Chloroplast genome sequencing in winged bean (Psophocarpus tetragonolobus L.) and comparative analysis with other legumes

Chloroplast genome sequencing in winged bean (Psophocarpus tetragonolobus L.) and comparative analysis with other legumes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chloroplast genome sequencing in winged bean (Psophocarpus tetragonolobus L.) and comparative analysis with other legumes Nikhil Kumar Singh, Binay K. Singh, Anupama Giddhi, Harsha Srivastava, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4615004/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The winged bean ( Psophocarpus tetragonolobus ) is a fast-growing, underutilized legume thriving in hot, humid regions. It forms symbiotic associations with a broad-spectrum cowpea rhizobial group, making it ideal for crop rotation or intercropping systems. Winged bean seeds are rich in protein, fiber, vitamins, minerals, fat, and carbohydrates, highlighting its potential as a valuable agricultural crop. In this study, we conducted whole-genome sequencing of the winged bean chloroplast using high-coverage short-read sequencing on the Illumina platform, generating over 1 billion paired-end raw reads. We utilized the GetOrganelle toolkit to assemble the chloroplast genome comprising 130 genes, including 85 protein-coding genes, 37 tRNAs, and eight rRNA genes. We also identified 84 perfect SSRs, two compound SSRs, and 15 VNTRs. Our analysis revealed the typical quadripartite structure of the chloroplast genome, along with insights into its functional classification and phylogenetic relationships with other legumes. Additionally, we identified possible genomic rearrangements through synteny analysis. Characterizing the winged bean chloroplast genome provides crucial resources for research and crop improvement. Comparative genomics of the chloroplast offers significant insights into the evolutionary and molecular biology of legumes. Biological sciences/Biotechnology Biological sciences/Computational biology and bioinformatics Biological sciences/Molecular biology Winged bean Plastome SSR AKWB-1 Phylogenetic tree Codon Usage Bias Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Winged bean ( Psophocarpus tetragonolobus (L.) DC.) (2n = 2× = 18; 782 Mbp) is an underutilised legume of the family Leguminosae 1, 2 . It grows in hot, humid, equatorial countries of Southern Asia, Melanesia, and the Pacific area 3 . It is a short-day self-pollinated species but can experience cross-pollination up to 16.0% 4 . This tropical legume, maturing in 4–5 months, yields up to 14 quintals of dry seeds and 115 quintal tubers per hectare, even with minimal management 5 . Its rapid growth and vining nature make it suitable for use as a cover crop 6, 7 . Moreover, it is highly suited for crop rotation or intercropping systems as it forms symbiotic associations with a broad-spectrum cowpea rhizobial group 8 . Winged bean is often referred to as the 'one-stalk supermarket' due to its versatile culinary applications worldwide. Various plant parts, including leaves, branches, flowers, seeds, fruits, and tubers, are utilised 3, 9 . Often dubbed the "soybean of the tropics," winged bean seeds offer crude protein (~ 34.0%), similar to soybean (~ 35.0%) 10 . Additionally, it is rich in fiber, vitamins, minerals, and carbohydrates. Winged bean seeds contain 15–18% fat, constituted by 30–40% saturated and 60–70% unsaturated fatty acids 11 . Because of its high oxidative stability, solid fat content, and good thermal conductivity, winged bean seed oil is considered superior to soybean oil 12 . Winged bean seed powder is a valuable flour that can be brewed to make a coffee-like drink 13 . The winged bean tubers have ivory flesh and contain 12–19% protein and 1–4% fat 14 . The origin of winged bean is subject to debate with two primary hypotheses. The first hypothesis suggests an African origin, proposing either in situ domestication followed by migration to the east or trans-domestication from an African progenitor species later in the Indian Ocean rim of Asia 2, 3 . The second hypothesis posits that winged bean is distinct from current African members of the genus and arose through allopatric speciation preceding any domestication processes 15 . However, the precise origin and progenitor of winged bean remain unresolved. Chloroplasts originated from a cyanobacterium through endosymbiosis with a eukaryotic host around a billion years ago, creating an autotrophic line of nucleus-containing cells 16 . Throughout evolution, they retained essential genes for replication, transcription, photosynthesis, and several other critical metabolic pathways while losing much of their original genome. Chloroplast genomes are mostly maternally inherited in angiosperms and paternally inherited in gymnosperms 17 . Angiosperm chloroplast genomes typically have a quadripartite structure, ranging in size from 120 kb to 200 kb, including a double-stranded closed loop with a long single-copy sequence (LSC, 80 kb-90 kb), a short single-copy sequence (SSC, 16 kb-27 kb), and two inverted repeats (IRs, 20 kb-28 kb) with roughly equal length. The IRs divide the chloroplast genome into LSC and SSC regions 18, 19 . Chloroplast genomes exhibit relative stability and conservation across species, with a lower mutation rate compared to the nuclear genome 20 . Nevertheless, diversification occurs in chloroplast genomes, resulting in variations in size and organisation. The expansion/contraction or loss of IRs and gene loss/duplication outside the IR are major factors contributing to size variation 21 . Loss of IRs leads to a dynamic arrangement of the chloroplast genome 22, 23 . Within the Leguminosae family, chloroplast genomes have undergone extensive rearrangements, with some legume species experiencing complete loss of one copy of the IR 24, 25 . The Genistoids, Dalbergioids, and the Old World clades of the Leguminosae species displayed several inversions, particularly a 50 kb inversion in the LSC region 26 . Additionally, during plant evolution, chloroplast genes have been lost, with some transferred to the nucleus, such as rpl22 , infA , and accD genes 27, 28, 29 . The loss of introns from rps12 and clpP is also reported in the legume genome 22, 27 . Chloroplast genomes have been extensively used in evolution, phylogeny, and phylogeography studies 30, 31, 32 . In the Leguminosae family, comprising approximately 751 genera and 19,500 species ranging from trees to herbaceous crop plants 33 , chloroplast-derived markers, particularly matK gene and the trnL-trnF intergenic spacer have played a crucial role in exploring evolutionary relationships 34, 35, 36 . Chloroplast genomic data have also been extensively employed to study gene expression and regulation, including RNA editing sites and codon usage bias 37, 38, 39 . Besides, simple sequence repeats (SSRs) within chloroplast genomes are potential DNA markers for species identification 40 . Chloroplast genetic engineering offers unique advantages such as high-level transgene expression 41 , multigene engineering in a single transformation event 42 , transgene containment via maternal inheritance 43 , lack of gene silencing 44 , absence of position effect 45 , pleiotropic effects 46 , and prevention of undesirable foreign DNA 47 . Complete chloroplast genome sequences are essential for identifying spacer regions for transgene integration at optimal sites through homologous recombination, as well as for determining endogenous regulatory sequences for optimal transgene expression 48 . Traditional methods for obtaining chloroplast genome sequences involve chloroplast DNA isolation, random shearing, cloning into large-insert size vectors, and shotgun sequencing. Recent advancements, such as whole-genome PCR amplification with universal primers and high-throughput sequencing, have introduced faster and cost-effective approaches 49, 50, 51 . Next Generation Sequencing (NGS) technology, particularly platforms like Illumina, has significantly accelerated chloroplast genome sequencing. Moore et al. 52 made the pioneering attempt to use NGS for chloroplast genome sequencing, leading to the sequencing of numerous chloroplast genomes, with Illumina being the most commonly used platform 53, 54, 55, 56 . Despite over 44 published chloroplast genomes within the Leguminosae family, the chloroplast genome of winged bean remains unexplored. In this study, genomic DNA from fresh young leaves of winged bean was sequenced using the Illumina HiSeq2500 platform, and the chloroplast genome was assembled using the embryophyta plastid database as reference. The study elucidated the winged bean chloroplast genome sequence and its characteristics and compared it with those of other Leguminosae species. This research aims to enhance our understanding of the winged bean chloroplast genome and provide valuable markers for phylogenetic and genetic studies. Materials and Methods Plant material The study utilised the dual-purpose cultivar AKWB-1 of winged bean, which serves both as a vegetable and a pulse. Given the significant level of cross-pollination reported in winged bean, the AKWB-1 plants were selfed for three successive generations to achieve homozygosity. The winged bean plants were grown at the experimental farm of the ICAR-Indian Institute of Agricultural Biotechnology, located in Ranchi, Jharkhand, India, with the geographical coordinates 23°16'27.6"N, 85°20'29.4"E. DNA isolation, library preparation, sequencing, and annotation The young leaves of winged bean were utilised to extract high molecular weight genomic DNA using the Cetyl Trimethyl Ammonium Bromide (CTAB) method 57 . To remove RNA contamination from the DNA, 2.0 µl of RNase A (10 mg/ml, HiMedia) was added to 20 µl of DNA dissolved in TE buffer (Tris–EDTA, pH = 8.0), followed by incubation at 37°C for 3–4 h. The quality of the purified DNA samples was analysed on a 1.0% agarose gel and quantified based on the absorbance at 260 nm using a NanoDrop™ OneC microvolume UV–Vis spectrophotometer (Thermo Scientific). The whole-genome sequencing library was constructed using the TruSeq DNA PCR-Free Library Prep Kit (Illumina, catalog no. 20015963) following the manufacturer’s instructions. The prepared library was quantified using a Qubit 3 fluorometer (Thermo Fisher Scientific, USA) with the DNA High Sensitivity kit, and the library size was verified using a Bioanalyzer 2100 (Agilent Technologies, CA, USA). After assessing the quality and quantity of the library, whole-genome sequencing was conducted on a Illumina HiSeq2500 instrument with paired-end (2 × 150 bp) sequencing strategy using the TruSeq Rapid SBS Kit (Illumina, catalog no. FC-402-4023). FastQC v0.11.9 software ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) was employed to evaluate the quality of sequencing data, including base qualities, GC content, adapter content, and overrepresentation analysis. Adapter sequences were trimmed using fastp v0.20.1 software 58 ( https://github.com/OpenGene/fastp ), with a minimum length of two bases, quality filtering disabled, and forced poly-G trimming. The high-quality reads were de novo assembled into the chloroplast genome using the GetOrganelle v1.5.1c toolkit 59 , specifying the embryophyta plastid database. Annotation of the complete chloroplast genome was performed with GeSeq and manual corrections 60 . The complete chloroplast genome sequence of the winged bean was submitted to GenBank with the accession number PP894786.1. Phylogenetic Analysis The nucleotide sequences of all predicted chloroplast genes of P. tetragonolobus and the reported chloroplast genes for nine other related species, including Vigna radiata , Phaseolus vulgaris , Lablab purpureus , Cyamopsis tetragonoloba , Glycine max , Cajanus cajan , Medicago truncatula , Cicer arietinum , and Arachis hypogaea , along with Arabidopsis thaliana as the outgroup, were obtained from NCBI. Alignment and subsequent phylogenetic analysis were conducted using the genes shared across the species. The sequences were aligned using MAFFT v7 61 with 1000 iterative refinement steps using "--maxiterate 1000". The resulting aligned sequences were saved in FASTA format. Phylogenetic trees for each aligned gene were inferred using RAxML-NG 62 with 1000 Bootstrap replicates and the "General Time Reversible (GTR) with gamma-distributed rate variation among sites (GTR + G)" model. The best genetree files were used to prepare a multigene-based species tree with ASTRAL