Bangladesh J. Plant Taxon. 31(2): 205-223, 2024 (December) DOI: https://doi.org/10.3329/bjpt.v31i2.78749 © 2024 Bangladesh Association of Plant Taxonomists COMPLETE CHLOROPLAST GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA (L.) DUNAL: COMPARATIVE GENOMICS AND PHYLOGENETIC INSIGHTS NADIA MOHAMMAD ALSUHAIMI1, MOHAMMAD AJMAL ALI1,*, MONA SOLAIMAN ALWAHIBI1, SHEIKH SUNZID AHMED2, M. OLIUR RAHMAN2*, SHANKAR KUMAR PANDEY3, MOHAMED S ELSHIKH1, SAYFI RASHED SAYFI ALSHALLALI4, JOONGKU LEE5 AND SOO-YONG KIM6 1Department of Botany and Microbiology, College of Science, King Saud University, Riyadh-11451, Saudi Arabia 2Department of Botany, Faculty of Biological Sciences, University of Dhaka, Dhaka 1000, Bangladesh 3Department of Botany, SSV College Kahalgaon, Tilka Manjhi Bhagalpur University, Bhagalpur, Bihar, India 4Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia 5Department of Environment and Forest Resources, Chungnam National University, Daejeon, Republic of Korea 6International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea Keywords: Chloroplast genome; Phylogeny; Solanaceae; Nucleotide diversity; Bioinformatics; Withania somnifera (L.) Dunal var. abhaica Nadia, A. Ali & M.S. Alwahibi, var. nov. Abstract This study introduces a novel variety of the highly esteemed medicinal plant Withania somnifera (L.) Dunal from the family Solanaceae. The new variety, Withania somnifera var. abhaica Nadia, A. Ali & M.S. Alwahibi, var. nov., is distributed at high altitudes in the Abha hills of Saudi Arabia. The distinct characteristics of the novel variety of W. somnifera include elliptic-elongated leaves that are thick and semi- succulent in nature, and a fruiting calyx with a bifurcated tip measuring approximately 0.5 mm in length, with each bifurcated tip being botuliform in shape. Using next- generation sequencing (NGS) techniques, we investigated the chloroplast genome of this variety. The complete chloroplast genome of W. somnifera var. abhaica from the Abha region, Saudi Arabia, spans 153,621 bp, with a GC content of 37.7%. It includes a large single-copy (LSC) region of 84,972 bp (GC 35.8%), a small single-copy (SSC) region of 18,400 bp (GC 31.7%), and two inverted repeats (IRs) of 50,249 bp (GC 43.2%). Annotation of the chloroplast genome identified 131 genes, comprising 86 protein- encoding genes (PCGs), eight ribosomal RNA genes, and 37 transfer RNA genes. Repeat analysis identified 38 simple sequence repeats (SSRs) and 50 longer repeat sequences in the plastome. A total of 66 RNA-editing sites were detected across 24 PCGs of the plastome. Comparative genomic studies including synteny analysis, supported and validated the assembled plastome. Nucleotide diversity analysis revealed psbJ, psbA, ndhF, and ycf1 as the most hypervariable barcodes. Phylogenetic analyses suggested the monophyly of the genus Withania. Moreover, the newly sequenced chloroplast genome of W. somnifera var. abhaica was found to be distinct from the typical W. somnifera. *Corresponding authors. Email: alimohammad@ksu.edu.sa ; oliur.bot@du.ac.bd https://doi.org/10.3329/bjpt.v31i2.78749 mailto:alimohammad@ksu.edu.sa mailto:oliur.bot@du.ac.bd 206 ALSUHAIMI et al. Introduction The genus Withania Pauquy, a member of the Solanaceae family, includes 23 species widely distributed across North Africa, West Asia, and Southern Europe (Olmstead et al., 2008). In Saudi Arabia, this genus is represented by Withania somnifera (L.) Dunal, a highly esteemed medicinal plant, commonly known as "Indian Ginseng" (Paul et al., 2021). W. somnifera has been extensively utilized in traditional medicine for centuries, either alone or in combination with other herbs (Visweswari et al., 2013). It thrives in a diverse range of habitats, from desert plains to altitudes as high as 2,995 meters in the Abha region, including Jabal Sawda (Rahman et al., 2004). This herb exhibits a broad spectrum of biological activities due to its diverse phytochemical composition, including anti-inflammatory, antimicrobial, anti-tumor, neuroprotective, cardioprotective, and antidiabetic properties (Dar et al., 2015). Additionally, studies on W. somnifera have demonstrated its efficacy to lower reactive oxygen species, alter mitochondrial operations, control apoptosis, lessen inflammation, as well as improve endothelial function. Due to these pharmacological properties, W. somnifera holds significant potential as a therapeutic option for various clinical disorders, specifically those affecting the nervous system (Kulkarni and Dhir, 2008). W. somnifera var. abhaica is characterized by reduced plant height, elliptic-elongated, thick and puberulous leaves that resemble semi-succulent morphological nature. Under a scanning electron microscope, the tip of the fruiting calyx appeared bifurcated, c. 0.5 mm in length, and each bifurcated tip being botuliform in shape (Fig. 1). These unique morphological characteristics, which differ from the typical features of W. somnifera, have sparked interest in conducting a