70, 662; 74, 580; 75, 746; 81, 096; 83,050; 101, 316; 109, 159; 121, 849; 123, 004; 123, 606; 123, 638; 145, 345 11 3 30, 883; 31, 611; 123, 292; 12 3 35, 533; 77, 108; 124, 143 14 2 50, 040; 54, 066; 13 2 106, 785; 123, 755  AT 6 1 7, 215 5 1 20, 206  TA 5 2 19, 175; 30, 628  TTC 4 1 34, 429  TAT 4 1 84991  TGA 4 1 90, 256  TCT 5 1 124, 548  ATC 4 1 143, 566  ATA 4 1 148, 832  TAA 4 1 62, 980  TTTC 3 1 5, 222  ATTG 3 1 5, 410  ATAA 3 1 58, 759  TAAA 4 1 66, 121  AAAC 3 1 67, 128  AATA 3 1 112, 973  AATC 3 1 118, 083  AATT 3 1 122, 503  CAATA 3 1 30, 293Figure 4
Frequency of different SSR motifs in different repeat types inJ. flava chloroplast genome.Figure 5
Number of SSR types in complete genome, protein coding regions, and noncoding genes.The frequency of SSR among the cp genome of the four species was also compared (Figure6); the comparison showed that mononucleotide occurs more frequently across all the genomes.E. longzhouensis had the highest number of mononucleotides and pentanucleotides with 115 and 9, respectively, but had the lowest number of tetranucleotides with 2. Pentanucleotide is not present in cp genome ofR. breedlovei while possessing hexanucleotide which is not present in the remaining 3 species.Figure 6
Number of different SSR types in the four chloroplast genome of Acanthaceae.


### 3.3. Comparative Analysis ofJ. flava Chloroplast to Other Acanthaceae Genomes

The complete chloroplast genome ofJ.flava was compared with three chloroplast genomes of Acanthaceae available in the Genbank, namely,R. breedlovei,S. cusia, andE.longzhouensis. To examine the degree of DNA sequence divergence among the species of Acanthaceae chloroplast genome, the programme mVISTA was used to align the sequences using annotation of Justicia flava as reference. The result of the alignment showed that the genomes highly conserved with some degree of divergence. The inverted repeat regions are more conserved than the single copy regions, the large single copy and small single copy; on the other hand the protein coding genes were found to be more conserved than the noncoding region, particularly the intergeneric spacer. The nonprotein coding regions that showed high rate of divergence across the genome aretrnH-GUG-psbA,rps16-trnQ,trnC-petN,accD-psaI,clpP intron,trnL-trnF,rps15-ycf1,rps12-trnV, andtrnL-trnA among others (Figure 7). For the protein coding genes the following genes showed a little sequence variation among the genomesatpE, atpF, rbcL, petA, psbL, petB, and ycf2.Figure 7
Sequence alignment of four chloroplast genomes in the Acanthaceae family performed with mVISTA using annotation ofJ. flavaas reference. The top arrow shows transcription direction, blue colour indicates protein coding, pink colour shows conserved noncoding sequence CNS, and light green indicates tRNAs and rRNAs. The x-axis represents the coordinates in the cp genome while y-axis represents percentage identity within 50-100%.The chloroplast genome of angiosperms is reported to be conserved in terms of structure and size [65]; despite the conserved nature there is slightly variation in size and the boundaries of inverted repeats and single copy regions due to evolutionary events such as expansion and contraction in the genome [66, 67]. The comparisons between IR-LCS and IR-SSC boundaries in the four cp genome of Acanthaceae (Justicia flava, Echinocactus longzhouensis, Ruellia breedlovei, andStrobilanthes cusia) are shown in (Figure 8). The result showed that there is slightly variation among the compared cp genomes (Figure 8); four genes, namely,rps19,ndhF,ycf1, andtrnH, were located in the junction of inverted repeats and single copy region ofJ. flava andE. longzhouensisgenome with slightly variation in number of base pairs in the borders (Figure 8).Two genes,ndhF andycf1, are found in the IRb/SSC border among all the four genomes. The IRb/LCS border ofR.breedlovei is unique by havingycf2 gene with 390 bp in LSC and 6800 bp in IRb whereas Strobilanthes cusia genome also has unique structural variation by having trnH in IRa and IRb. ThendhF was found to have 70 bp, 59 bp, and 44 bp in the IRb region inJ. flava, E. longzhouensis, R. breedlovei, and S. cusia, respectively, whereas thetrnH ofJ. flava andE. longzhouensis starts at exactly IRa/LSC border while the tRNA is 76 bp away the border inR. breedlovei genome.Figure 8
Comparison of the borders of the IR, SSC, and LSC regions among four chloroplast genome of Acanthaceae.


### 3.4. Divergence of Protein Coding Gene Sequence

The rates of synonymous (dS) and nonsynonymous (dN) substitution and dN/dS ratio were calculated to detect the selective pressure among the 78 protein coding genes in the cp genome of four Acanthaceae species. The results showed that the dN/dS ration is less than 1 in most of the paired genes exceptpetB,psaI, andycf1 ofJ. flava vs.S. cusia having 1.52, 1.24, and 1.12, respectively (Figure 9). Two genes are also found to be greater than 1 inJ. flava vs.R. breedlovei atpF,clpP,psaI, andrpl32.petB,rpl16, andpsaIgenes ofJ. flava vs.E. longzhouensis are greater than 1 as well. This indicates that most of the genes were under negative selection; only few undergo positive selection. The synonymous (dS) values in all the genes range from 0.02 to 0.44 (Figure 9). The genes,atpH, petG, petN, psaC, psaJ, psbE, psbF, psbI, psbT, andrps12,showed no nonsynonymous change occurs in the cp genome of the four species of Acanthaceae.Figure 9
The synonymous (dS) and dN/dS ration values of 78 protein coding genes from four Acanthaceae cp genomes (Jf:J.flava; Rb:R. breedlovei;El:E. longzhouensis).