v5.7.8 63 . Finally, the phylogenetic tree was visualised using FigTree v1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ). Synteny and genome rearrangement analysis We downloaded the chloroplast genomes of G. max , C. cajan , and L. purpureus , along with A. thaliana as an outgroup species from CpGDB ( https://www.gndu.ac.in/CpGDB/ ) and analysed the synteny and rearrangement in the chloroplast genomes of P. tetragonolobus and related legumes using two different methods. For synteny plots, pairwise BLASTN results were generated using the whole chloroplast genome of each species in R using a custom script. Information on BLAST hits was visualised in R using the genoPlotR package 64 . Subsequently, the alignments were annotated using species-specific GFF3 specifications. Information on BLAST hits among homologous segments was also visualised in R using the genoplotR package 64 . As a complementary approach, we performed global alignment of the chloroplast genomes with P. tetragonolobus as the reference. The alignment was conducted on the online platform mVISTA in Shuffle-LAGAN mode 65 . Estimation of adaptive evolution of protein-coding genes To analyse the degree of non-synonymous (Ka) and synonymous (Ks) substitutions, as well as their ratio (dN/dS), the coding DNA sequences (CDS) of P. tetragonolobus were compared with G. max , C. cajan , and L. purpureus , along with A. thaliana. For this, we performed pairwise alignments using MAFFT v7 66 , and then Ka, Ks substitutions, and their ratio were calculated using KaKs_Calculator 3 67 with the MA model. Codon usage analysis The codon usage within the coding part of the chloroplast was calculated using CodonW v1.4.4 available at https://codonw.sourceforge.net using universal codon standards. RSCU (relative synonymous codon usage) values were plotted using R package ggplot 68 . Repeat Analysis The long repeats in the winged bean chloroplast genome was analysed using REPuter 69 . Repeats were reported as either F (forward), R (reverse), P (palindromic), or C (complement), with parameters set at a Hamming Distance of 3 and a Minimal Repeat Size of 30. The chloroplast genome was also screened for perfect SSRs (pSSRs), compound SSRs (cSSRs), and variable number tandem repeats (VNTRs) using Krait v1.3.3 software 70 . The screening was conducted based on specific criteria: mono-nucleotide repeat motifs were required to have a minimum of 10 repeats, di-nucleotide repeat motifs required a minimum of five repeats, tri-nucleotide repeat motifs needed at least five repeats, while tetra-, penta-, and hexa-nucleotide repeat motifs were required to have a minimum of four repeats. If the distance between two SSRs was < 10 bp, they were considered as cSSR. Results Assembly and annotation of winged bean chloroplast genome We used the Illumina HiSeq2500 next-generation sequencing platform for whole-genome sequencing of winged bean, generating 1,030,974,930 paired-end raw reads. After cleaning for adaptors and low-quality reads, 1,030,150,624 clean reads were obtained. Subsequently, the GetOrganelle v1.5.1c toolkit was employed to assemble the chloroplast genome from the winged bean whole-genome sequencing data. Using a modified baiting and iterative mapping approach, GetOrganelle successfully recruited 36,123,587 chloroplast-associated clean reads, constituting 3.51% of the total clean reads. This approach facilitated the assembly of the complete winged bean chloroplast genome, totaling 151,571 bp, with > 35,000X coverage ( Supplementary Table S1 ). The chloroplast genome of winged bean exhibited a typical quadripartite structure, consisting of an LSC region of 82,736 bp, an SSC region of 17,777 bp, and a pair of equal-sized IRs each measuring 25,529 bp. The winged bean chloroplast genome had an overall GC content of 35.26%, with the LSC, SSC, and IR regions showing GC contents of 32.63%, 28.55%, and 41.86%, respectively (Table 1 ). Table 1 Characteristics of the winged bean chloroplast genome. Attribute Start End Size (bp) Adenine (A) Thymine (T) Guanine (G) Cytosine (C) AT (%) GC (%) Whole chloroplast genome (bp) 1 151571 151571 49135 (32.42%) 48995 (32.32%) 26888 (17.74%) 26553 (17.52%) 64.74 35.26 Large single-copy region (bp) 68836 151571 82736 27979 (33.82%) 27763 (33.56%) 13833 (16.72%) 13159 (15.90%) 67.37 32.63 Small single-copy region (bp) 25530 43306 17777 6313 (35.51%) 6388 (35.93%) 2368 (13.32%) 2707 (15.23%) 71.45 28.55 Inverted repeat B (IRb) (bp) 1 25529 25529 7379 (28.90%) 7462 (29.23%) 5547 (21.72%) 5140 (20.13%) 58.14 41.86 Inverted repeat A (IRa) (bp) 43307 68835 25529 7462 (29.23%) 7380 (28.90%) 5140 (20.13%) 5547 (21.73%) 58.14 41.86 Prt. coding genes - - 78004 24940 (31.97%) 24929 (31.95%) 14292 (18.32%) 13843 (17.75%) 63.93 36.07 tRNA - - 2746 654 (23.82%) 639 (23.27%) 718 (26.15%) 735 (26.77%) 47.09 53.52 rRNA - - 8667 1951 (22.51%) 1952 (22.52%) 2381 (27.47%) 2383 (27.49%) 45.03 54.96 The comparison of the chloroplast genome of winged bean with the chloroplast genomes of related legume species, including V. radiata , P. vulgaris , L. purpureus , C. tetragonoloba , G. max , C. cajan , M. truncatula , C. arietinum , and A. hypogaea , along with A. thaliana indicated that although the sizes of the overall genome had differences, the GC content was similar in LSC, SSC, and IR regions of different species. We observed a little difference in total genes, CDS and tRNAs among the ten legume species. C. tetragonoloba exhibited the maximum number of protein coding genes, CDS and tRNAs and C. arietinum showed the least ( Supplementary Table S2 ). The chloroplast genome of winged bean consisted of 130 genes, including 85 protein-coding genes, 37 tRNAs, and 8 rRNAs genes. Individually, LSC contained 83 genes, including 61 protein-coding and 22 tRNAs genes while SSC contained 13 genes, including 12 protein-coding and one tRNA genes. The IR regions contained 17 duplicated copies of 6 protein-coding genes, 7 tRNAs, and 4 rRNAs genes ( Fig. 1 ). Overall, 24 intron-containing genes (14 protein-coding genes, 8 tRNA genes, and 2 rRNA genes) were found. Among these, 22 genes had one intron, and clpP and pafI had two introns each. trnK-UUU had the largest intron (2585 bp) and rrn23 had the smallest intron (198 bp) ( Supplementary Table S3 ). The functional classification of winged bean chloroplast genes indicated that 47 genes had photosynthesis-related functions, including genes for photosystem I, photosystem II, Cytochrome b/f complex, ATP synthase, NADH dehydrogenase, Rubisco large subunit, and Photosystem assembly stability factor. Similarly, 75 genes were categorised for transcription and translational related functions, which included RNA polymerases, ribosomal protein small subunit, ribosomal protein large subunit, tRNA, and rRNAs. The remaining 8 genes, including maturase RNA processing, protease gene, c-type cytochrome synthesis gene, subunit of acetyl-CoA-carboxylase (fatty acid synthesis) gene, envelope membrane protein (carbon metabolism), and hypothetical chloroplast reading frames, displayed other functions (Table 2 ). Table 2 Chloroplast encoding genes in Psophocarpus tetragonolobus Category Group Genes Photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ Cytochrome b/f complex atpA, atpB, atpE, atpF, atpH, atpI ATP synthase petA, petB, petD, petL, petG, petN NADH dehydrogenase ndhA, ndhB # , ndhB # , ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK Rubisco large subunit rbcL Photosystem assembly stability factor pafI, pafII, pbf1 Transcription and Translation RNA polymerase rpoA, rpoB, rpoC1, rpoC2 Ribosomal protein (SSU) rps7, rps12 # , rps15, rps12 # , rps7, rps4, rps14, rps2, rps16, rps18, rps12, rps11, rps8, rps3, rps22, rps19 Ribosomal protein (LSU) rpl2 # , rpl23 # , rpl32, rpl23 # , rpl2 # , rpl33, rpl20, rpl36, rpl14, rpl16 Transfer RNA trnI-CAU # , trnL-CAA # , trnV-GAC # , trnI_GAU # , trnA-UGC # , trnR-ACG # , trnN-GUU # , trnL-UAG, trnN-GUU # , trnR-ACG # , trnA-UGC # , trnI-GAU, trnV-GAC # , trnL-CAA # , trnI-CAU # , trnH-GUG, trnK-UUU, trnM-CAU, trnV-UAC, trnF-GAA, trnL-UAA, trnT-UGU, trnS-GGA, TrnfM-CAU, trnG-GCC, trnS-UGA, trnT-GGU, trnE-UUC, trnY-GUA, trnD-GUC, trnC-GCA, trnR-UCU, trnG-UCC, trnS-GCU, trnQ-UUG, trnW-CCA, trnP-UGG Ribosomal RNAs rrn16 # , rrn23 # , rrn4.5 # , rrn5 # , rrn5 # , rrn4.5 # , rrn23 # , rrn16 # Other genes Maturase RNA processing matK Protease clpP1 c-type cytochrome synthesis gene ccsA Subunit of acetyl-CoA-carboxylase (fatty acid synthesis) accD Envelope membrane protein (carbon metabolism) cemA hypothetical chloroplast reading frames ycf1, ycf2, ycf2 # The present in the IR region has duplicate copies Phylogenetic relationships with other legumes The phylogenetic position of P. tetragonolobus within the Leguminosae family was determined by aligning chloroplast gene sequences with related species, including V. radiata , P. vulgaris , L. purpureus , C. tetragonoloba , G. max , C. cajan , M. truncatula , C. arietinum , and A. hypogaea , with A. thaliana serving as the outgroup. The resulting multigene-based phylogenetic tree showed robust support (bootstrap value 1.0) for all resolved nodes. The analysis revealed two major clades among the studied legume species. A. hypogaea was affiliated with the Dalbergioid clade, while the remaining species formed an Old World clade. C. tetragonoloba , belonging to the Indigoferoid group (Tribe Indigofereae), clustered closely with C. cajan , P. tetragonolobus , G. max , L. purpureus , P. vulgaris , and V. radiata , all of which are part of the Millettioid group under Tribe Phaseoleae within the Phaseoloid clade. Additionally, the Old World clade included C. arietinum (Tribe Cicereae) and M. truncatula (Tribe Trifolieae), which clustered together as part of the Inverted Repeat Lacking Clade (IRLC) within the major Hologalegina clade (Fig. 2 ). Analysis of synteny and genomic rearrangements To identify possible occurrences of rearrangements in the chloroplast genomes, we analysed the synteny of whole chloroplast genome sequences of C. cajan , G. max , and L. purpureus along with A. thaliana as an outgroup species (Fig. 3 ). We used two approaches: a) pairwise and b) global alignment. The pairwise alignments presented high synteny but a strong signature of rearrangements and inversions involving the IR and SSC regions. Specifically, the genomic segment of the SSC region appeared to be inverted in P. tetragonolobus compared to C. cajan and G. max . The IR region flanking the SSC showed high synteny with other legumes. Since the chloroplast is a circular genome, the visible rearrangement was also confirmed with gene order analysis using P. tetragonolobus as a reference. The global alignment, using the shuffle-LAGAN algorithm incorporated in the m-Vista pipeline and winged bean chloroplast genome annotations, indicated that within the region between 24.5 to 46 kb, the exons were comparatively less conserved than the rest of the chloroplast (Fig. 4 ). This was the same region where the inversion appeared to occur in the pairwise synteny analysis. Analysing selective pressure on protein-coding genes To assess selective pressures acting on protein-coding genes in the chloroplast genome, the Ka/Ks ratio was calculated among P. tetragonolobus , C. cajan , G. max , and L. purpureus , along with A. thaliana (Fig. 5 ). The Ka/Ks ratios ranged between 0.001 to 1.4. The highest and only gene with a ratio > 1 was rpl23 in pairwise comparison to A. thaliana . Similarly, rpl23 and an additional gene, rpl2 , exhibited Ka/Ks values of 0.87 and 0.96, respectively, in comparison to C. cajan , suggesting significant evolutionary divergence. When compared with L. purpureus , the only gene with a Ka/Ks value close to 1.0 was ndhB, with a value of 0.8. The remaining genes had Ka/Ks values ≤ 0.5. Comparative analysis of codon usage frequency Sixty-one distinct codons for 20 different amino acids were identified in the chloroplast genome of winged bean (Fig. 6 ). The termination codons were excluded from the analysis. Among the 61 distinct codons, 6 individual codons encoded Arginine, Leucine and Serine; 4 codons encoded Alanine, Glycine, Proline, Threonine, and Valine; and 3 codons encoded Isoleucine; and 2 codons encoded Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Histidine, Lysine, Phenylalanine, and Tyrosine. Methionine and Tryptophan were encoded by 1 codon each. The RSCU values ranged between 0.47 and 4.38, with CGC coding for alanine having the lowest RSCU, while TTT for phenylalanine had