chloroplast (Cp) genome-based systematic investigation. Unveiling the Cp genome of W. somnifera var. abhaica holds significant importance, even though the Cp genome of W. somnifera is already available (GenBank Accession MK142783) (Mehmood et al., 2020). While the plastome of W. somnifera provides a comprehensive reference for the species, studying the plastome of W. somnifera var. abhaica is essential to clarify taxonomic boundaries and confirm its classification as a distinct variety. Variations at the genomic level, particularly within the chloroplast DNA, can offer critical insights into the evolutionary relationships and genetic divergence between the variety and the typical species. These genomic differences can help resolve ambiguities in taxonomy, ensuring that W. somnifera var. abhaica is accurately classified and distinguished from other closely related taxa. Moreover, such studies can reveal unique genetic features of the variety, which may have implications for its ecological adaptation, medicinal properties, and conservation strategies (Dobrogojski et al., 2020). Chloroplasts (Cp) are essential cellular components of angiosperms involved in photosynthesis and the synthesis of important macromolecules, including amino acid and fatty acid. In molecular systematics research, plastome is a crucial element because of its distinctive characteristics and adaptive processes (Ahmed and Rahman, 2024). Typically, the Cp genome represents a quadripartite structure, comprising two inverted repeats separated by the large single- copy (LSC) and small single-copy (SSC) regions. In angiosperms, plastome sizes typically range from 107 kb to 218 kb (Wang et al., 2020). Diversity in gene content and organization can result from dynamic shifting in this genomic configuration, including contractions, expansions, and even unfolding (Ravi et al., 2008). In angiosperms, the plastome is typically inherited through the maternal line, whereas, in certain gymnosperms, it is inherited through the paternal line. The Cp genome encodes several groups of genes, including transfer RNAs (tRNAs), protein-coding genes (PCGs), and ribosomal RNAs (rRNAs), all of which are necessary for chloroplast function. Due to the inherent diversity found in Cp genomes, expressed through structural variants and polymorphisms, phylogenetic analysis provides new opportunities to resolve systematic COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 207 relationships, track the evolution of species, and investigate how species adapt to certain environments (Daniell et al., 2016; Dobrogojski et al., 2020). Advancements in bioinformatics and next-generation sequencing (NGS) technologies have revolutionized genomics, significantly enriching the GenBank repository with plastomes (Jongsun et al., 2020). Bioinformatic tools have streamlined the analysis and assembly of large-scale genomic data, while NGS has accelerated the sequencing process, making it faster, more cost- effective, and widely accessible. These advancements have resulted in a swift rise in the number of complete plastomes deposited in the GenBank database, providing a valuable resource for comparative genomics, phylogenetics, and evolutionary research. The availability of full plastome sequences has also elevated their role as a "superbarcode" for resolving phylogeny (Zhang et al., 2019). Unlike traditional barcoding methods that rely on a few gene regions, the full plastome offers a comprehensive genetic blueprint, enabling more accurate and reliable phylogenetic analysis of W. somnifera var. abhaica. This enhanced resolution is particularly crucial for distinguishing closely related species, resolving complex evolutionary relationships, and improving the accuracy of species identification within the Solanaceae family (Olmstead et al., 2008). Fig. 1. Morphology of Withania somnifera var. abhaica collected from the hilly terrain of Abha region of Saudi Arabia. A. Habit, B. Flower, C. Fruit. Given the intriguing morphological features observed in W. somnifera var. abhaica from the Abha region, a chloroplast genome-based approach is crucial for uncovering the genetic foundations and elucidating the phylogenetic relationships within this species. In this study, we aim to deepen the understanding of the complete chloroplast genome of this novel variety, focusing on the unique ecological context of the Abha hills in Saudi Arabia, by utilizing a comprehensive NGS-driven bioinformatics approach. 