### 3.5. Phylogenetic Analysis

The phylogenetic relationship within the four species of Acanthaceae was reconstructed using the complete chloroplast genome. The tree from the Bayesian Inference and Maximum Parsimony is congruent with strong support in all the nodes PP, 1.00, and MP, 100. All the species of Acanthaceae sampled clustered in one clade with strong support (Figure10), as reported by [68]. The result showed that the tribe Justicieae is sister to Ruellieae; this relationship has also been reported by [69] and they should be regarded as independent tribe not as Justiceae under the tribe Ruellieae as proposed by [11]Figure 10
Phylogenetic tree reconstruction of 9 taxa based on the complete chloroplast genome using Bayesian Inference (BI) and Maximum Parsimony (MP) methods showing relationship within the four species of Acanthaceae. The numbers in the branch nodes represent bootstrap percentage (BP)/posterior probability (PP).


## 3.1. Characteristics ofJ. flava Chloroplast Genome

The complete chloroplast genome ofJ.flava is a circular molecule and has quadripartite structure; the genome was found to have 150, 888bp in length. The genome is divided into four regions, namely, large single copy (LSC), small single copy (SSC), and two inverted repeats (IRa and IRb). The coding region is 98, 671bp in length and constitutes 65.39% of the genome; the rest of 52, 217 bp is the intergenic spacer region including intron (34.60%). The LSC and SSC regions possessed 82, 995bp and 16, 893bp, respectively, the inverted repeats IRa and IRb have 25, 500bp and are separated by SSC region (Figure 1 and Table 1). The organization and structure of theJ.flava cp genome are similar to other sequenced Acanthaceae cp genomes [47, 48]. The percentage of occurrence of AT and GC content in the genome showed that the LSC, SSC, IRA, and IRB regions possessed 63.8% and 36.2%, 67.8% and 32.2%, 56.7% and 43.4%, and 56.7% and 3.4, respectively. The whole chloroplast genome has AT content of 61.8% and GC content of 38.2%; this is similar to cp genome ofStrobilanthes cusia [49]. The GC content in the inverted repeat is found to be higher than the single copy regions both the LSC and SSC.Table 1
Base composition in theJ. flavachloroplast genome.
Region  T(U) (%) C (%) A (%) G (%) Total (bp) cp genome  31 19 31 19 150888 LSC  32 19 31 18 82995 SSC  34 17 34 15 16893 IRA  28 23 28 21 25500 IRB  28 21 28 23 25500 1st Position 31 20 30 19 50296 2nd Position 31 19 31 19 50296 3rd Position 31 20 30 19 50296Figure 1
Gene map of theJ. flava chloroplast genome. Genes outside the circles are transcribed in counterclockwise direction and those inside in clockwise direction. Known functional genes are indicated in the coloured bar. The GC and AT content are denoted by the dark grey and light grey colour in the inner circle, respectively. LSC indicates large single copy; SSC, indicates small single copy, and IR indicates inverted repeat.The result of the genes annotation in the chloroplast genome ofJ. flava revealed a total of 132 genes among which 113 are unique; the remaining 19 are duplicated in the inverted region. The genome harbored 80 protein coding genes, 30 tRNA genes, and 4 rRNA genes (Figure 1 and Table 2). The numbers and orientation of the genes in the cp genome are the same as other cp genomes of Acanthaceae [47, 48]. The inverted repeat region contained eight protein coding genes, seven tRNA, and four rRNA while in the single copy region, the LSC contained 61 protein coding genes and 22 tRNA genes; the rest of 12 protein coding genes and 1 tRNA are located within the SSC region. Almost all the protein coding genes start with the ATG codon that code for methionine whereas some of the genes start with codon like ATC, GTG and ACG; this is common in most flowering plant (angiosperms) chloroplast genome [49–51].Table 2
Genes present in the chloroplast genome ofJ. flava.
Category Group of genes Name of genes RNA genes ribosomal RNA genes (rRNA) rrn5, rrn4.5, rrn16, rrn23  Transfer RNA genes (tRNA) trnH-GUG, trnK-UUU +, trnQ-UUG, trnS-GCU, trnS-CGA+, trnR-UCU,trnC-GCA, trnD-GUC, trnY-GUA, trnE-UUC, trnT-GGU, trnS-UGA, trnfM-CAU, trnG-GCC, trnS-GGA, trnL-UAA+, trnT-UGU, trnF-GAA, trnV-UAC+, trnM-CAU, trnW-CCA, trnP-UGG, trnI-CAUa, trnL-CAAa,trnV-GACa, trnI-GAU+,a,trnA-UGC+,a, trnR-ACGa, trnN-GUUa, trnL-UAG,  Ribosomal proteins Small subunit of ribosome rps2, rps3, rps4, r p s 7 a, rps8, rps11,rps12a, rps14, rps15,rps,16+, rps18,rps19  Transcription Large subunit of ribosome r p l 2 + , a, rpl14, rpl16, rpl20, rpl22,rpl23a, rpl32, rpl33, rpl36  DNA dependent RNA polymerase rpoA, rpoB, rpoC1 +, rpoC2  Protein genes Photosystem I psaA, psaB, psaC, psaI,psaJ,ycf3 ++  Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ  Subunit of cytochrome petA, petB, petD, petG, petL, petN  Subunit of synthase atpA, atpB, atpE, atpF +, atpH, atpI  Large subunit of rubisco rbcL  NADH dehydrogenase ndhA +,ndhB+a, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK  ATP dependent protease subunit P clpP ++  Chloroplast envelope membrane protein cemA  Other genes Maturase matK  Subunit acetyl-coA carboxylase accD  C-type cytochrome synthesis ccsA  Hypothetical proteins y c f 2 a, ycf4, ycf15a  Component of TIC complex y c f 1 a G e n e   + with one intron. Gene++ with two intron. a Gene with copies.TheJ. flava chloroplast genome is found to contain intron in some of the protein coding and tRNA genes, like other chloroplast genomes of angiosperms [49, 50]. There are 14 genes that contain intron out of the 113 different genes (Table 3); among the 14 genes 8 are protein coding genes while the remaining six are tRNA genes (Table 3). Four genes that have the intron, namely,rpl2,ndhB,trnI-GAU,andtrnA-UGCare located in the IR region while the remaining 12 are located in the LSC region. Only two genesycf3 andclpP have two introns, the other 12 have only one intron, and this is also seen inS. cusia [49]. The tRNA,trnK-UUUhas the longest intron of 2460 bp (Table 3); this is as a result of position of thematK gene in the intron.Table 3