the highest. Phenylalanine, lysine, and asparagine were the most abundant amino acids coded by the winged bean genome. Long-repeats and SSRs The analysis of long repeats in the winged bean chloroplast genome showed 38 palindromic, 16 forward, three reverse and two complement repeats, thus in total 59 long repeats (Fig. 7 b). Among them, 38 repeats were in the 30–35 bp range while 5 and 7 repeats were in the 36–40 bp and 41–45 bp range, respectively. Nine repeats were found to be more than 45 bp in length (Fig. 7 a ). The longest repeat was of length 287 bp. Among the 59 long repeats, LSC contained 35 repeats while SSC and IRs contained 6 and 18 repeats, respectively. In total 68% (n = 40) of the 59 repeats were present in the intergenic spacers while the rest 32% (n = 19) were found to be overlapping with gene ycf2, ndhA, ndhF, rps12, rpl22, pafI and psaA ( Supplementary Table S4 ). We identified 84 perfect SSRs, two compound SSRs, and 15 VNTRs (Fig. 7 c; Supplementary Table S5 ) accounting for one perfect SSR per 1.8 kbp, one compound SSR per 75.79 kbp, and one VNTR per 10.10 kbp of the winged bean chloroplast genome. Among the 84 perfect SSR loci analysed, 49 (58.33%) were mononucleotides while the remaining 35 (41.67%) were dinucleotides (Fig. 7 e). A/T repeats were the most frequent mononucleotide repeats (43, 51.19%) followed by G/C (6, 7.14%). Ten bp SSRs were most frequent (53, 63.09%) followed by 11 bp (14, 16.67%), 12 bp (10, 11.90%), 14 bp (4, 4.76%), 16 bp (2, 2.38%) and 15 bp (1, 1.19%). Dinucleotide repeats contained four types of repeat motifs (AG/CT, TA, TC, and AT), of which AT accounted for 22.61% ( 19 ) followed by TA (13, 15.48%), AG/CT (2, 2.38%), and TC (1, 1.19%) (Fig. 7 d). The frequency of the repeat motifs varied from 33.33% (n = 10) to 1.19% (n = 14 &15). A total of six distinct repeat motif types were identified in the study. Different repeat motifs were reiterated 5 to 16 times (Table 3 ). Table 3 Frequency distribution of perfect SSR repeat motifs in winged bean chloroplast genome. S. No. Repeat motif Number of reiterations of the motif Total 5 6 7 10 11 12 14 15 16 1 A/T - - - 23 14 3 - 1 2 43 2 G/C - - - 5 - - 1 - - 6 3 AG/CT 2 - - - - - - - - 2 4 AT 13 3 3 - - - - - - 19 5 TA 9 4 - - - - - - - 13 6 TC 1 - - - - - - - - 1 Total 25 7 3 28 14 3 1 1 2 84 Among the 84 perfect SSRs, two compound SSRs, and 15 VNTRs identified in the study, LSC contained the maximum number of perfect SSRs (57, 67.86%), compound SSRs (02, 100%), and VNTRs (09, 60%) followed by SSC with 18 (21.43%) perfect SSRs and 03 (20%) VNTRs, IRs with 09 (10.71%) perfect SSRs and 03 (20%) VNTRs. Regarding the distribution of SSRs and VNTRs in the genic and non-genic regions, the maximum number of perfect SSRs (57, 67.86%), compound SSRs (02, 100%), and VNTRs (10, 66.67%) were located in the intergenic spacers followed by exon with 16 (19.05%) perfect SSRs and 04 (26.67%) VNTRs. The introns accounted for the lowest number with 11 (13.10%) perfect SSRs and 01 (6.67%) VNTRs. The perfect SSR containing genes were rpl2 , ndhA , ycf1 , rpl2 , trnK-UUU , atpB , pafI , rpoB , rpoC1 , rpoC2 , rps2 , atpF , and rps18 . ycf1 contained 11 perfect SSRs. The VNTR containing genes were ycf2 , rps7 , ccsA , and psbB ( Supplementary Table S5 ). Discussion The legume family (Leguminosae) is economically one of the most successful lineages among flowering plants 33 . It exhibits a significantly higher species diversification rate over the last 60 million years compared to angiosperms as a whole 71 . Within this family, winged bean distinguishes itself with its nutrient-rich components and effective symbiotic associations with a broad spectrum of rhizobia strains, making it suitable for low-input and self-resilient agricultural systems 72 . Chloroplast genomes, with their conserved nature, are crucial resources for studying evolutionary dynamics and phylogenetic relationships across plant taxa 73, 74 . The present study reports the sequencing and characterisation of the chloroplast genome of P. tetragonolobus , along with its comparative analysis with other legumes, including V. radiata , P. vulgaris , L. purpureus , C. tetragonoloba , G. max , C. cajan , M. truncatula , C. arietinum , and A. hypogaea , with A. thaliana serving as the outgroup. Leguminosae is divided into three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae. Caesalpinioideae, a paraphyletic group, is the ancestral base for the monophyletic subfamilies Mimosoideae and Papilionoideae 75 . Papilionoideae, the largest subfamily, comprises 13,800 species across 28 tribes in 478 genera 33 . It is further divided into Swartzioid and Aldinoid lineages and other genera within a larger monophyletic group marked by a 50 kb inversion in the chloroplast genome 24 . The 50 kb inversion group includes three major clades: Genistoids, Dalbergioids, and the Old World clade. The Genistoid clade is characterised by the accumulation of quinolizidine while the Dalbergioid clade typically exhibits "aeschynomenoid" root nodule morphology 76 . The Old World clade further segregates into the Indigoferoid/Millettioid and Hologalegina clades, with the latter splitting into the Robinioid and Inverted Repeat-Lacking Clade (IRLC). Indigofereae is sister to the Millettioid group, comprising Phaseoloid and core Millettieae clades and allies 77, 78 . In recent years, there has been a growing interest in the legume systematics community to combine expertise and data to capitalise on new approaches in genetics and bioinformatics.With the advancement of sequencing technologies, an increasing number of chloroplast genomes have been sequenced and used for phylogenetic analysis. We used the nucleotide sequences of all predicted chloroplast genes of P. tetragonolobus and the reported chloroplast genes for nine other legumes, including V. radiata , P. vulgaris , L. purpureus , C. tetragonoloba , G. max , C. cajan , M. truncatula , C. arietinum , and A. hypogaea , along with A. thaliana as the outgroup, to delineate the phylogenetic position of P. tetragonolobus in relation to the related genera. The multigene-based phylogenetic tree resolved the cladistic position of all the legumes considered for the study with robust bootstrap support. P. tetragonolobus clustered closely with C. cajan , G. max, L. purpureus , P. vulgaris , and V. radiata of the Millettioid group under Tribe Phaseoleae within the Phaseoloid clade. Moreover, all the other legume species considered for the study showed affiliation with their respective clades consistent with the current state of legume phylogeny 79, 80, 81 , reinforcing the utility of chloroplast genome sequences in deep phylogenetic analysis. The comparative analysis of the sequences of the chloroplast genomes of P. tetragonolobus and other legumes revealed clade-wise general conservation in genome size, length of IR, LSC, and SSC regions, along with their GC contents and gene content. As expected, C. arietinum and M. truncatula , belonging to the Hologalegina/ Inverted Repeat Lacking Clade (IRLC), exhibited the smallest genome size and gene contents, attributed to the presence of only a single copy of IR 82, 83, 84 . A. hypogaea , belonging to the Dalbergioid clade, exhibited the largest genome size, whereas the genome sizes for Indigoferoid/Millettioids, comprising P. tetragonolobus , V. radiata , P. vulgaris , L. purpureus , C. tetragonoloba , G. max , and C. cajan , were slightly smaller than the Dalbergioids, ranging from 151,294 bp to 152,530 bp, varying by only 1236 bp. These results indicate a notable degree of genomic homogeneity among the leguminous species under investigation. Moreover, they highlight the strength of the cladistic approach to biological classification based on the hypotheses of most recent common ancestry. We observed a notable uniformity in GC content across the LSC, SSC, and IR regions among various legume species. Furthermore, the GC content of tRNAs and rRNAs was significantly higher than that of protein-coding genes. Notably, a proportionately higher number of GC-rich tRNAs and rRNAs in the IR regions contributed to their overall higher GC content compared to the LSC and SSC regions. These GC-rich regions ensure structural integrity and functional resilience across diverse taxa 85, 86, 87 . The synteny analysis of whole chloroplast sequences from P. tetragonolobus , C. cajan , G. max , and L. purpureus , along with A. thaliana , provided valuable insights into the structural variations within these genomes. Pairwise alignments revealed high synteny among the chloroplast genomes but also unveiled a notable signature of rearrangements and inversions, particularly within the IR and SSC regions. Notably, the SSC segment exhibited an inversion in P. tetragonolobus compared to C. cajan and G. max , indicating a structural deviation specific to this species. Complementing the pairwise analysis, global alignment using the shuffle-LAGAN algorithm highlighted a region of reduced exon conservation spanning from 24.5 to 46 kb within the chloroplast genomes. This region corresponded to the site of inversion observed in the pairwise synteny analysis, reinforcing the presence of rearrangements within this segment of the chloroplast genome. The observed rearrangements may be attributed to flip-flop intramolecular recombination in the plastome, a mechanism proposed by Ogihara et al. 88 . While such events are rare, recent studies 89, 90 highlight their significance as evolutionary drivers, potentially conferring adaptive advantages. Identifying structural variations within chloroplast genomes, such as inversions and rearrangements, underscores the dynamic nature of plastome evolution. Understanding the mechanistic underpinnings and functional implications of these rearrangements offers valuable insights into the evolutionary trajectories of plant species within the Leguminosae family. The Ka/Ks ratio value, which infers the rate of gene divergence between species, serves as an indicator to identify genes undergoing different selection pressures 91, 92 . In protein-coding genes, synonymous substitutions occur more frequently than non-synonymous substitutions 91, 93 . In this study, since the majority of genes had values between 0 and 0.5, this is a strong signature representing that in P. tetragonolobus , nonsynonymous mutations are being removed from the population at a faster rate than synonymous mutations. Thus, the genes are under purifying selection and tend to maintain their required functions. Meanwhile, the only gene observed to be under diversifying selection is rpl23 . This gene has been reported to be deleted, duplicated, and accumulate mutations not only in legumes but also in other plant species, including cereal crops 29, 94–100 . Among the 64 codons directing protein synthesis, 61 encode standard amino acids, while 3 serve as translation stop signals. Most amino acids have multiple synonymous codons, except for tryptophan and methionine, typically encoded by one codon each 101 . The degeneracy of the genetic code allows the same amino acid to be encoded by different codons 102, 103 . However, codon usage varies among organisms, genes, and even the same gene from different species, resulting in codon usage bias 104, 105 . Codon usage bias leads to non-random appearance of synonymous codons with different frequencies 106, 107 . Codon bias impacts numerous cellular processes, such as mRNA stability, transcription, translation efficiency, and protein expression and cotranslation folding 108–110 . It influences chromatin structure and mRNA folding, thereby regulating transcription levels and translation efficiency by modulating the elongation rate of translation 108–111 . Codon bias analysis aids in revealing horizontal gene transfer and evolutionary relationships between closely related organisms 112, 113 . The RSCU value compares the observed frequency of a specific synonymous codon to the expected frequency (no codon usage bias). A value of 1.0 suggests no bias, with equal codon usage for that amino acid. Values above 1.0 indicate positive bias, while those below 1.0 indicate negative bias. RSCU values exceeding 1.6 or falling below 0.6 indicate overrepresented and underrepresented codons, respectively 38, 114 . Excluding the termination codon, we found 61 codons for 20 amino acids in the winged bean chloroplast genome, with RSCU values ranging from 0.47 to 4.38. Notably, CGC, encoding alanine, exhibited the lowest RSCU, while TTT, encoding phenylalanine, showed