208 ALSUHAIMI et al. Materials and Methods Specimen collection The plant specimen was collected from the hilly regions of the Abha area in Saudi Arabia (coordinates: 18º17ʹ44ʹʹN, 42º25ʹ40ʹʹE; altitude: 2,491 m). The voucher specimen is preserved at the King Saud University Herbarium (KSUH) in Riyadh, Saudi Arabia, under the collection code Nadia, M.A. & Ali, M.A. 2021-1, ecotype (Abha hills, Saudi Arabia). Genome sequencing Total genomic DNA was isolated from silica gel-dried leaves utilizing the Qiagen DNA Extraction Kit. Paired-end reads of 151 bp were generated with a NextSeq 500 sequencer. The NGS (Next-generation sequencing) reads were assessed using FASTQC tool v.0.12.1 to evaluate Phred quality scores (Ahmed and Rahman, 2024). The raw sequencing data are publicly accessible on NCBI under the SRA accession ID SRR27753935. Construction of the plastome and its annotation The high-quality SRA data were configured into the Cp genome using Unipro UGENE v45.1 (Okonechnikov et al., 2012). Annotation of the plastome was carried out employing CPGAVAS2 server and subsequently verified with CPGView (Shi et al., 2019; Liu et al., 2023). The manually curated annotation was used to construct the circular plastome diagram employing OGDraw server (Greiner et al., 2019). The assembled plastome has been deposited in GenBank under the accession number OR166175. Evaluation of longer repeats and SSRs The REPuter server was used to identify longer repeat structures, while SSRs (simple sequence repeats) were identified utilizing the MISA-Web server (Kurtz et al., 2001; Beier et al., 2017). For the analysis of longer repeats in REPuter, all matching directions were considered. SSRs were analyzed using the default settings of the MISA-Web server. Assessment of RNA editing sites and GC skewness The plastome was examined for RNA editing sites using the PREPACT 3.0 server (Lenz et al., 2018). The BLASTx module was utilized to identify forward editing sites (C→U), with Nicotiana tabacum L. (Solanaceae) as the reference database and an e-value threshold of 0.001. For GC content skewness analysis, the assembled plastome was uploaded in FASTA format to the Proksee server (Grant et al., 2023). After initial processing, GC content and GC skew analysis options were applied to visualize the circular map. Genome rearrangement and collinearity analysis The assembled plastome of W. somnifera var. abhaica was subjected to comparative genomic analysis using the Mauve v.20150226 tool to identify gene order similarities with other taxa (Darling et al., 2004). GenBank flat files of the relevant taxa were initially imported into the Java console to run the progressive Mauve module. The analysis employed the HOXD scoring matrix, with gap opening and gap extension penalties set to -400 and -30, respectively. These parameters were optimized to maximize alignment accuracy and synteny detection before initiating the final comparative analysis. For collinearity analysis, the plastome was analyzed using the Circoletto server (Darzentas, 2010). Nucleotide diversity analysis The nucleotide diversity analysis commenced with the alignment of chloroplast genome sequences utilizing the MAFFT online tool to ensure precise sequence alignment across the studied genomes (Katoh et al., 2005). Subsequently, nucleotide diversity was assessed with COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 209 DnaSP v.5 tool. A sliding window approach was employed, with a window length of 600 base pairs and a step size of 200 base pairs, allowing for a detailed examination of nucleotide diversity across the genome (Librado and Rozas, 2009). The genomic coordinates of each window were then compared with the annotated gene regions of the chloroplast genome to identify and characterize patterns of nucleotide diversity. Plastome-wide molecular phylogeny To conduct a comprehensive plastome-wide molecular phylogenetic analysis within the Solanaceae family, 36 taxa, including W. somnifera var. abhaica, were chosen and retrieved from the NCBI GenBank database. Mentha spicata L. and Phyla nodiflora (L.) Greene were included as outgroup taxa to root the phylogenetic tree and provide context for evolutionary relationships. The sequences were compiled into a multi-FASTA file and subsequently aligned using the MAFFT server to ensure accurate sequence alignment across all taxa. The aligned sequences were then analyzed in MEGA v.11, where a maximum-likelihood (ML) tree was constructed. The Tamura 3- parameter model was employed as the nucleotide substitution model, which effectively captures the evolutionary dynamics within the plastomes (Ahmed and Rahman, 2024). The substitution rates were set to uniform to maintain a consistent rate of nucleotide changes across the sequences. A partial deletion approach was adopted for gap treatment, allowing for the exclusion of gaps that could potentially skew the results. To evaluate the robustness of the phylogenetic relationships, the ML tree was generated with 1,000 bootstrap replicates, providing a measure of confidence for each branch (Tamura et al., 2021). Results and Discussion Quality evaluation of NGS reads The sequencing run using the NextSeq 500 platform generated approximately 5.6 GB of high- quality, adapter-removed reads. Analysis of these raw reads revealed a GC content of 43% and an AT content of 57%, indicating a balanced nucleotide composition that reflects the genomic characteristics of the sample. Quality control metrics further confirmed the reliability of the sequencing process, with 