Genes with intron theJ. flava chloroplast genome and length of introns and exons.
Gene Location Exon I (bp) Intron I (bp) Exon II (bp) Intron II (bp) Exon III (bp) atpF LSC 143 659 467 rpoC1 LSC 443 769 1628 ycf3 LSC 128 682 227 721 152 clpP LSC 68 763 293 635 227 rpl2 IR 392 664 434 ndhB IR 776 680 755 ndhA SSC 551 954 539 trnK-UU LSC 36 2460 37 trnS-CGA LSC 31 667 59 trnL-UAA LSC 36 501 49 trnV-UAC LSC 37 593 36 trnI-GAU IR 41 938 34 trnA-UGC IR 37 818 34The codon usage bias in the plastome was computed using the protein coding genes and tRNA genes nucleotide sequences 89, 377bp. The relative synonymous codon usage of each codon in the genome is presented in (Table4); the result revealed that all the genes are encoded by 29, 790 codons. Codons, coding for the amino acids Leucine, are the most frequent codons 3,329 (11.7%) (Figure 2), similar to that ofAilanthus altissima [52], whereas codons coding for Trp are the least 570 (1.91%) in the genome. G- and C-ending are found to be more frequent than their counterpart A and T; this is not the case in other plastomes sequences [53–55]. The result of the analysis (Table 4) showed that codon usage bias is low in the chloroplast genome ofJ.flava. The RSCU values of 29 codons were >1 and all of them have A/T ending while for 30 codons were <1 and are all of G/C ending. Only two amino acids Tryptophan and Methionine have RSCU value of 1; therefore they are the only amino acids with no codon bias.Table 4
Codon-anticodon recognition patterns and codon usage of theJ. flava chloroplast genome.
Codon Amino Acid RSCU tRNA Codon Amino Acid RSCU tRNA UUU Phe 1.18 trnF-GAA UAU Tyr 1.38 trnY-GUA UUC Phe 0.82  UAC Tyr 0.62 UUA Leu 1.31 trnL-UAA UAA Stop 1.01 UUG Leu 1.31 trnL-CAA UAG Stop 1.03 CUU Leu 1.23 trnL-UAG CAU His 1.26 trnH-GUG CUC Leu 0.65  CAC His 0.74 CUA Leu 0.92  CAA Gln 1.37 trnQ-UUG CUG Leu 0.59  CAG Gln 0.63 AUU Ile 1.22 trnI-GAU AAU Asn 1.34 trnG-GUU AUC Ile 0.82  AAC Asn 0.66 AUA Ile 0.95 trnI-CAU AAA Lys 1.29 trnK-UUU AUG Met 1 trnM-CAU AAG Lys 0.71 GUU Val 1.45 trnV-GAC GAU Asp 1.44 trnD-GUC GUC Val 0.65  GAC Asp 0.56 GUG Val 0.74  GAA Glu 1.38 trnE-UUC GUA Val 1.16 trnV-UAC GAG Glu 0.62 UCU Ser 1.46 trnS-GGA UGU Cys 1.14 trnC-GCA UCC Ser 0.95  UGC Cys 0.86 UCG Ser 0.76  UGA Stop 0.96 UCA Ser 1.33 trnS-UGA UGG Trp 1 trnW-CCA CCU Pro 1.21 trnP-UGG CGU Arg 0.84 trnR-ACG CCC Pro 0.87  CGC Arg 0.35 trnR-UCU CCA Pro 1.13  CGA Arg 1.16 CCG Pro 0.79  CGG Arg 0.77 ACU Thr 1.22  AGA Arg 1.8 ACC Thr 0.87  AGG Arg 1.07 ACG Thr 0.72 trnT-GGU AGU Ser 0.92 trnS-GCU ACA Thr 1.2 trnT-UGU AGC Ser 0.58 GCU Ala 1.33 trnA-UGC GGU Gly 1.18 trnG-GCC GCC Ala 0.76  GGC Gly 0.54 GCA Ala 1.18  GGA Gly 1.28 GCG Ala 0.72  GGG Gly 0.99 trnG-UCCFigure 2
Amino acids frequencies inJ. flava chloroplast genome protein coding sequences.The RNA editing site in theJ. flavachloroplast genomes was predicted using the program PREP suite; all the analysis was done using the first codon position of the first nucleotide. The result (Table 5) shows that the majority of the conversion in the codon positions is from the amino acid Serine to Leucine (Table 5). In all, the programme revealed 61 editing sites in the genome; the editing sites are distributed among 18 protein coding genes. As reported in previous researches [56–58] thendhB gene has the highest number of editing sites (9 sites) followed byrpoB (7 site) andndhG,atpF,rpl2,rpl20, andrps2 have the least 1 site each. Among all the conversion in the RNA editing site, one site changed the amino acid from apolar group to polar group (Proline to Serine). The following genes do not have RNA predicting site in their first codon of the first nucleotidesatpB, ccsA, clpP, ndhC, ndhE psaB, petD, petG, rpoA, and petL, among others.Table 5
Predicted RNA editing site in theJ. flava chloroplast genome.
gene Nucleotide Position Amino Acid Position Codon Conversion Amino Acid Conversion Score accD 800 267 TCG => TTG S => L 0.8 844 282 CCC => TCC P => S 0.8 atpA 776 259 ACC => ATC T => I 1 914 305 TCA => TTA S => L 1 1270 424 CCC => TCC P => S 1 atpF 92 31 CCA => CTA P => L 0.86 atpI 404 135 GCT => GTT A => V 1 620 207 TCA => TTA S => L 1 matK 640 214 CAT => TAT H => Y 1 1249 417 CAT => TAT H => Y 1 ndhA 326 109 ACT => ATT T => I 1 566 189 TCA => TTA S => L 1 922 308 CTT => TTT L => F 1 ndhB 149 50 TCA => TTA S => L 1 467 156 CCA => CTA P => L 1 586 196 CAT => TAT H => Y 1 737 246 CCA => CTA P => L 1 746 249 TCT => TTT S => F 1 830 277 TCA => TTA S => L 1 836 279 TCA => TTA S => L 1 1292 431 TCC => TTC S => F 1 1481 494 CCA => CTA P => L 1 ndhD 2 1 ACG => ATG T => M 1 32 11 GCA => GTA A => V 1 878 293 TCA => TTA S => L 1 1445 482 GCT => GTT A => V 1 ndhF 124 42 CTT => TTT L => F 1 671 224 CCA => CTA P => L 1 713 238 GCT => GTT A => V 0.8 1505 502 TCT => TTT S => F 1 1667 556 CCC => CTC P => L 1 2173 725 CTC => TTC L => F 1 ndhG 314 105 ACA => ATA T => I 0.8 petB 617 206 CCA=> CTA P => L 1 psaI 22 8 CCT => TCT P => S 1 28 10 CTT => TTT L => F 0.86 rpl2 596 199 GCG => GTG A => V 0.86 rpl20 308 103 TCA => TTA S => L 0.86 rpoB 338 113 TCT => TTT S => F 1 473 158 TCA => TTA S => L 0.86 551 184 TCA => TTA S => L 1 566 189 TCG => TTG S => L 1 593 198 GCT => GTT A => V 0.86 2000 667 TCT => TTT S => F 1 2426 809 TCA => TTA S => L 0.86 rpoC2 2290 764 CGG => TGG R => W 1 3202 1068 CTT => TTT L => F 0.86 3719 1240 TCA => TTA S => L 0.86 rps2 248 83 TCA => TTA S => L 1 266 89 ACA => ATA T => I 0.86 rps14 80 27 TCA => TTA S => L 1