the highest RSCU. Phenylalanine, lysine, and asparagine were the most abundant amino acids encoded by the winged bean genome. Among the various synonymous codons for amino acids such as arginine, asparagine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, and tyrosine in the winged bean chloroplast genome, codons ending with either A or U were overrepresented. This bias towards codons ending with A or U may be attributed to the higher AT content of chloroplast genomes, resulting from mutation and natural selection processes 38, 115 . Understanding repeat sequences within genomes is critical for deciphering evolutionary patterns and genetic diversity 116, 117 . In the present study, we identified 59 repeats, primarily concentrated in intergenic spacers, consistent with patterns in other legumes studied recently 82, 118, 119 . A large proportion of repeats were also found in the genes namely ycf2, ndhA, ndhF, rps12, rpl22, pafI and psaA , indicating potential functional implications. The prevalence of repeats in intergenic regions highlights their role in genomic rearrangements and evolutionary dynamics 120, 121 . Leveraging these conserved patterns as genetic markers can enhance phylogenetic and population studies in legumes, providing insights into chloroplast genome evolution and plant adaptation 120–122 . We identified 84 perfect SSRs, two compound SSRs, and 15 VNTRs in the chloroplast genome of winged bean. Their distribution in the LSC, SSC, and IR regions was generally similar to that observed in other legumes 99, 82, 118, 123, 124,125 . Typically, a significant proportion of SSRs in genic regions consist of trinucleotide repeats, which help mitigate the detrimental effects of frame-shift mutations 126, 127 . However, in our study, we found only mono- and dinucleotide repeats, predominantly concentrated in intergenic spacers and introns rather than exons. This may help counteract the detrimental effects of frame-shift mutations caused by mono- and dinucleotide repeats in genic regions 128–130 . The SSR markers identified in the chloroplast genome of the winged bean are of significant advantage in evolutionary and taxonomic research due to their maternal inheritance and lower mutation rates 126, 127 . In conclusion, the study of the chloroplast genome of P. tetragonolobus and its comparative analysis with other legumes has provided valuable insights into the evolutionary dynamics and phylogenetic relationships within the legume family. The findings highlight the Andrews, utility of chloroplast genome sequences in deep phylogenetic analysis and support the strength of the cladistic approach to biological classification based on the hypotheses of most recent common ancestry. The observed genomic homogeneity among the leguminous species under investigation and the uniformity in GC content across different regions underscores the conserved nature of chloroplast genomes within this plant family. These results contribute to our understanding of legume systematics and emphasise the importance of combining expertise in genetics and bioinformatics to capitalise on new approaches for studying plant evolutionary biology. Declarations Acknowledgment We acknowledge the funding and logistic support provided by Director, ICAR-IIAB, Ranchi. Authors Contribution KUT – Sequencing, data analysis, NKS – Data analysis, AG – Data analysis, HS - Data analysis and submission of sequences to NCBI, AP- Data analysis, SK- Generated the plant material, VPB-Coordinated the project, SR-Coordinated the project and manuscript editing, APT- Coordinated the project, BKS- Planning the experiment, sequencing and writing the manuscript. 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BMC Plant Biol. 23 , 15; 10.1186/s12870-022-04028-3 (2023). Yang, S., Gun, L. & Hao, L. Molecular characterisations of genes in chloroplast genomes of the genus Arachis L. (Fabaceae) based on the codon usage divergence. PloS One 18 (3), e0281843; 10.1371/journal.pone.0281843 (2023). Xin, D. et al. Identification and characterization of SSRs from soybean ( Glycine max ) ESTs. Mol. Biol. Rep. 39 , 9047-9057 (2012). Thumilan, M. et al. Development and characterization of genic SSR markers from Indian Mulberry transcriptome and their transferability to related species of Moraceae. PLoS One 11 (9), e0162909; 10.1371/journal.pone.0162909 (2016). Weisschuh, N., Elena, B.-A. & Bernd W. Splicing mutations in inherited retinal diseases. Prog. Retin Eye Res. 80 , 100874; 10.1016/j.preteyeres.2020.100874 (2021). Zhu, L. et al. Short tandem repeats in plants: Genomic distribution and function prediction. Electron. J. Biotechnol. 50 , 37–44 (2021). Xu, Y., Miaomiao, X., Lixiao, S., Jiyong, Y., Wenjiang, L. & Aisong, Z. Genome-wide analysis of simple sequence repeats in cabbage ( Brassica Oleracea L.). Front. Plant Sci. 12 , 726084; 10.3389/fpls.2021.726084 (2021). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.docx SupplementaryTableS2.docx SupplementaryTableS3.docx SupplementaryTableS4.csv SupplementaryTableS5.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4615004","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":326644486,"identity":"83b8391c-e662-4757-81e1-6f234e393202","order_by":0,"name":"Nikhil Kumar Singh","email":"","orcid":"","institution":"ICAR-Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Nikhil","middleName":"Kumar","lastName":"Singh","suffix":""},{"id":326644487,"identity":"6d41d5ab-0ab0-425a-864e-3a15aca48a68","order_by":1,"name":"Binay K. 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Tribhuvan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie3SMUvEMBTA8VcetEt6XV+o9DNECj0E8bsEQTcXl4JaC4V27FrBDyEITg6BQLucOLudHHRyUJzcLJx3HJK74zaR/KeQ5EcSCIDN9gcjBHc+QjYfBLhcdPJNhJaEF9sILAj8EKG2XIxXXj/9eIRsXPntzC+PzuIOXz9TuIqEwnJqICGy8X7TA+3p0Wnsl8fniXZjPoEuFsqphIFEwxNCpoAIWRKyCcoHzYDn0Mqb3CnJSLx+lVzL+4Lh1yYSIiQrJNXybjh3OOVS1mAmvGAJbxTxBkcn/DbtZKPd5CAXKg7QTOi56+ldHQYUPLX0Ji5kXevZS55mketVvYks5O8JoYf/sH6/sWzH/TabzfaP+wbpFU8nibVsCwAAAABJRU5ErkJggg==","orcid":"","institution":"ICAR-Indian Institute of Agricultural Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Kishor","middleName":"U.","lastName":"Tribhuvan","suffix":""}],"badges":[],"createdAt":"2024-06-21 05:25:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4615004/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4615004/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60680101,"identity":"fdb50fff-2e3a-4229-b6e6-02338ac3ee59","added_by":"auto","created_at":"2024-07-19 12:08:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":492036,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome map of \u003cem\u003ePsophocarpus tetragonolobus\u003c/em\u003e. Genes showed on the inner side are transcribed anti-clockwise while genes presented on the outer side are transcribed clockwise. Genes are color coded according to their functions as mentioned within the figure. The genome is divided into Inverted repeat B (IRB), Inverted repeat A (IRA), Small Single Copy (SSC) and Large Single Copy (LSC) region\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/f3ca99ca81fb8ff27226e09d.png"},{"id":60680106,"identity":"0662b842-d813-4e6d-9dfe-0cdb1b5ba259","added_by":"auto","created_at":"2024-07-19 12:08:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177990,"visible":true,"origin":"","legend":"\u003cp\u003eGene-based phylogeny construction. Chloroplast genes (n=70) based on maximum likelihood Phylogenetic tree of 11 species. Respective node lengths are written above the branches, while bootstrap values are represented with weighted circular bubbles. The respective clade names are mentioned at the node of diversion, highlighted with dark blue circles.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/542bb9bfa4d564810ac21dab.png"},{"id":60680105,"identity":"0a37d3c3-1368-4236-91a5-3bdf851f909c","added_by":"auto","created_at":"2024-07-19 12:08:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":880183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynteny analysis between five species including \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP.tetragonolobus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Chloroplast synteny block analysis performed using BLASTn with the default parameters and visualised using genoplotR (Guy et al., 2010). Only syntenic regions with ≥100 bp are shown. Traces connecting cDNA genomes represent synteny blocks, while the gene arrowhead represents the forward or reverse direction of the gene. The genes share the same gene colour code as in genome figure 1. The red gradient segments represent the percentage of sequence identity from BLASTN alignments; thus, darker colors indicate higher identity.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/625459e0288b86280914766f.png"},{"id":60680592,"identity":"bba69955-48e9-4efb-9ed1-9b9a6d572e19","added_by":"auto","created_at":"2024-07-19 12:16:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1346180,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of \u003cem\u003eArabidopsis thalina\u003c/em\u003e and three other legume species using whole chloroplast genome alignment. \u003cem\u003eP. tetragonolobus \u003c/em\u003ewas used as the reference for the alignment. The arrows indicate the length and direction of the gene. The color legends have been included within the figure.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/ee8c3fd0a2df8d9fba86269b.png"},{"id":60680102,"identity":"80d8c370-048c-4fc3-a10e-c561ae140c6f","added_by":"auto","created_at":"2024-07-19 12:08:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":441625,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of non-synonymous and synonymous substitution within the gene coding sequences of winged bean when compared to three other legume species. \u003cem\u003eA.thaliana\u003c/em\u003e was taken as an outgroup. Species are color coded and described within the figure.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/3e1622dbc69364eef744f446.png"},{"id":60680591,"identity":"8e10f546-8597-4d4f-9f9f-4e721af6b383","added_by":"auto","created_at":"2024-07-19 12:16:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":337979,"visible":true,"origin":"","legend":"\u003cp\u003eRelative synonymous codon usage (RSCU) for codons for coding their respective amino acids\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/a39732e6a23500147fe8bbc7.png"},{"id":60680593,"identity":"686a7ed4-80bd-4f91-9001-1fc2190d2c27","added_by":"auto","created_at":"2024-07-19 12:16:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":232351,"visible":true,"origin":"","legend":"\u003cp\u003eRepeat content and SSR analysis analysis in \u003cem\u003eP. tetragonolobus\u003c/em\u003e chloroplast\u003cstrong\u003e. \u003c/strong\u003ea) Count distribution of repeats found in different length brackets b) Repeats count based on their type F (forward), R (reverse), P (palindromic) or C (complement) c) distribution of perfect and compound SSRs along with variable number tandem repeats (VNTRs) d-e) Difference in the presence of nucleotides defined in the SSRs.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/7fdf2ac7b995953254934a55.png"},{"id":74351963,"identity":"96351afc-50b1-4c6f-b5c2-7547f062493b","added_by":"auto","created_at":"2025-01-21 11:02:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5679183,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/70afa0e9-5eb8-4b92-8752-ea7dd757d506.pdf"},{"id":60680590,"identity":"703ea921-4e49-4d4f-97e2-74939e1d6aef","added_by":"auto","created_at":"2024-07-19 12:16:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14178,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/52c4311433a1299da1439a03.docx"},{"id":60680099,"identity":"64408978-535a-4506-9755-7c9a5beac335","added_by":"auto","created_at":"2024-07-19 12:08:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22674,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/afdb6106201986ad1c7bd7df.docx"},{"id":60680103,"identity":"997dae74-dcde-40f7-8fbc-8b7bbf18374b","added_by":"auto","created_at":"2024-07-19 12:08:39","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":52849,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/8244a06b7f5f97617248d34b.docx"},{"id":60681049,"identity":"6b262417-4457-4190-a059-ed6cbb25c79e","added_by":"auto","created_at":"2024-07-19 12:24:40","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2807,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS4.csv","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/de8995b6ee984a3ab8b6bdb3.csv"},{"id":60680110,"identity":"9f799927-7695-4d3b-873a-7ea0a5ad8308","added_by":"auto","created_at":"2024-07-19 12:08:40","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":54661,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-4615004/v1/6c36fbd57bcc2495b1585991.