96.6% of the bases achieving a Q20 score, indicating a 99% base call accuracy. Additionally, 90.8% of the bases reached the Q30 threshold, corresponding to a 99.9% base call accuracy, underscoring the overall robustness and precision of the sequencing data obtained. The quality of the raw reads in this study was found to be consistent with a recently published plastome of Tribulus macropterus variety, where the Phred quality scores were 95.9% and 89.7% for the Q20 and Q30 indices, respectively (Albediwi et al., 2024). Genome structure and contents The orbicular quadripartite plastome spanned a total length of 153,621 bp, comprising 84,972 bp in the LSC region, 18,400 bp in the SSC region, and 50,249 bp in the IRs regions (Fig. 2). The present investigation demonstrated a remarkable consistency with the plastome of W. somnifera as reported by Mehmood et al. (2020). The plastome of W. somnifera (MK142783) was characterized by a total length of 154,386 bp, with LSC, SSC, and IR regions measuring 85,688 bp, 18,464 bp, and 50,234 bp, respectively (Mehmood et al., 2020). This strong correlation reinforces the validity of the plastome structure for W. somnifera var. abhaica constructed in this study. The comparative analysis of the guanine-cytosine (GC) ratio and adenine-thymine (AT) ratio across different compartments of the plastome revealed distinct patterns (Table 1). The overall plastome exhibited an AT content of 62.26% and a GC content of 37.74%. The LSC region had the highest AT content of 64.20%, with a correspondingly lower GC content of 35.80%, reflecting 210 ALSUHAIMI et al. a higher proportion of adenine and thymine bases. The SSC region further amplified this trend, showing the highest AT content of 68.22% and the lowest GC content of 31.78%. In contrast, the inverted repeats (IRA and IRB) displayed a higher GC content, both around 43.2%, with corresponding AT contents of approximately 56.8%. These differences underscore the variability in nucleotide composition across the plastome. Fig. 2. Complete chloroplast genome of Withania somnifera var. abhaica representing gene orders and quadripartite junction sites. An elevated AT ratio in the SSC and LSC regions, in contrast to the IRs indicates an evolutionary trend where these protein-coding gene-rich regions show a selection for AT-rich codons to potentially enhance gene expression and protein function (Qian et al., 2013). This increased AT content is likely a consequence of higher recombination rates in these dynamic regions, driving nucleotide variability. In contrast, the IR regions, characterized by fewer recombination events, exhibit a lower AT content and greater structural stability, resulting in a more preserved nucleotide pattern. This contrast underscores how distinct evolutionary pressures COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 211 have shaped the structure and composition of the Withania somnifera var. abhaica plastome, reflecting both functional requirements and genomic stability (Saina et al., 2018). Table 1. Proportion of nucleotides in the quadripartite sites of Withania somnifera var. abhaica. Zones A (%) T (U) (%) C (%) G (%) C + G (%) A + T (%) Plastome 30.74 31.52 19.19 18.55 37.74 62.26 LSC 31.43 32.77 18.32 17.48 35.80 64.20 SSC 33.86 34.36 16.65 15.14 31.78 68.22 IRA 28.42 28.35 22.43 20.80 43.23 56.77 IRB 28.43 28.37 20.77 22.44 43.21 56.79 Annotation of genes The annotation of the W. somnifera var. abhaica Cp genome revealed a total of 131 genes, comprising 86 PCGs, 37 tRNA genes, and eight were rRNA genes (Fig. 2). Among the 19 genes associated with photosystems, 14 genes encode photosystem II, whereas 5 genes are involved with photosystem I. The genes encoding the small subunit of the ribosome (15 genes) outnumbered those encoding the large subunit (11 genes) (Table 2). Most PCGs were localized in the single- copy zones, while the inverted repeats predominantly contained RNA genes. The SSC region was notable for its high concentration of genes encoding subunits of NADH dehydrogenases. Furthermore, the LSC region featured two key DNA barcodes, rbcL and matK. In addition, the LSC harbored the cemA gene, which encodes the chloroplast envelope membrane protein. The high concentration of PCGs in the single-copy regions highlights their role in essential processes like photosynthesis and metabolism, contributing to functional diversity. The predominance of RNA genes in the IRs emphasizes their importance in maintaining genomic stability and supporting efficient protein synthesis. Additionally, the abundance of NADH dehydrogenase genes in the SSC region underscores its crucial role in energy metabolism and photosynthetic efficiency (Dobrogojski et al., 2020). Table 2. Classification of the protein-coding genes present in Withania somnifera var. abhaica plastome. Categories Gene groups Gene names Genes for photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbM, psbN, psbT, psbZ ATP synthase atpA, atpB, atpE, atpF, atpH, atpI Cytochrome b/f complex