## 3.2. Repeat Analysis

### 3.2.1. Long Repeats

REPuter programme was used to identify the repeat sequence in the chloroplast genome ofJ. flava using default settings; the result obtained showed that all the four types of repeats (palindromic, forward, reverse, and complement) were present in the genome (Table 6). The analysis showed 16 palindromic repeats, 23 forward repeats, 9 reverse repeats, and only one complement repeat (Table 6). Majority of the repeats size is between 20 and 29bp (46.93%), followed by 10-19bp (40.81%), whereas 30-39bp and 40-49bp are the least with 8.16% and 4.08%, respectively. In total, there are 49 repeats in the chloroplast genome ofJ.flava. In the first location the intergenic spacer harbored 65.30% of the repeats; this has also been reported in cp genome ofFagopyrum dibotrys [59]. tRNA contained 8 repeats (16.32%); the remaining 9 repeats (18.36%) are located in the protein coding genes specificallyatpB,psaB,ndhC,ycf1, andycf2. Within the protein coding genesycf2 contained 1 reverse and 2 palindromic and forward repeats.Table 6
Repeat sequences present in theJ. flava chloroplast genome.
S/N Repeat Size Repeat Position 1 Repeat Type Repeat Location 1 Repeat Position 2 Repeat Location 2 E-Value 1 41 97201 F IGS 117423 IGS 1.32E-15 2 41 117423 P IGS 136591 IGS 1.32E-15 3 39 42922 F ycf3 Intron 97203 IGS 2.12E-14 4 39 42922 F ycf3 Intron 117425 IGS 2.12E-14 5 39 42922 P ycf3 Intron 136591 IGS 2.12E-14 6 30 7623 P IGS-trnS-GCU 44420 IGS-trnSGCA 5.55E-09 7 26 86664 P ycf2 86664 ycf2 1.42E-06 8 26 86664 F ycf2 147143 ycf2 1.42E-06 9 26 120785 F IGS 120808 IGS 1.42E-06 10 26 147143 P ycf2 147143 ycf2 1.42E-06 11 25 118049 F ndhA Intron 118074 ndhA Intron 5.69E-06 12 24 58638 F IGS 58661 IGS 2.27E-05 13 23 31057 R IGS 31057 IGS 9.10E-05 14 22 9120 F trnG-GCC 35746 trnG-UCC 3.64E-04 15 21 7629 F trnS-GCU-IGS 34812 trnS-UGA 1.46E-03 16 21 11762 R IGS 11762 IGS 1.46E-03 17 21 34812 P trnS-UGA 44423 trnS-GGA 1.46E-03 18 21 45956 R trnT-UGU 45956 trnT-UGU 1.46E-03 19 21 106777 F IGS 123747 ycf1 1.46E-03 20 21 123747 P ycf1 127035 IGS 1.46E-03 21 20 29704 R IGS 29704 IGS 5.82E-03 22 20 30622 P IGS 30622 IGS 5.82E-03 23 20 49490 R ndhC 49490 ndhC 5.82E-03 24 20 51290 P trnV-UAC 101924 trnA-UGC 5.82E-03 25 20 51290 F trnV-UAC 131889 trnA-UGC 5.82E-03 26 20 92988 F IGS 93005 IGS 5.82E-03 27 20 92988 P IGS 140808 IGS 5.82E-03 28 20 93005 P IGS 140825 IGS 5.82E-03 29 20 140808 F IGS 140825 IGS 5.82E-03 30 19 11760 P IGS 80056 IGS 2.33E-02 31 19 43979 F IGS 43997 IGS 2.33E-02 32 19 58511 R IGS 58511 IGS 2.33E-02 33 19 108471 R ycf2 108471 ycf2 2.33E-02 34 19 120905 R IGS 120905 IGS 2.33E-02 35 19 123419 F ycf1 123443 ycf1 2.33E-02 36 18 235 P IGS 269 IGS 9.32E-02 37 18 5052 F IGS 5069 IGS 9.32E-02 38 18 7694 F trnS-GCU 34882 trnS-UGAIGS 9.32E-02 39 18 7758 P IGS 67000 IGS 9.32E-02 40 18 11761 F atpF Intron 54061 IGS 9.32E-02 41 18 21857 F rpoC1 Intron 27884 IGS 9.32E-02 42 18 30254 P IGS 30254 IGS 9.32E-02 43 18 30895 R IGS 30895 IGS 9.32E-02 44 18 37376 F psaB 39591 psaA 9.32E-02 45 18 37925 F psaB 40149 psaA 9.32E-02 46 18 46008 P IGS 46008 IGS 9.32E-02 47 18 50038 C IGS 80055 IGS 9.32E-02 48 18 53019 F atpB 125148 ycf1 9.32E-02 49 18 54060 F IGS 106779 IGS 9.32E-02We compared the frequency of repeats among four Acanthaceae cp genomes and found that all the types of repeats (palindromic, forward, reverse, and complement) are present in all the genomes (Figure3).S.cusiahas the highest frequency of palindromic repeats (23) whileJ. flava has the lowest with (16).R. breedlovei andS. cusia have the same number of forward repeats 15 each, and number of reverse repeats is the same in the genome ofJ. flava andS. cusia(Figure 3). Complement repeats are found to be the less type of repeat in the genome withE. longzhouensis andJ. flava having 1 and the other two species having 3 each.Figure 3