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chloroplast genome sequencing in winged bean (Psophocarpus tetragonolobus L.) and comparative analysis with other legumes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWinged bean (\u003cem\u003ePsophocarpus tetragonolobus\u003c/em\u003e (L.) DC.) (2n\u0026thinsp;=\u0026thinsp;2\u0026times; = 18; 782 Mbp) is an underutilised legume of the family Leguminosae \u003csup\u003e1, 2\u003c/sup\u003e. It grows in hot, humid, equatorial countries of Southern Asia, Melanesia, and the Pacific area \u003csup\u003e3\u003c/sup\u003e. It is a short-day self-pollinated species but can experience cross-pollination up to 16.0% \u003csup\u003e4\u003c/sup\u003e. This tropical legume, maturing in 4\u0026ndash;5 months, yields up to 14 quintals of dry seeds and 115 quintal tubers per hectare, even with minimal management \u003csup\u003e5\u003c/sup\u003e. Its rapid growth and vining nature make it suitable for use as a cover crop \u003csup\u003e6, 7\u003c/sup\u003e. Moreover, it is highly suited for crop rotation or intercropping systems as it forms symbiotic associations with a broad-spectrum cowpea rhizobial group \u003csup\u003e8\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eWinged bean is often referred to as the 'one-stalk supermarket' due to its versatile culinary applications worldwide. Various plant parts, including leaves, branches, flowers, seeds, fruits, and tubers, are utilised \u003csup\u003e3, 9\u003c/sup\u003e. Often dubbed the \"soybean of the tropics,\" winged bean seeds offer crude protein (~\u0026thinsp;34.0%), similar to soybean (~\u0026thinsp;35.0%) \u003csup\u003e10\u003c/sup\u003e. Additionally, it is rich in fiber, vitamins, minerals, and carbohydrates. Winged bean seeds contain 15\u0026ndash;18% fat, constituted by 30\u0026ndash;40% saturated and 60\u0026ndash;70% unsaturated fatty acids \u003csup\u003e11\u003c/sup\u003e. Because of its high oxidative stability, solid fat content, and good thermal conductivity, winged bean seed oil is considered superior to soybean oil \u003csup\u003e12\u003c/sup\u003e. Winged bean seed powder is a valuable flour that can be brewed to make a coffee-like drink \u003csup\u003e13\u003c/sup\u003e. The winged bean tubers have ivory flesh and contain 12\u0026ndash;19% protein and 1\u0026ndash;4% fat \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe origin of winged bean is subject to debate with two primary hypotheses. The first hypothesis suggests an African origin, proposing either \u003cem\u003ein situ\u003c/em\u003e domestication followed by migration to the east or trans-domestication from an African progenitor species later in the Indian Ocean rim of Asia \u003csup\u003e2, 3\u003c/sup\u003e. The second hypothesis posits that winged bean is distinct from current African members of the genus and arose through allopatric speciation preceding any domestication processes \u003csup\u003e15\u003c/sup\u003e. However, the precise origin and progenitor of winged bean remain unresolved.\u003c/p\u003e \u003cp\u003eChloroplasts originated from a cyanobacterium through endosymbiosis with a eukaryotic host around a billion years ago, creating an autotrophic line of nucleus-containing cells \u003csup\u003e16\u003c/sup\u003e. Throughout evolution, they retained essential genes for replication, transcription, photosynthesis, and several other critical metabolic pathways while losing much of their original genome. Chloroplast genomes are mostly maternally inherited in angiosperms and paternally inherited in gymnosperms \u003csup\u003e17\u003c/sup\u003e. Angiosperm chloroplast genomes typically have a quadripartite structure, ranging in size from 120 kb to 200 kb, including a double-stranded closed loop with a long single-copy sequence (LSC, 80 kb-90 kb), a short single-copy sequence (SSC, 16 kb-27 kb), and two inverted repeats (IRs, 20 kb-28 kb) with roughly equal length. The IRs divide the chloroplast genome into LSC and SSC regions \u003csup\u003e18, 19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChloroplast genomes exhibit relative stability and conservation across species, with a lower mutation rate compared to the nuclear genome \u003csup\u003e20\u003c/sup\u003e. Nevertheless, diversification occurs in chloroplast genomes, resulting in variations in size and organisation. The expansion/contraction or loss of IRs and gene loss/duplication outside the IR are major factors contributing to size variation \u003csup\u003e21\u003c/sup\u003e. Loss of IRs leads to a dynamic arrangement of the chloroplast genome \u003csup\u003e22, 23\u003c/sup\u003e. Within the Leguminosae family, chloroplast genomes have undergone extensive rearrangements, with some legume species experiencing complete loss of one copy of the IR \u003csup\u003e24, 25\u003c/sup\u003e. The Genistoids, Dalbergioids, and the Old World clades of the Leguminosae species displayed several inversions, particularly a 50 kb inversion in the LSC region \u003csup\u003e26\u003c/sup\u003e. Additionally, during plant evolution, chloroplast genes have been lost, with some transferred to the nucleus, such as \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003einfA\u003c/em\u003e, and \u003cem\u003eaccD\u003c/em\u003e genes \u003csup\u003e27, 28, 29\u003c/sup\u003e. The loss of introns from \u003cem\u003erps12\u003c/em\u003e and \u003cem\u003eclpP\u003c/em\u003e is also reported in the legume genome \u003csup\u003e22, 27\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChloroplast genomes have been extensively used in evolution, phylogeny, and phylogeography studies \u003csup\u003e30, 31, 32\u003c/sup\u003e. In the Leguminosae family, comprising approximately 751 genera and 19,500 species ranging from trees to herbaceous crop plants \u003csup\u003e33\u003c/sup\u003e, chloroplast-derived markers, particularly \u003cem\u003ematK\u003c/em\u003e gene and the \u003cem\u003etrnL-trnF\u003c/em\u003e intergenic spacer have played a crucial role in exploring evolutionary relationships \u003csup\u003e34, 35, 36\u003c/sup\u003e. Chloroplast genomic data have also been extensively employed to study gene expression and regulation, including RNA editing sites and codon usage bias \u003csup\u003e37, 38, 39\u003c/sup\u003e. Besides, simple sequence repeats (SSRs) within chloroplast genomes are potential DNA markers for species identification \u003csup\u003e40\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eChloroplast genetic engineering offers unique advantages such as high-level transgene expression \u003csup\u003e41\u003c/sup\u003e, multigene engineering in a single transformation event \u003csup\u003e42\u003c/sup\u003e, transgene containment \u003cem\u003evia\u003c/em\u003e maternal inheritance \u003csup\u003e43\u003c/sup\u003e, lack of gene silencing \u003csup\u003e44\u003c/sup\u003e, absence of position effect \u003csup\u003e45\u003c/sup\u003e, pleiotropic effects \u003csup\u003e46\u003c/sup\u003e, and prevention of undesirable foreign DNA \u003csup\u003e47\u003c/sup\u003e. Complete chloroplast genome sequences are essential for identifying spacer regions for transgene integration at optimal sites through homologous recombination, as well as for determining endogenous regulatory sequences for optimal transgene expression \u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTraditional methods for obtaining chloroplast genome sequences involve chloroplast DNA isolation, random shearing, cloning into large-insert size vectors, and shotgun sequencing. Recent advancements, such as whole-genome PCR amplification with universal primers and high-throughput sequencing, have introduced faster and cost-effective approaches \u003csup\u003e49, 50, 51\u003c/sup\u003e. Next Generation Sequencing (NGS) technology, particularly platforms like Illumina, has significantly accelerated chloroplast genome sequencing. Moore et al.\u003csup\u003e52\u003c/sup\u003e made the pioneering attempt to use NGS for chloroplast genome sequencing, leading to the sequencing of numerous chloroplast genomes, with Illumina being the most commonly used platform \u003csup\u003e53, 54, 55, 56\u003c/sup\u003e. Despite over 44 published chloroplast genomes within the Leguminosae family, the chloroplast genome of winged bean remains unexplored. In this study, genomic DNA from fresh young leaves of winged bean was sequenced using the Illumina HiSeq2500 platform, and the chloroplast genome was assembled using the embryophyta plastid database as reference. The study elucidated the winged bean chloroplast genome sequence and its characteristics and compared it with those of other Leguminosae species. This research aims to enhance our understanding of the winged bean chloroplast genome and provide valuable markers for phylogenetic and genetic studies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eThe study utilised the dual-purpose cultivar AKWB-1 of winged bean, which serves both as a vegetable and a pulse. Given the significant level of cross-pollination reported in winged bean, the AKWB-1 plants were selfed for three successive generations to achieve homozygosity. The winged bean plants were grown at the experimental farm of the ICAR-Indian Institute of Agricultural Biotechnology, located in Ranchi, Jharkhand, India, with the geographical coordinates 23\u0026deg;16'27.6\"N, 85\u0026deg;20'29.4\"E.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA isolation, library preparation, sequencing, and annotation\u003c/h2\u003e \u003cp\u003eThe young leaves of winged bean were utilised to extract high molecular weight genomic DNA using the Cetyl Trimethyl Ammonium Bromide (CTAB) method \u003csup\u003e57\u003c/sup\u003e. To remove RNA contamination from the DNA, 2.0 \u0026micro;l of RNase A (10 mg/ml, HiMedia) was added to 20 \u0026micro;l of DNA dissolved in TE buffer (Tris\u0026ndash;EDTA, pH\u0026thinsp;=\u0026thinsp;8.0), followed by incubation at 37\u0026deg;C for 3\u0026ndash;4 h. The quality of the purified DNA samples was analysed on a 1.0% agarose gel and quantified based on the absorbance at 260 nm using a NanoDrop\u0026trade; OneC microvolume UV\u0026ndash;Vis spectrophotometer (Thermo Scientific). The whole-genome sequencing library was constructed using the TruSeq DNA PCR-Free Library Prep Kit (Illumina, catalog no. 20015963) following the manufacturer\u0026rsquo;s instructions. The prepared library was quantified using a Qubit 3 fluorometer (Thermo Fisher Scientific, USA) with the DNA High Sensitivity kit, and the library size was verified using a Bioanalyzer 2100 (Agilent Technologies, CA, USA). After assessing the quality and quantity of the library, whole-genome sequencing was conducted on a Illumina HiSeq2500 instrument with paired-end (2 \u0026times; 150 bp) sequencing strategy using the TruSeq Rapid SBS Kit (Illumina, catalog no. FC-402-4023). FastQC v0.11.9 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to evaluate the quality of sequencing data, including base qualities, GC content, adapter content, and overrepresentation analysis. Adapter sequences were trimmed using fastp v0.20.1 software \u003csup\u003e58\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with a minimum length of two bases, quality filtering disabled, and forced poly-G trimming. The high-quality reads were \u003cem\u003ede novo\u003c/em\u003e assembled into the chloroplast genome using the GetOrganelle v1.5.1c toolkit \u003csup\u003e59\u003c/sup\u003e, specifying the embryophyta plastid database. Annotation of the complete chloroplast genome was performed with GeSeq and manual corrections \u003csup\u003e60\u003c/sup\u003e. The complete chloroplast genome sequence of the winged bean was submitted to GenBank with the accession number PP894786.