petA, petB, petD, petG, petL, petN Rubisco rbcL NADH-dehydrogenase ndhA, ndhB(×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK Self-replication Small subunit of ribosome rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×3), rps14, rps15, rps16, rps18, rps19 Large subunit of ribosome rpl2(×2), rpl14, rpl16, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36 DNA dependent RNA polymerase rpoA, rpoB, rpoC1, rpoC2 Other genes Maturase matK Envelop membrane protein cemA Acetyl-CoA-carboxylase accD C-type cytochrome synthesis gene ccsA Protease clpP Unknown Conserved open reading frames ycf1, ycf2(×2), ycf3, ycf4, ycf15(×2) 212 ALSUHAIMI et al. In a recent study, Mehmood et al. (2020) identified 86 PCGs, 37 tRNAs, and eight rRNAs in the plastome of W. somnifera. The gene content, organization, and localization in their investigation were congruent with the findings of the present study, showcasing the genetic similarity between W. somnifera and W. somnifera var. abhaica. Exon-intron distribution and cis-trans splicing The exon-intron distribution in the plastome of W. somnifera var. abhaica revealed a diverse and intricate gene structure across different regions (Table 3). In the LSC region, several genes, such as trnK-UUU, rps16, and atpF, have two exons separated by a single intron. The genes ycf3 and clpP exhibit more complex structures, each containing three exons and two introns. Notably, the ycf3 gene stands out with its relatively long intron II of 745 bp. The IRA and IRB regions display a mirrored arrangement of genes, such as rpl2 and ndhB, both containing two exons separated by a substantial intron. The SSC region includes the ndhA gene, which also features two exons and a notably a long intron of 1159 bp. This distribution highlights the complex organization of the plastome, with multiple genes featuring introns, contributing to the regulatory mechanisms of gene expression and the potential for alternative splicing in the plastid genome. The pattern of exon-intron distribution observed in this study aligns closely with that of the plastome of Capparis decidua (Forsk) Edgew (Alzahrani and Albokhari, 2022). Similar to our findings in W. somnifera var. abhaica, C. decidua also exhibited complex structures in the ycf3 and clpP genes within the LSC region, with both genes containing three exons separated by two introns. This resemblance corroborated the accuracy of the exon-intron prediction in the plastome of W. somnifera var. abhaica. Table 3. Exons-introns distribution in the plastome of Withania somnifera var. abhaica. Location Genes Exon I (bp) Intron I (bp) Exon II (bp) Intron II (bp) Exon III (bp) IRB trnA-UGC 37 811 36 IRA ndhB 775 679 758 SSC ndhA 553 1159 539 IRB rpl2 391 666 434 IRB trnE-UUC 32 723 40 IRB ndhB 775 679 758 IRA trnE-UUC 32 723 40 IRA trnA-UGC 37 811 36 LSC rpl16 9 1028 396 IRA rpl2 391 666 434 LSC rpoC1 453 737 1614 LSC ycf3 124 727 232 745 151 LSC trnK-UUU 37 2477 36 LSC petB 6 746 642 LSC petD 8 745 475 LSC trnL-UAA 35 492 50 LSC trnV-UAC 36 552 56 LSC rps16 40 855 227 LSC trnS-CGA 31 674 60 LSC atpF 145 700 410 LSC clpP 71 790 294 632 244 COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 213 The plastome of W. somnifera var. abhaica unveiled several cis-splicing genes, such as rps16, atpF, rpoC1, ycf3, clpP, petB, petD, rpl16, rpl2, ndhB, and ndhA (Fig. 3). These genes feature diverse intron-exon structures, with some containing multiple introns and others having a single intron, exhibiting significant variability in intron lengths. This diversity in intron structures and lengths suggests a range of splicing requirements and potential impacts on gene expression and chloroplast function, reflecting adaptations to specific functional needs and environmental conditions (Dobrogojski et al., 2020). The rps12 is a trans-spliced gene with exons located in separate regions of the genome (Fig. 4). Exon 1 is located in the LSC, while Exons 2 and 3 are situated in the IRs. These exons were transcribed separately and then spliced together to form a mature mRNA, which facilitates the correct assembly of the rps12 coding sequence. This trans- splicing mechanism is essential for the gene's proper expression, ensuring that exons from distinct genomic regions are combined to produce a functional protein. The plastome of Mandragora caulescens C. B. Clarke (tribe Solaneae) revealed a similar structural organization of cis- and trans-spliced genes, supporting the findings of the present investigation (Ma et al., 2024). Fig. 3. W. somnifera var. abhaica plastome showing genes responsible for cis-splicing. 