Number of different repeats in four chloroplast genome of Acanthaceae. P= palindromic, F = forward, R=reverse, and C= complement.


### 3.2.2. Simple Sequence Repeats (SSRs)

There are short repeats of nucleotide series (1-6 bp) that are dispensed all through genome called microsatellites (SSRs). This short repeat in plastid genome is passed from a single parent. As a result, they are used as molecular indicators in developmental studies such as genetic heterogeneity and also contribute in recognition of species [60–62]. The sums of 98 microsatellites were found in plastid genome ofJ. flava in this study (Table 7). Majority of SSRs in the cp genome are mononucleotide (83.67%) of which most are poly T and A (Figure 4). Poly T (polythymine) constituted 58.53% whereas poly A (polyadenine) 40.24%; this is consistent in previous studies [63, 64]. Only a single poly C (polycytosine) 1.21% is present in the genome whereas 2 poly G (polyguanine) is found in the genome. Among the dinucleotide only AT/AT is found in the genome. Reflecting series complementary, three trinucleotide AAG/CTT, ATC/ATG, and AAT/ATT, five tetra AAAC/GTTT, AAAG/CTTT, AAAT/ATTT, AATC/ATTG, and AATT/AATT, and two penta AATAC/ATTGT and AATTC/AATTG were discovered in the genome while no hexanucleotide repeat is present (Figure 4). The intergenic spacer region harbored most of the microsatellite (62.24%) than the coding region (33.67%) (Figure 5). Most but not all the repeats (70.40%) were detected in the LSC region and the SSC region incorporates the least number of repeats in the genome.Table 7
Simple sequence repeats in the chloroplast genome ofJ. flava.
Repeat Length (bp) Number Start position A 8 17 1, 941; 4,077; 7, 866; 11, 688; 13, 037; 17, 946; 41, 684; 43, 752; 51, 259; 54, 200; 68, 846; 66, 730; 95, 631; 110, 063; 111, 010; 113, 860; 118, 261; 150, 742; 150, 809 11 3 7, 592; 14, 787; 15, 599 10 3 14, 665; 21, 867; 28, 269; 9 8 27, 894; 43, 551; 45, 670; 63, 290; 88, 481; 114, 354; 132, 510; 150, 784 14 1 80, 057; 13 1 127, 037;  C 12  4, 487  G 8 2 57, 723; 74, 644  T 8 25 7, 383; 25, 533; 32, 688; 59, 519; 60, 372; 65, 495; 66, 271; 66, 377; 68, 096; 68, 556; 74, 497; 81, 050; 82, 410; 82, 710; 83, 018; 83, 085; 109, 106; 109, 136; 112, 173; 112, 563; 113, 364; 123, 499; 123, 541; 125, 326; 138, 196 10 4 9, 482; 30, 515; 53, 621; 123, 624; 15 1 11, 766; 9 15 12, 488; 15, 906; 17, 805; 70, 662; 74, 580; 75, 746; 81, 096; 83,050; 101, 316; 109, 159; 121, 849; 123, 004; 123, 606; 123, 638; 145, 345 11 3 30, 883; 31, 611; 123, 292; 12 3 35, 533; 77, 108; 124, 143 14 2 50, 040; 54, 066; 13 2 106, 785; 123, 755  AT 6 1 7, 215 5 1 20, 206  TA 5 2 19, 175; 30, 628  TTC 4 1 34, 429  TAT 4 1 84991  TGA 4 1 90, 256  TCT 5 1 124, 548  ATC 4 1 143, 566  ATA 4 1 148, 832  TAA 4 1 62, 980  TTTC 3 1 5, 222  ATTG 3 1 5, 410  ATAA 3 1 58, 759  TAAA 4 1 66, 121  AAAC 3 1 67, 128  AATA 3 1 112, 973  AATC 3 1 118, 083  AATT 3 1 122, 503  CAATA 3 1 30, 293Figure 4
Frequency of different SSR motifs in different repeat types inJ. flava chloroplast genome.Figure 5
Number of SSR types in complete genome, protein coding regions, and noncoding genes.The frequency of SSR among the cp genome of the four species was also compared (Figure6); the comparison showed that mononucleotide occurs more frequently across all the genomes.E. longzhouensis had the highest number of mononucleotides and pentanucleotides with 115 and 9, respectively, but had the lowest number of tetranucleotides with 2. Pentanucleotide is not present in cp genome ofR. breedlovei while possessing hexanucleotide which is not present in the remaining 3 species.Figure 6
Number of different SSR types in the four chloroplast genome of Acanthaceae.