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe nucleotide sequences of all predicted chloroplast genes of \u003cem\u003eP. tetragonolobus\u003c/em\u003e and the reported chloroplast genes for nine other related species, including \u003cem\u003eVigna radiata\u003c/em\u003e, \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e, \u003cem\u003eLablab purpureus\u003c/em\u003e, \u003cem\u003eCyamopsis tetragonoloba\u003c/em\u003e, \u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003eCajanus cajan\u003c/em\u003e, \u003cem\u003eMedicago truncatula\u003c/em\u003e, \u003cem\u003eCicer arietinum\u003c/em\u003e, and \u003cem\u003eArachis hypogaea\u003c/em\u003e, along with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e as the outgroup, were obtained from NCBI. Alignment and subsequent phylogenetic analysis were conducted using the genes shared across the species. The sequences were aligned using MAFFT v7 \u003csup\u003e61\u003c/sup\u003e with 1000 iterative refinement steps using \"--maxiterate 1000\". The resulting aligned sequences were saved in FASTA format. Phylogenetic trees for each aligned gene were inferred using RAxML-NG \u003csup\u003e62\u003c/sup\u003e with 1000 Bootstrap replicates and the \"General Time Reversible (GTR) with gamma-distributed rate variation among sites (GTR\u0026thinsp;+\u0026thinsp;G)\" model. The best genetree files were used to prepare a multigene-based species tree with ASTRAL v5.7.8 \u003csup\u003e63\u003c/sup\u003e. Finally, the phylogenetic tree was visualised using FigTree v1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSynteny and genome rearrangement analysis\u003c/h2\u003e \u003cp\u003eWe downloaded the chloroplast genomes of \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, and \u003cem\u003eL. purpureus\u003c/em\u003e, along with \u003cem\u003eA. thaliana\u003c/em\u003e as an outgroup species from CpGDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gndu.ac.in/CpGDB/\u003c/span\u003e\u003cspan address=\"https://www.gndu.ac.in/CpGDB/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and analysed the synteny and rearrangement in the chloroplast genomes of \u003cem\u003eP. tetragonolobus\u003c/em\u003e and related legumes using two different methods. For synteny plots, pairwise BLASTN results were generated using the whole chloroplast genome of each species in R using a custom script. Information on BLAST hits was visualised in R using the genoPlotR package \u003csup\u003e64\u003c/sup\u003e. Subsequently, the alignments were annotated using species-specific GFF3 specifications. Information on BLAST hits among homologous segments was also visualised in R using the genoplotR package \u003csup\u003e64\u003c/sup\u003e. As a complementary approach, we performed global alignment of the chloroplast genomes with \u003cem\u003eP. tetragonolobus\u003c/em\u003e as the reference. The alignment was conducted on the online platform mVISTA in Shuffle-LAGAN mode \u003csup\u003e65\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of adaptive evolution of protein-coding genes\u003c/h2\u003e \u003cp\u003eTo analyse the degree of non-synonymous (Ka) and synonymous (Ks) substitutions, as well as their ratio (dN/dS), the coding DNA sequences (CDS) of \u003cem\u003eP. tetragonolobus\u003c/em\u003e were compared with \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, and \u003cem\u003eL. purpureus\u003c/em\u003e, along with \u003cem\u003eA. thaliana.\u003c/em\u003e For this, we performed pairwise alignments using MAFFT v7 \u003csup\u003e66\u003c/sup\u003e, and then Ka, Ks substitutions, and their ratio were calculated using KaKs_Calculator 3 \u003csup\u003e67\u003c/sup\u003e with the MA model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCodon usage analysis\u003c/h2\u003e \u003cp\u003eThe codon usage within the coding part of the chloroplast was calculated using CodonW v1.4.4 available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://codonw.sourceforge.net\u003c/span\u003e\u003cspan address=\"https://codonw.sourceforge.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e using universal codon standards. RSCU (relative synonymous codon usage) values were plotted using R package ggplot \u003csup\u003e68\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRepeat Analysis\u003c/h2\u003e \u003cp\u003eThe long repeats in the winged bean chloroplast genome was analysed using REPuter \u003csup\u003e69\u003c/sup\u003e. Repeats were reported as either F (forward), R (reverse), P (palindromic), or C (complement), with parameters set at a Hamming Distance of 3 and a Minimal Repeat Size of 30. The chloroplast genome was also screened for perfect SSRs (pSSRs), compound SSRs (cSSRs), and variable number tandem repeats (VNTRs) using Krait v1.3.3 software \u003csup\u003e70\u003c/sup\u003e. The screening was conducted based on specific criteria: mono-nucleotide repeat motifs were required to have a minimum of 10 repeats, di-nucleotide repeat motifs required a minimum of five repeats, tri-nucleotide repeat motifs needed at least five repeats, while tetra-, penta-, and hexa-nucleotide repeat motifs were required to have a minimum of four repeats. If the distance between two SSRs was \u0026lt;\u0026thinsp;10 bp, they were considered as cSSR.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssembly and annotation of winged bean chloroplast genome\u003c/h2\u003e \u003cp\u003eWe used the Illumina HiSeq2500 next-generation sequencing platform for whole-genome sequencing of winged bean, generating 1,030,974,930 paired-end raw reads. After cleaning for adaptors and low-quality reads, 1,030,150,624 clean reads were obtained. Subsequently, the GetOrganelle v1.5.1c toolkit was employed to assemble the chloroplast genome from the winged bean whole-genome sequencing data. Using a modified baiting and iterative mapping approach, GetOrganelle successfully recruited 36,123,587 chloroplast-associated clean reads, constituting 3.51% of the total clean reads. This approach facilitated the assembly of the complete winged bean chloroplast genome, totaling 151,571 bp, with \u0026gt;\u0026thinsp;35,000X coverage (\u003cb\u003eSupplementary Table S1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe chloroplast genome of winged bean exhibited a typical quadripartite structure, consisting of an LSC region of 82,736 bp, an SSC region of 17,777 bp, and a pair of equal-sized IRs each measuring 25,529 bp. The winged bean chloroplast genome had an overall GC content of 35.26%, with the LSC, SSC, and IR regions showing GC contents of 32.63%, 28.55%, and 41.86%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the winged bean chloroplast genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAttribute\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStart\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSize (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdenine (A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThymine (T)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGuanine (G)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCytosine\u003c/p\u003e \u003cp\u003e(C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAT (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGC (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWhole chloroplast genome (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e151571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49135 (32.42%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48995 (32.32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e26888 (17.74%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e26553 (17.52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e64.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e35.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLarge single-copy region (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e82736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27979 (33.82%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27763 (33.56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13833 (16.72%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13159 (15.90%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e67.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e32.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSmall single-copy region (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6313 (35.51%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6388 (35.93%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2368 (13.32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2707 (15.23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e71.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e28.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInverted repeat B (IRb) (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7379 (28.90%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7462 (29.23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5547 (21.72%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5140 (20.13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e41.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInverted repeat A (IRa) (bp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7462 (29.23%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7380 (28.90%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5140 (20.13%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5547 (21.73%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e41.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrt. coding genes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24940 (31.97%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e24929 (31.95%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14292 (18.32%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13843 (17.75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e63.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e36.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003etRNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2746\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e654 (23.82%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e639 (23.27%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e718 (26.15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e735 (26.77%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e47.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e53.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003erRNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1951 (22.51%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1952 (22.52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2381 (27.47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2383 (27.49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e45.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e54.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe comparison of the chloroplast genome of winged bean with the chloroplast genomes of related legume species, including \u003cem\u003eV. radiata\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eC. tetragonoloba\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eC. arietinum\u003c/em\u003e, and \u003cem\u003eA. hypogaea\u003c/em\u003e, along with \u003cem\u003eA. thaliana\u003c/em\u003e indicated that although the sizes of the overall genome had differences, the GC content was similar in LSC, SSC, and IR regions of different species. We observed a little difference in total genes, CDS and tRNAs among the ten legume species. \u003cem\u003eC. tetragonoloba\u003c/em\u003e exhibited the maximum number of protein coding genes, CDS and tRNAs and \u003cem\u003eC. arietinum\u003c/em\u003e showed the least (\u003cb\u003eSupplementary Table S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe chloroplast genome of winged bean consisted of 130 genes, including 85 protein-coding genes, 37 tRNAs, and 8 rRNAs genes. Individually, LSC contained 83 genes, including 61 protein-coding and 22 tRNAs genes while SSC contained 13 genes, including 12 protein-coding and one tRNA genes. The IR regions contained 17 duplicated copies of 6 protein-coding genes, 7 tRNAs, and 4 rRNAs genes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, 24 intron-containing genes (14 protein-coding genes, 8 tRNA genes, and 2 rRNA genes) were found. Among these, 22 genes had one intron, and \u003cem\u003eclpP\u003c/em\u003e and \u003cem\u003epafI\u003c/em\u003e had two introns each. \u003cem\u003etrnK-UUU\u003c/em\u003e had the largest intron (2585 bp) and \u003cem\u003errn23\u003c/em\u003e had the smallest intron (198 bp) (\u003cb\u003eSupplementary Table S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe functional classification of winged bean chloroplast genes indicated that 47 genes had photosynthesis-related functions, including genes for photosystem I, photosystem II, Cytochrome b/f complex, ATP synthase, NADH dehydrogenase, Rubisco large subunit, and