214 ALSUHAIMI et al. Fig. 4. W. somnifera var. abhaica plastome showing rps12 responsible for trans-splicing. Longer repeats and SSRs In the analysis of longer repeats, W. somnifera var. abhaica exhibited 15 forward, eight reverse, 26 palindromic, and one complement repeats (Fig. 5A). Compared to other species, W. somnifera var. abhaica had fewer forward and reverse repeats than W. coagulans, W. riebeckii, and Dunalia obovata. However, W. somnifera var. abhaica had more palindromic repeats than W. riebeckii and W. coagulans, and fewer complement repeats compared to most species, except for D. obovata, which had none. Physalis peruviana had one longer repeat, similar to W. somnifera var. abhaica. Fig. 5. Comparative overview of the repeat structures present in the plastome of W. somnifera var. abhaica and allied genera. A. Longer repeats, B. Simple sequence repeats. The evaluation of SSR profile revealed W. somnifera var. abhaica had 31 mononucleotide repeats, zero dinucleotide and trinucleotide repeats, and seven tetranucleotide repeats (Fig. 5B). In comparison, W. somnifera and W. coagulans had more mononucleotide repeats, with 40 each, and D. obovata had the highest number of 43. Unlike W. somnifera var. abhaica, which had no dinucleotide or trinucleotide repeats, P. peruviana showed one dinucleotide repeat, and W. COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 215 riebeckii had two trinucleotide repeats. W. somnifera var. abhaica was unique in having seven tetranucleotide repeats, a characteristic feature not found in the other species studied. These findings suggest that W. somnifera var. abhaica possesses a unique profile compared to the other analyzed species. SSRs in the plastome exhibit lower mutation rates compared to nuclear SSRs, enhancing their stability and reliability in phylogenetic studies (Albediwi et al., 2024). RNA-editing sites and GC skewness The RNA-editing analysis of the W. somnifera var. abhaica cp genome revealed a varied distribution of editing sites across different regions, with the LSC region comprising 40% of the sites, the IR region 21%, and the SSC region 39% (Fig. 6). A total of 66 RNA-editing sites were identified in the plastome across various compartments. The SSC region exhibited the highest concentration of RNA-editing sites, with the ycf1 gene containing 12 sites, followed by the ndhD gene with 7 sites. In contrast, the LSC region, covering the largest portion of the genome, had a wider range of genes with editing sites, though the rpoB gene had the highest count at 5 sites. The IR region showed significant editing activity in the ycf2 gene with 5 sites. Overall, the SSC region, despite having slightly fewer editing sites than the LSC, showed a higher frequency in specific genes, such as ycf1 and ndhD compared to the more evenly distributed editing sites among various genes in the LSC and IR regions. Fig. 6. Distribution of RNA editing loci in the plastome of W. somnifera var. abhaica. A. Distribution across PCGs, B. Distribution across compartments. A high concentration of RNA-editing sites in the SSC region, as observed in our findings, has also been reported in the chloroplast genome of Capparis decidua (Alzahrani and Albokhari, 2022), where 46 RNA-editing sites were detected across 18 genes, including nine sites within the ndhD gene. This pattern of RNA-editing is further supported by findings in the plastome of Solanum dulcamara L., which exhibited a similar tendency, reinforcing the significance of our results (Amiryousefi et al., 2018). Analyzing RNA-editing sites in the W. somnifera var. abhaica plastome is crucial for uncovering the complex regulatory mechanisms that govern gene expression and photosynthesis in this medicinal plant. Variations in RNA-editing efficiency across different genes, or even at different loci within the same gene, suggest intricate layers of control that can impact protein structure and function. These modifications have the potential to regulate key physiological processes, including photosynthesis, which is vital for the survival and metabolic activities of this variety (Amiryousefi et al., 2018). 216 ALSUHAIMI et al. Skewness analysis revealed a highly similar pattern of GC content and GC skew across all species examined (Fig. 7). A positive GC skew indicated that guanine (G) was more abundant than cytosine (C) in the analyzed genome region, whereas a negative GC skew suggested the opposite. The consistent patterns in GC skew and content across these species suggest a conserved DNA structure and stability, implying that the mechanisms governing guanine and cytosine distribution have been preserved within the Solanaceae family (Wang et al., 2023). These metrics provide insights into DNA density, as regions with high GC content are more stable and denser due to the stronger hydrogen bonding between G and C pairs. The uniformity observed between W. somnifera var. abhaica and other closely related taxa further supports the evolutionary stability of these species and reinforces the accuracy of the assembled plastome of W. somnifera var. abhaica. Fig. 7. GC content and skewness analysis of the W. somnifera var. abhaica along with closely related species. COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 217 Comparative genomic assessments Plastome-wide alignment revealed locally collinear blocks (LCBs) with high similarities (Fig. 8). Gene orders and arrangements