## 3.2.1. Long Repeats

REPuter programme was used to identify the repeat sequence in the chloroplast genome ofJ. flava using default settings; the result obtained showed that all the four types of repeats (palindromic, forward, reverse, and complement) were present in the genome (Table 6). The analysis showed 16 palindromic repeats, 23 forward repeats, 9 reverse repeats, and only one complement repeat (Table 6). Majority of the repeats size is between 20 and 29bp (46.93%), followed by 10-19bp (40.81%), whereas 30-39bp and 40-49bp are the least with 8.16% and 4.08%, respectively. In total, there are 49 repeats in the chloroplast genome ofJ.flava. In the first location the intergenic spacer harbored 65.30% of the repeats; this has also been reported in cp genome ofFagopyrum dibotrys [59]. tRNA contained 8 repeats (16.32%); the remaining 9 repeats (18.36%) are located in the protein coding genes specificallyatpB,psaB,ndhC,ycf1, andycf2. Within the protein coding genesycf2 contained 1 reverse and 2 palindromic and forward repeats.Table 6
Repeat sequences present in theJ. flava chloroplast genome.
S/N Repeat Size Repeat Position 1 Repeat Type Repeat Location 1 Repeat Position 2 Repeat Location 2 E-Value 1 41 97201 F IGS 117423 IGS 1.32E-15 2 41 117423 P IGS 136591 IGS 1.32E-15 3 39 42922 F ycf3 Intron 97203 IGS 2.12E-14 4 39 42922 F ycf3 Intron 117425 IGS 2.12E-14 5 39 42922 P ycf3 Intron 136591 IGS 2.12E-14 6 30 7623 P IGS-trnS-GCU 44420 IGS-trnSGCA 5.55E-09 7 26 86664 P ycf2 86664 ycf2 1.42E-06 8 26 86664 F ycf2 147143 ycf2 1.42E-06 9 26 120785 F IGS 120808 IGS 1.42E-06 10 26 147143 P ycf2 147143 ycf2 1.42E-06 11 25 118049 F ndhA Intron 118074 ndhA Intron 5.69E-06 12 24 58638 F IGS 58661 IGS 2.27E-05 13 23 31057 R IGS 31057 IGS 9.10E-05 14 22 9120 F trnG-GCC 35746 trnG-UCC 3.64E-04 15 21 7629 F trnS-GCU-IGS 34812 trnS-UGA 1.46E-03 16 21 11762 R IGS 11762 IGS 1.46E-03 17 21 34812 P trnS-UGA 44423 trnS-GGA 1.46E-03 18 21 45956 R trnT-UGU 45956 trnT-UGU 1.46E-03 19 21 106777 F IGS 123747 ycf1 1.46E-03 20 21 123747 P ycf1 127035 IGS 1.46E-03 21 20 29704 R IGS 29704 IGS 5.82E-03 22 20 30622 P IGS 30622 IGS 5.82E-03 23 20 49490 R ndhC 49490 ndhC 5.82E-03 24 20 51290 P trnV-UAC 101924 trnA-UGC 5.82E-03 25 20 51290 F trnV-UAC 131889 trnA-UGC 5.82E-03 26 20 92988 F IGS 93005 IGS 5.82E-03 27 20 92988 P IGS 140808 IGS 5.82E-03 28 20 93005 P IGS 140825 IGS 5.82E-03 29 20 140808 F IGS 140825 IGS 5.82E-03 30 19 11760 P IGS 80056 IGS 2.33E-02 31 19 43979 F IGS 43997 IGS 2.33E-02 32 19 58511 R IGS 58511 IGS 2.33E-02 33 19 108471 R ycf2 108471 ycf2 2.33E-02 34 19 120905 R IGS 120905 IGS 2.33E-02 35 19 123419 F ycf1 123443 ycf1 2.33E-02 36 18 235 P IGS 269 IGS 9.32E-02 37 18 5052 F IGS 5069 IGS 9.32E-02 38 18 7694 F trnS-GCU 34882 trnS-UGAIGS 9.32E-02 39 18 7758 P IGS 67000 IGS 9.32E-02 40 18 11761 F atpF Intron 54061 IGS 9.32E-02 41 18 21857 F rpoC1 Intron 27884 IGS 9.32E-02 42 18 30254 P IGS 30254 IGS 9.32E-02 43 18 30895 R IGS 30895 IGS 9.32E-02 44 18 37376 F psaB 39591 psaA 9.32E-02 45 18 37925 F psaB 40149 psaA 9.32E-02 46 18 46008 P IGS 46008 IGS 9.32E-02 47 18 50038 C IGS 80055 IGS 9.32E-02 48 18 53019 F atpB 125148 ycf1 9.32E-02 49 18 54060 F IGS 106779 IGS 9.32E-02We compared the frequency of repeats among four Acanthaceae cp genomes and found that all the types of repeats (palindromic, forward, reverse, and complement) are present in all the genomes (Figure3).S.cusiahas the highest frequency of palindromic repeats (23) whileJ. flava has the lowest with (16).R. breedlovei andS. cusia have the same number of forward repeats 15 each, and number of reverse repeats is the same in the genome ofJ. flava andS. cusia(Figure 3). Complement repeats are found to be the less type of repeat in the genome withE. longzhouensis andJ. flava having 1 and the other two species having 3 each.Figure 3
Number of different repeats in four chloroplast genome of Acanthaceae. P= palindromic, F = forward, R=reverse, and C= complement.


## 3.2.2. Simple Sequence Repeats (SSRs)

There are short repeats of nucleotide series (1-6 bp) that are dispensed all through genome called microsatellites (SSRs). This short repeat in plastid genome is passed from a single parent. As a result, they are used as molecular indicators in developmental studies such as genetic heterogeneity and also contribute in recognition of species [60–62]. The sums of 98 microsatellites were found in plastid genome ofJ. flava in this study (Table 7). Majority of SSRs in the cp genome are mononucleotide (83.67%) of which most are poly T and A (Figure 4). Poly T (polythymine) constituted 58.53% whereas poly A (polyadenine) 40.24%; this is consistent in previous studies [63, 64]. Only a single poly C (polycytosine) 1.21% is present in the genome whereas 2 poly G (polyguanine) is found in the genome. Among the dinucleotide only AT/AT is found in the genome. Reflecting series complementary, three trinucleotide AAG/CTT, ATC/ATG, and AAT/ATT, five tetra AAAC/GTTT, AAAG/CTTT, AAAT/ATTT, AATC/ATTG, and AATT/AATT, and two penta AATAC/ATTGT and AATTC/AATTG were discovered in the genome while no hexanucleotide repeat is present (Figure 4). The intergenic spacer region harbored most of the microsatellite (62.24%) than the coding region (33.67%) (Figure 5). Most but not all the repeats (70.40%) were detected in the LSC region and the SSC region incorporates the least number of repeats in the genome.Table 7
Simple sequence repeats in the chloroplast genome ofJ. flava.
Repeat Length (bp) Number Start position A 8 17 1, 941; 4,077; 7, 866; 11, 688; 13, 037; 17, 946; 41, 684; 43, 752; 51, 259; 54, 200; 68, 846; 66, 730; 95, 631; 110, 063; 111, 010; 113, 860; 118, 261; 150, 742; 150, 809 11 3 7, 592; 14, 787; 15, 599 10 3 14, 665; 21, 867; 28, 269; 9 8 27, 894; 43, 551; 45, 670; 63, 290; 88, 481; 114, 354; 132, 510; 150, 784 14 1 80, 057; 13 1 127, 037;  C 12  4, 487  G 8 2 57, 723; 74, 644  T 8 25 7, 383; 25, 533; 32, 688; 59, 519; 60, 372; 65, 495; 66, 271; 66, 377; 68, 096; 68, 556; 74, 497; 81, 050; 82, 410; 82, 710; 83, 018; 83, 085; 109, 106; 109, 136; 112, 173; 112, 563; 113, 364; 123, 499; 123, 541; 125, 326; 138, 196 10 4 9, 482; 30, 515; 53, 621; 123, 624; 15 1 11, 766; 9 15 12, 488; 15, 906; 17, 805; 70, 662; 74, 580; 75, 746; 81, 096; 83,050; 101, 316; 109, 159; 121, 849; 123, 004; 123, 606; 123, 638; 145, 345 11 3 30, 883; 31, 611; 123, 292; 12 3 35, 533; 77, 108; 124, 143 14 2 50, 040; 54, 066; 13 2 106, 785; 123, 755  AT 6 1 7, 215 5 1 20, 206  TA 5 2 19, 175; 30, 628  TTC 4 1 34, 429  TAT 4 1 84991  TGA 4 1 90, 256  TCT 5 1 124, 548  ATC 4 1 143, 566  ATA 4 1 148, 832  TAA 4 1 62, 980  TTTC 3 1 5, 222  ATTG 3 1 5, 410  ATAA 3 1 58, 759  TAAA 4 1 66, 121  AAAC 3 1 67, 128  AATA 3 1 112, 973  AATC 3 1 118, 083  AATT 3 1 122, 503  CAATA 3 1 30, 293Figure 4
Frequency of different SSR motifs in different repeat types inJ. flava chloroplast genome.Figure 5
Number of SSR types in complete genome, protein coding regions, and noncoding genes.The frequency of SSR among the cp genome of the four species was also compared (Figure6); the comparison showed that mononucleotide occurs more frequently across all the genomes.E. longzhouensis had the highest number of mononucleotides and pentanucleotides with 115 and 9, respectively, but had the lowest number of tetranucleotides with 2. Pentanucleotide is not present in cp genome ofR. breedlovei while possessing hexanucleotide which is not present in the remaining 3 species.Figure 6
Number of different SSR types in the four chloroplast genome of Acanthaceae.