were represented by multi-colored mini blocks: white for PCGs, black for tRNAs, green for intron-containing tRNAs, and red for rRNAs. The high similarity of W. somnifera var. abhaica with other closely related taxa supports the accuracy of its plastome assembly and annotation. The results of the Mauve whole-genome alignment are congruent with findings from other similar studies (Henriquez et al., 2020; Munyao et al., 2020). The collinearity analysis unveiled a high synteny of W. somnifera var. abhaica with other closely related taxa (Fig. 9). No significant rearrangements were detected among the taxa studied, highlighting their structural similarity and integrity. Sequence identity was notably similar among W. somnifera, W. coagulans, and W. riebeckii. Unlike other Withania species, P. peruviana and D. obovata exhibited syntenic blocks (red) with over 75% sequence similarity (Fig. 9). This high synteny in P. peruviana and D. obovata suggests that, despite belonging to different genera, these species share significant conserved genomic regions. This conservation could be due to evolutionary constraints or functional necessities that have maintained these sequences (Ding et al., 2022). Table 4 presents a detailed comparative account of the plastomes of various taxa examined in the present study, underscoring differences in chloroplast genome length, GC content, and gene count. Fig. 8. Mauve progressive alignment of the complete plastome of W. somnifera var. abhaica showing resemblances with other closely related taxa within Solanaceae. 218 ALSUHAIMI et al. Fig. 9. Synteny analysis of W. somnifera var. abhaica with other closely related taxa within Solanaceae. Nucleotide diversity assessments The nucleotide diversity analysis of the W. somnifera var. abhaica plastome identified several hypervariable sites (Fig. 10). The mean Pi value across all genomic positions was 0.0048. The most hypervariable site was found in the psbJ gene (Pi = 0.04844), followed by psbA (Pi = 0.04222), both located in the LSC region. In the SSC region, the ndhF gene showed the highest variability (Pi = 0.3389), followed by ycf1 (Pi = 0.3044). Nucleotide diversity was lower in the IRs compared to the LSC and SSC, reflecting the more conserved nature of the IR regions. Our results align with earlier findings for Chlorophytum comosum, C. gallabatense, and Tribulus macropterus var. arabicus (Munyao et al., 2020; Albediwi et al., 2024). Identifying hypervariable genes in the plastome of W. somnifera var. abhaica is important for developing genetic markers or barcodes. Due to their high variability, these genes can serve as accurate genetic identifiers for distinguishing closely related species or even different subspecies within the same species. These hypervariable barcodes enable precise identification and classification of Withania species. Moreover, these markers can enhance the understanding of unusual mutations within a lineage and help to elucidate evolutionary relationships (Breen et al., 2009). COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 219 Table 4. Comparison of plastome features of the taxa analyzed in the current investigation. Taxa GenBank ID Total length GC content (%) PCGs tRNAs rRNAs Total genes Withania somnifera var. abhaica Nadia, A. Ali & M.S. Alwahibi, var. nov. OR166175.1 153,621 37.74 86 37 8 131 W. somnifera (L.) Dunal MK142783.1 154,386 37.67 84 39 8 131 W. frutescens (L.) Pauquy ON153173.1 153,771 37.73 89 44 8 141 Discopodium penninervium Hochst. OR400640.1 155,033 37.52 93 38 8 139 Nothocestrum latifolium A. Gray OR400642.1 155,669 37.53 93 38 8 139 Physalis peruviana L. NC_026570.1 156,706 37.54 91 37 8 136 P. cordata Houst. ex Mill. NC_072167.1 157,000 37.51 92 38 8 138 Dunalia obovata (Ruiz & Pav.) Dammer NC_026563.1 156,559 37.69 88 36 8 132 Capsicum baccatum L. NC_072696.1 157,475 37.64 87 37 8 132 C. lycianthoides Bitter NC_026551.1 156,583 37.76 87 36 8 131 Jaltomata sinuosa (Miers) Mione NC_062863.1 156,163 37.91 89 36 8 133 J. bicolor (Ruiz & Pav.) Mione NC_062862.1 155,459 38.03 89 36 8 133 Solanum corneliomulleri J.F. Macbr. NC_062080.1 155,544 37.85 93 37 8 138 S. huaylasense Peralta NC_062081.1 155,571 37.83 93 37 8 138 S. americanum Mill. NC_062693.1 155,266 37.95 91 37 8 136 S. scabrum Mill. MT621038.1 155,552 37.90 91 37 8 136 S. villosum Mill. MT621039.1 155,529 37.89 91 37 8 136 S. nigrum L. MT621037.1 155,446 37.90 91 37 8 136 Brugmansia arborea (L.) Sweet NC_081500.1 155,939 37.83 86 37 8 131 Datura stramonium L. MT610897.1 155,884 37.86 86 37 8 131 D. metel L. OK040953.1 155,934 37.86 86 38 8 132 Nicandra physalodes (L.) Gaertn. MN165114.1 156,729 37.78 86 38 8 132 Mandragora caulescens C.B. Clarke NC_086882.1 154,810 37.98 94 40 8 142 Atropa bella-donna L. NC_004561.1 156,687 37.56 85 37 8 130 Lycium ferocissimum Miers MN866909.1 155,894 37.85 86 37 8 131 L. chinense Mill. MN102357.1 155,736 37.84 89 37 8 134 L. ruthenicum Murray MT955897.1 154,911 37.91 89 37 8 134 L. qingshuiheense Jiang & Li NC_084119.1 154,945 37.92 87 37 8 132 Nicotiana tomentosiformis Goodsp. NC_007602.1 155,745 37.79 82 37 8 127 N. tabacum L. NC_001879.2 155,943 37.85 84 37 8 129 N. sylvestris Speg. NC_007500.1 155,941 37.85 81 38 8 127 N. attenuata Torr. ex Watson MG182422.1 155,914 37.86 90 37 8 135 N. undulata Ruiz & Pav. NC_016068.1 155,863 37.88 89 37 8 134 Petunia exserta Stehmann MT644125.1 156,597 37.81 88 37 8 133 Mentha spicata L. OM617844.1 152,048 37.85 88 37 8 133 Phyla nodiflora (L.) Greene OQ673174.1 154,341 39.19 87 34 8 129 Molecular phylogenetics A plastome-wide molecular phylogeny within the Solanaceae family was reconstructed that supported the systematic position of W. somnifera var. abhaica as a novel variety of W. somnifera (Fig. 11). The maximum-likelihood (ML) tree showed a strong bootstrap support across most of the clades and subclades. The family Solanaceae consists of seven subfamilies, such as Cestroideae, Goetzeoideae, Nicotianoideae, Petunioideae, Schizanthoideae, Schwenckioideae, and Solanoideae (Olmstead et al., 2008). However, Cp genomes in the NCBI GenBank database were 220 ALSUHAIMI et al. available for only three subfamilies, viz. Solanoideae, Nicotianoideae, and Petunioideae. The reconstructed ML tree provided a well-resolved phylogeny for these three subfamilies, all of which exhibited a monophyletic origin (Fig. 11). W. somnifera var. abhaica was found to be closely related to other W. somnifera accessions and grouped with other members of the tribe Physaleae within the Solanoideae subfamily. Tribe Physaleae displayed a monophyletic origin with 100% bootstrap support and showed a close relationship with the member taxa of the tribe Capsiceae. The tribe Solaneae, represented by eight taxa, also demonstrated a monophyletic nature with 100% bootstrap support. Similarly, tribe Datureae exhibited strong bootstrap support with its two representative genera, Brugmansia and Datura. The tribes Nicandreae and Mandragoreae, each represented by one species, formed a cluster. The remaining two tribes, Hyoscyameae and Lycieae grouped together with robust bootstrap support. The subfamily Nicotianoideae was represented solely by the tribe Nicotianeae, which depicted a well-resolved monophyletic nature. Petunia exserta was the only representative of the subfamily Petunioideae, occupying a distinct position in the ML tree. Fig. 10. Nucleotide diversity of W. somnifera var. abhaica Cp genome elucidating hypervariable barcodes across LSC, SSC and IRs compartments. The accurate systematic positioning of the W. somnifera var. abhaica plastome justifies its assembly. Mehmood et al. (2020) constructed ML-based phylogenetic tree with 23 taxa of Solanaceae to validate the assembly of W. somnifera (MK142783). In their analysis, the tribe Physaleae exhibited a close affinity with Capsiceae tribe, while the tribe Hyoscyameae clustered with the tribe Lycieae. These tribal relationships are further supported by our current investigation (Fig. 11). Mehmood et al. (2020) included two species of Nicotianoideae, viz. N. sylvestris and N. tabacum in their ML tree. In the present study, we have included five species of Nicotiana, where N. tabacum clustered with N. sylvestris, and these consistent findings reinforce the well-resolved phylogeny (Fig. 11). Given the distinctive characteristics of this novel variety of W. somnifera, including reduced plant height, elliptic-elongated, thick and puberulous leaves resembling semi-succulent morphological nature, a bifurcated fruiting calyx tip, c. 0.5 mm in length, with each bifurcation being botuliform in shape, and robust phylogenetic evidence supporting its uniqueness, we herein COMPLETE CP GENOME SEQUENCE OF A NOVEL WITHANIA SOMNIFERA 221 propose that the collected ecotype accession be recognized as a new variety: Withania somnifera (L.) Dunal var. abhaica Nadia, A. Ali & M.S. Alwahibi, var. nov. This new variety is named after its ‘Type’ locality. Fig. 11. Maximum-likelihood (ML) tree representing plastome-wide phylogenetic relationships of W. somnifera var. abhaica. The complete chloroplast genome presented in this study will contribute valuable new data to the GenBank repository, providing an essential resource for comprehensive molecular phylogenetic and dating analyses. The identified hypervariable barcodes will advance DNA barcoding efforts, offering precise tools for the taxonomic identification of the medicinally significant taxon. Additionally, this study lays the foundation for deeper insights into the evolutionary processes and genetic diversity within the Solanaceae family. 222 ALSUHAIMI et al. Acknowledgements The authors extend their appreciation to the Researchers Supporting Project number (RSP2025R306), King Saud University, Riyadh, Saudi Arabia. This research was also funded by the Korean Research Institute of Bioscience and Biotechnology (KRIBB) Initiative Program of the Republic of Korea (KGM4582423). References Ahmed, S.S. and Rahman, M.O. 2024. 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