## 3.3. Comparative Analysis ofJ. flava Chloroplast to Other Acanthaceae Genomes

The complete chloroplast genome ofJ.flava was compared with three chloroplast genomes of Acanthaceae available in the Genbank, namely,R. breedlovei,S. cusia, andE.longzhouensis. To examine the degree of DNA sequence divergence among the species of Acanthaceae chloroplast genome, the programme mVISTA was used to align the sequences using annotation of Justicia flava as reference. The result of the alignment showed that the genomes highly conserved with some degree of divergence. The inverted repeat regions are more conserved than the single copy regions, the large single copy and small single copy; on the other hand the protein coding genes were found to be more conserved than the noncoding region, particularly the intergeneric spacer. The nonprotein coding regions that showed high rate of divergence across the genome aretrnH-GUG-psbA,rps16-trnQ,trnC-petN,accD-psaI,clpP intron,trnL-trnF,rps15-ycf1,rps12-trnV, andtrnL-trnA among others (Figure 7). For the protein coding genes the following genes showed a little sequence variation among the genomesatpE, atpF, rbcL, petA, psbL, petB, and ycf2.Figure 7
Sequence alignment of four chloroplast genomes in the Acanthaceae family performed with mVISTA using annotation ofJ. flavaas reference. The top arrow shows transcription direction, blue colour indicates protein coding, pink colour shows conserved noncoding sequence CNS, and light green indicates tRNAs and rRNAs. The x-axis represents the coordinates in the cp genome while y-axis represents percentage identity within 50-100%.The chloroplast genome of angiosperms is reported to be conserved in terms of structure and size [65]; despite the conserved nature there is slightly variation in size and the boundaries of inverted repeats and single copy regions due to evolutionary events such as expansion and contraction in the genome [66, 67]. The comparisons between IR-LCS and IR-SSC boundaries in the four cp genome of Acanthaceae (Justicia flava, Echinocactus longzhouensis, Ruellia breedlovei, andStrobilanthes cusia) are shown in (Figure 8). The result showed that there is slightly variation among the compared cp genomes (Figure 8); four genes, namely,rps19,ndhF,ycf1, andtrnH, were located in the junction of inverted repeats and single copy region ofJ. flava andE. longzhouensisgenome with slightly variation in number of base pairs in the borders (Figure 8).Two genes,ndhF andycf1, are found in the IRb/SSC border among all the four genomes. The IRb/LCS border ofR.breedlovei is unique by havingycf2 gene with 390 bp in LSC and 6800 bp in IRb whereas Strobilanthes cusia genome also has unique structural variation by having trnH in IRa and IRb. ThendhF was found to have 70 bp, 59 bp, and 44 bp in the IRb region inJ. flava, E. longzhouensis, R. breedlovei, and S. cusia, respectively, whereas thetrnH ofJ. flava andE. longzhouensis starts at exactly IRa/LSC border while the tRNA is 76 bp away the border inR. breedlovei genome.Figure 8
Comparison of the borders of the IR, SSC, and LSC regions among four chloroplast genome of Acanthaceae.


## 3.4. Divergence of Protein Coding Gene Sequence

The rates of synonymous (dS) and nonsynonymous (dN) substitution and dN/dS ratio were calculated to detect the selective pressure among the 78 protein coding genes in the cp genome of four Acanthaceae species. The results showed that the dN/dS ration is less than 1 in most of the paired genes exceptpetB,psaI, andycf1 ofJ. flava vs.S. cusia having 1.52, 1.24, and 1.12, respectively (Figure 9). Two genes are also found to be greater than 1 inJ. flava vs.R. breedlovei atpF,clpP,psaI, andrpl32.petB,rpl16, andpsaIgenes ofJ. flava vs.E. longzhouensis are greater than 1 as well. This indicates that most of the genes were under negative selection; only few undergo positive selection. The synonymous (dS) values in all the genes range from 0.02 to 0.44 (Figure 9). The genes,atpH, petG, petN, psaC, psaJ, psbE, psbF, psbI, psbT, andrps12,showed no nonsynonymous change occurs in the cp genome of the four species of Acanthaceae.Figure 9
The synonymous (dS) and dN/dS ration values of 78 protein coding genes from four Acanthaceae cp genomes (Jf:J.flava; Rb:R. breedlovei;El:E. longzhouensis).


## 3.5. Phylogenetic Analysis

The phylogenetic relationship within the four species of Acanthaceae was reconstructed using the complete chloroplast genome. The tree from the Bayesian Inference and Maximum Parsimony is congruent with strong support in all the nodes PP, 1.00, and MP, 100. All the species of Acanthaceae sampled clustered in one clade with strong support (Figure10), as reported by [68]. The result showed that the tribe Justicieae is sister to Ruellieae; this relationship has also been reported by [69] and they should be regarded as independent tribe not as Justiceae under the tribe Ruellieae as proposed by [11]Figure 10
Phylogenetic tree reconstruction of 9 taxa based on the complete chloroplast genome using Bayesian Inference (BI) and Maximum Parsimony (MP) methods showing relationship within the four species of Acanthaceae. The numbers in the branch nodes represent bootstrap percentage (BP)/posterior probability (PP).



---

*Source: 4370258-2019-08-06.xml*