Photosystem assembly stability factor. Similarly, 75 genes were categorised for transcription and translational related functions, which included RNA polymerases, ribosomal protein small subunit, ribosomal protein large subunit, tRNA, and rRNAs. The remaining 8 genes, including maturase RNA processing, protease gene, c-type cytochrome synthesis gene, subunit of acetyl-CoA-carboxylase (fatty acid synthesis) gene, envelope membrane protein (carbon metabolism), and hypothetical chloroplast reading frames, displayed other functions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChloroplast encoding genes in \u003cem\u003ePsophocarpus tetragonolobus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003ePhotosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsaA, psaB, psaC, psaI, psaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCytochrome b/f complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatpA, atpB, atpE, atpF, atpH, atpI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epetA, petB, petD, petL, petG, petN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNADH dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003endhA, ndhB\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003endhB\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003endhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRubisco large subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem assembly stability factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epafI, pafII, pbf1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eTranscription and Translation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpoA, rpoB, rpoC1, rpoC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal protein (SSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps7, rps12\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003erps15, rps12\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003erps7, rps4, rps14, rps2, rps16, rps18, rps12, rps11, rps8, rps3, rps22, rps19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal protein (LSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl2\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003erpl23\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003erpl32, rpl23\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003erpl2\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003erpl33, rpl20, rpl36, rpl14, rpl16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransfer RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrnI-CAU\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnL-CAA\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnV-GAC\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnI_GAU\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnA-UGC\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnR-ACG\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnN-GUU\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnL-UAG, trnN-GUU\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnR-ACG\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnA-UGC\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnI-GAU, trnV-GAC\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnL-CAA\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnI-CAU\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrnH-GUG, trnK-UUU, trnM-CAU, trnV-UAC, trnF-GAA, trnL-UAA, trnT-UGU, trnS-GGA, TrnfM-CAU, trnG-GCC, trnS-UGA, trnT-GGU, trnE-UUC, trnY-GUA, trnD-GUC, trnC-GCA, trnR-UCU, trnG-UCC, trnS-GCU, trnQ-UUG, trnW-CCA, trnP-UGG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn16\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn23\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn4.5\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn5\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn5\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn4.5\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn23\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003errn16\u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOther genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase RNA processing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclpP1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec-type cytochrome synthesis gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunit of acetyl-CoA-carboxylase (fatty acid synthesis)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvelope membrane protein (carbon metabolism)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehypothetical chloroplast reading frames\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eycf1, ycf2, ycf2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e#\u003c/sup\u003e The present in the IR region has duplicate copies\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic relationships with other legumes\u003c/h2\u003e \u003cp\u003eThe phylogenetic position of \u003cem\u003eP. tetragonolobus\u003c/em\u003e within the Leguminosae family was determined by aligning chloroplast gene sequences with related species, including \u003cem\u003eV. radiata\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eC. tetragonoloba\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eC. arietinum\u003c/em\u003e, and \u003cem\u003eA. hypogaea\u003c/em\u003e, with \u003cem\u003eA. thaliana\u003c/em\u003e serving as the outgroup. The resulting multigene-based phylogenetic tree showed robust support (bootstrap value 1.0) for all resolved nodes. The analysis revealed two major clades among the studied legume species. \u003cem\u003eA. hypogaea\u003c/em\u003e was affiliated with the Dalbergioid clade, while the remaining species formed an Old World clade. \u003cem\u003eC. tetragonoloba\u003c/em\u003e, belonging to the Indigoferoid group (Tribe Indigofereae), clustered closely with \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eP. tetragonolobus\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, and \u003cem\u003eV. radiata\u003c/em\u003e, all of which are part of the Millettioid group under Tribe Phaseoleae within the Phaseoloid clade. Additionally, the Old World clade included \u003cem\u003eC. arietinum\u003c/em\u003e (Tribe Cicereae) and \u003cem\u003eM. truncatula\u003c/em\u003e (Tribe Trifolieae), which clustered together as part of the Inverted Repeat Lacking Clade (IRLC) within the major Hologalegina clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of synteny and genomic rearrangements\u003c/h2\u003e \u003cp\u003eTo identify possible occurrences of rearrangements in the chloroplast genomes, we analysed the synteny of whole chloroplast genome sequences of \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eL. purpureus\u003c/em\u003e along with \u003cem\u003eA. thaliana\u003c/em\u003e as an outgroup species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We used two approaches: a) pairwise and b) global alignment. The pairwise alignments presented high synteny but a strong signature of rearrangements and inversions involving the IR and SSC regions. Specifically, the genomic segment of the SSC region appeared to be inverted in \u003cem\u003eP. tetragonolobus\u003c/em\u003e compared to \u003cem\u003eC. cajan\u003c/em\u003e and \u003cem\u003eG. max\u003c/em\u003e. The IR region flanking the SSC showed high synteny with other legumes. Since the chloroplast is a circular genome, the visible rearrangement was also confirmed with gene order analysis using \u003cem\u003eP. tetragonolobus\u003c/em\u003e as a reference.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe global alignment, using the shuffle-LAGAN algorithm incorporated in the m-Vista pipeline and winged bean chloroplast genome annotations, indicated that within the region between 24.5 to 46 kb, the exons were comparatively less conserved than the rest of the chloroplast (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This was the same region where the inversion appeared to occur in the pairwise synteny analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysing selective pressure on protein-coding genes\u003c/h2\u003e \u003cp\u003eTo assess selective pressures acting on protein-coding genes in the chloroplast genome, the Ka/Ks ratio was calculated among \u003cem\u003eP. tetragonolobus\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eL. purpureus\u003c/em\u003e, along with \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Ka/Ks ratios ranged between 0.001 to 1.4. The highest and only gene with a ratio\u0026thinsp;\u0026gt;\u0026thinsp;1 was \u003cem\u003erpl23\u003c/em\u003e in pairwise comparison to \u003cem\u003eA. thaliana\u003c/em\u003e. Similarly, \u003cem\u003erpl23\u003c/em\u003e and an additional gene, \u003cem\u003erpl2\u003c/em\u003e, exhibited Ka/Ks values of 0.87 and 0.96, respectively, in comparison to \u003cem\u003eC. cajan\u003c/em\u003e, suggesting significant evolutionary divergence. When compared with \u003cem\u003eL. purpureus\u003c/em\u003e, the only gene with a Ka/Ks value close to 1.0 was ndhB, with a value of 0.8. The remaining genes had Ka/Ks values\u0026thinsp;\u0026le;\u0026thinsp;0.5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eComparative analysis of codon usage frequency\u003c/h2\u003e \u003cp\u003eSixty-one distinct codons for 20 different amino acids were identified in the chloroplast genome of winged bean (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The termination codons were excluded from the analysis. Among the 61 distinct codons, 6 individual codons encoded Arginine, Leucine and Serine; 4 codons encoded Alanine, Glycine, Proline, Threonine, and Valine; and 3 codons encoded Isoleucine; and 2 codons encoded Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Histidine, Lysine, Phenylalanine, and Tyrosine. Methionine and Tryptophan were encoded by 1 codon each. The RSCU values ranged between 0.47 and 4.38, with CGC coding for alanine having the lowest RSCU, while TTT for phenylalanine had the highest. Phenylalanine, lysine, and asparagine were the most abundant amino acids coded by the winged bean genome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLong-repeats and SSRs\u003c/h2\u003e \u003cp\u003eThe analysis of long repeats in the winged bean chloroplast genome showed 38 palindromic, 16 forward, three reverse and two complement repeats, thus in total 59 long repeats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Among them, 38 repeats were in the 30\u0026ndash;35 bp range while 5 and 7 repeats were in the 36\u0026ndash;40 bp and 41\u0026ndash;45 bp range, respectively. Nine repeats were found to be more than 45 bp in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e The longest repeat was of length 287 bp. Among the 59 long repeats, LSC contained 35 repeats while SSC and IRs contained 6 and 18 repeats, respectively. In total 68% (n\u0026thinsp;=\u0026thinsp;40) of the 59 repeats were present in the intergenic spacers while the rest 32% (n\u0026thinsp;=\u0026thinsp;19) were found to be overlapping with gene \u003cem\u003eycf2, ndhA, ndhF, rps12, rpl22, pafI and psaA\u003c/em\u003e (\u003cb\u003eSupplementary Table S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWe identified 84 perfect SSRs, two compound SSRs, and 15 VNTRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec; \u003cb\u003eSupplementary Table S5\u003c/b\u003e) accounting for one perfect SSR per 1.8 kbp, one compound SSR per 75.79 kbp, and one VNTR per 10.10 kbp of the winged bean chloroplast genome. Among the 84 perfect SSR loci analysed, 49 (58.33%) were mononucleotides while the remaining 35 (41.67%) were dinucleotides (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). A/T repeats were the most frequent mononucleotide repeats (43, 51.19%) followed by G/C (6, 7.14%). Ten bp SSRs were most frequent (53, 63.09%) followed by 11 bp (14, 16.67%), 12 bp (10, 11.90%), 14 bp (4, 4.76%), 16 bp (2, 2.38%) and 15 bp (1, 1.19%). Dinucleotide repeats contained four types of repeat motifs (AG/CT, TA, TC, and AT), of which AT accounted for 22.61% (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) followed by TA (13, 15.48%), AG/CT (2, 2.38%), and TC (1, 1.19%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). The frequency of the repeat motifs varied from 33.33% (n\u0026thinsp;=\u0026thinsp;10) to 1.19% (n\u0026thinsp;=\u0026thinsp;14 \u0026amp;15).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of six distinct repeat motif types were identified in the study. Different repeat motifs were reiterated 5 to 16 times (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency distribution of perfect SSR repeat motifs in winged bean chloroplast genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eS. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRepeat\u003c/p\u003e \u003cp\u003emotif\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"9\" nameend=\"c11\" namest=\"c3\"\u003e \u003cp\u003eNumber of reiterations of the motif\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG/C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAG/CT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong the 84 perfect SSRs, two compound SSRs, and 15 VNTRs identified in the study, LSC contained the maximum number of perfect SSRs (57, 67.86%), compound SSRs (02, 100%), and VNTRs (09, 60%) followed by SSC with 18 (21.43%) perfect SSRs and 03 (20%) VNTRs, IRs with 09 (10.71%) perfect SSRs and 03 (20%) VNTRs. Regarding the distribution of SSRs and VNTRs in the genic and non-genic regions, the maximum number of perfect SSRs (57, 67.86%), compound SSRs (02, 100%), and VNTRs (10, 66.67%) were located in the intergenic spacers followed by exon with 16 (19.05%) perfect SSRs and 04 (26.67%) VNTRs. The introns accounted for the lowest number with 11 (13.10%) perfect SSRs and 01 (6.67%) VNTRs. The perfect SSR containing genes were \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003endhA\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003etrnK-UUU\u003c/em\u003e, \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003epafI\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e, \u003cem\u003erpoC2\u003c/em\u003e, \u003cem\u003erps2\u003c/em\u003e, \u003cem\u003eatpF\u003c/em\u003e, and \u003cem\u003erps18\u003c/em\u003e. \u003cem\u003eycf1\u003c/em\u003e contained 11 perfect SSRs. The VNTR containing genes were \u003cem\u003eycf2\u003c/em\u003e, \u003cem\u003erps7\u003c/em\u003e, \u003cem\u003eccsA\u003c/em\u003e, and \u003cem\u003epsbB\u003c/em\u003e (\u003cb\u003eSupplementary Table S5\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe legume family (Leguminosae) is economically one of the most successful lineages among flowering plants \u003csup\u003e33\u003c/sup\u003e. It exhibits a significantly higher species diversification rate over the last 60\u0026nbsp;million years compared to angiosperms as a whole \u003csup\u003e71\u003c/sup\u003e. Within this family, winged bean distinguishes itself with its nutrient-rich components and effective symbiotic associations with a broad spectrum of rhizobia strains, making it suitable for low-input and self-resilient agricultural systems \u003csup\u003e72\u003c/sup\u003e. Chloroplast genomes, with their conserved nature, are crucial resources for studying evolutionary dynamics and phylogenetic relationships across plant taxa \u003csup\u003e73, 74\u003c/sup\u003e. The present study reports the sequencing and characterisation of the chloroplast genome of \u003cem\u003eP. tetragonolobus\u003c/em\u003e, along with its comparative analysis with other legumes, including \u003cem\u003eV. radiata\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eC. tetragonoloba\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eC. arietinum\u003c/em\u003e, and \u003cem\u003eA. hypogaea\u003c/em\u003e, with \u003cem\u003eA. thaliana\u003c/em\u003e serving as the outgroup.\u003c/p\u003e \u003cp\u003eLeguminosae is divided into three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae. Caesalpinioideae, a paraphyletic group, is the ancestral base for the monophyletic subfamilies Mimosoideae and Papilionoideae \u003csup\u003e75\u003c/sup\u003e. Papilionoideae, the largest subfamily, comprises 13,800 species across 28 tribes in 478 genera \u003csup\u003e33\u003c/sup\u003e. It is further divided into Swartzioid and Aldinoid lineages and other genera within a larger monophyletic group marked by a 50 kb inversion in the chloroplast genome \u003csup\u003e24\u003c/sup\u003e. The 50 kb inversion group includes three major clades: Genistoids, Dalbergioids, and the Old World clade. The Genistoid clade is characterised by the accumulation of quinolizidine while the Dalbergioid clade typically exhibits \"aeschynomenoid\" root nodule morphology \u003csup\u003e76\u003c/sup\u003e. The Old World clade further segregates into the Indigoferoid/Millettioid and Hologalegina clades, with the latter splitting into the Robinioid and Inverted Repeat-Lacking Clade (IRLC). Indigofereae is sister to the Millettioid group, comprising Phaseoloid and core Millettieae clades and allies \u003csup\u003e77, 78\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, there has been a growing interest in the legume systematics community to combine expertise and data to capitalise on new approaches in genetics and bioinformatics.With the advancement of sequencing technologies, an increasing number of chloroplast genomes have been sequenced and used for phylogenetic analysis. We used the nucleotide sequences of all predicted chloroplast genes of \u003cem\u003eP. tetragonolobus\u003c/em\u003e and the reported chloroplast genes for nine other legumes, including \u003cem\u003eV. radiata\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eC. tetragonoloba\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eC. arietinum\u003c/em\u003e, and \u003cem\u003eA. hypogaea\u003c/em\u003e, along with \u003cem\u003eA. thaliana\u003c/em\u003e as the outgroup, to delineate the phylogenetic position of \u003cem\u003eP. tetragonolobus\u003c/em\u003e in relation to the related genera. The multigene-based phylogenetic tree resolved the cladistic position of all the legumes considered for the study with robust bootstrap support. \u003cem\u003eP. tetragonolobus\u003c/em\u003e clustered closely with \u003cem\u003eC. cajan\u003c/em\u003e, G. max, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, and \u003cem\u003eV. radiata\u003c/em\u003e of the Millettioid group under Tribe Phaseoleae within the Phaseoloid clade. Moreover, all the other legume species considered for the study showed affiliation with their respective clades consistent with the current state of legume phylogeny \u003csup\u003e79, 80, 81\u003c/sup\u003e, reinforcing the utility of chloroplast genome sequences in deep phylogenetic analysis.\u003c/p\u003e \u003cp\u003eThe comparative analysis of the sequences of the chloroplast genomes of \u003cem\u003eP. tetragonolobus\u003c/em\u003e and other legumes revealed clade-wise general conservation in genome size, length of IR, LSC, and SSC regions, along with their GC contents and gene content. As expected, \u003cem\u003eC. arietinum\u003c/em\u003e and \u003cem\u003eM. truncatula\u003c/em\u003e, belonging to the Hologalegina/ Inverted Repeat Lacking Clade (IRLC), exhibited the smallest genome size and gene contents, attributed to the presence of only a single copy of IR \u003csup\u003e82, 83, 84\u003c/sup\u003e. \u003cem\u003eA. hypogaea\u003c/em\u003e, belonging to the Dalbergioid clade, exhibited the largest genome size, whereas the genome sizes for Indigoferoid/Millettioids, comprising \u003cem\u003eP. tetragonolobus\u003c/em\u003e, \u003cem\u003eV. radiata\u003c/em\u003e, \u003cem\u003eP. vulgaris\u003c/em\u003e, \u003cem\u003eL. purpureus\u003c/em\u003e, \u003cem\u003eC. tetragonoloba\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eC. cajan\u003c/em\u003e, were slightly smaller than the Dalbergioids, ranging from 151,294 bp to 152,530 bp, varying by only 1236 bp. These results indicate a notable degree of genomic homogeneity among the leguminous species under investigation. Moreover, they highlight the strength of the cladistic approach to biological classification based on the hypotheses of most recent common ancestry.\u003c/p\u003e \u003cp\u003eWe observed a notable uniformity in GC content across the LSC, SSC, and IR regions among various legume species. Furthermore, the GC content of tRNAs and rRNAs was significantly higher than that of protein-coding genes. Notably, a proportionately higher number of GC-rich tRNAs and rRNAs in the IR regions contributed to their overall higher GC content compared to the LSC and SSC regions. These GC-rich regions ensure structural integrity and functional resilience across diverse taxa \u003csup\u003e85, 86, 87\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe synteny analysis of whole chloroplast sequences from \u003cem\u003eP. tetragonolobus\u003c/em\u003e, \u003cem\u003eC. cajan\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eL. purpureus\u003c/em\u003e, along with \u003cem\u003eA. thaliana\u003c/em\u003e, provided valuable insights into the structural variations within these genomes. Pairwise alignments revealed high synteny among the chloroplast genomes but also unveiled a notable signature of rearrangements and inversions, particularly within the IR and SSC regions. Notably, the SSC segment exhibited an inversion in \u003cem\u003eP. tetragonolobus\u003c/em\u003e compared to \u003cem\u003eC. cajan\u003c/em\u003e and \u003cem\u003eG. max\u003c/em\u003e, indicating a structural deviation specific to this species. Complementing the pairwise analysis, global alignment using the shuffle-LAGAN algorithm highlighted a region of reduced exon conservation spanning from 24.5 to 46 kb within the chloroplast genomes. This region corresponded to the site of inversion observed in the pairwise synteny analysis, reinforcing the presence of rearrangements within this segment of the chloroplast genome. The observed rearrangements may be attributed to flip-flop intramolecular recombination in the plastome, a mechanism proposed by Ogihara et al.\u003csup\u003e88\u003c/sup\u003e. While such events are rare, recent studies \u003csup\u003e89, 90\u003c/sup\u003e highlight their significance as evolutionary drivers, potentially conferring adaptive advantages. Identifying structural variations within chloroplast genomes, such as inversions and rearrangements, underscores the dynamic nature of plastome evolution. Understanding the mechanistic underpinnings and functional implications of these rearrangements offers valuable insights into the evolutionary trajectories of plant species within the Leguminosae family.\u003c/p\u003e \u003cp\u003eThe Ka/Ks ratio value, which infers the rate of gene divergence between species, serves as an indicator to identify genes undergoing different selection pressures \u003csup\u003e91, 92\u003c/sup\u003e. In protein-coding genes, synonymous substitutions occur more frequently than non-synonymous substitutions \u003csup\u003e91, 93\u003c/sup\u003e. In this study, since the majority of genes had values between 0 and 0.5, this is a strong signature representing that in \u003cem\u003eP. tetragonolobus\u003c/em\u003e, nonsynonymous mutations are being removed from the population at a faster rate than synonymous mutations. Thus, the genes are under purifying selection and tend to maintain their required functions. Meanwhile, the only gene observed to be under diversifying selection is \u003cem\u003erpl23\u003c/em\u003e. This gene has been reported to be deleted, duplicated, and accumulate mutations not only in legumes but also in other plant species, including cereal crops \u003csup\u003e29, 94\u0026ndash;100\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the 64 codons directing protein synthesis, 61 encode standard amino acids, while 3 serve as translation stop signals. Most amino acids have multiple synonymous codons, except for tryptophan and methionine, typically encoded by one codon each \u003csup\u003e101\u003c/sup\u003e. The degeneracy of the genetic code allows the same amino acid to be encoded by different codons \u003csup\u003e102, 103\u003c/sup\u003e. However, codon usage varies among organisms, genes, and even the same gene from different species, resulting in codon usage bias \u003csup\u003e104, 105\u003c/sup\u003e. Codon usage bias leads to non-random appearance of synonymous codons with different frequencies \u003csup\u003e106, 107\u003c/sup\u003e. Codon bias impacts numerous cellular processes, such as mRNA stability, transcription, translation efficiency, and protein expression and cotranslation folding \u003csup\u003e108\u0026ndash;110\u003c/sup\u003e. It influences chromatin structure and mRNA folding, thereby regulating transcription levels and translation efficiency by modulating the elongation rate of translation \u003csup\u003e108\u0026ndash;111\u003c/sup\u003e. Codon bias analysis aids in revealing horizontal gene transfer and evolutionary relationships between closely related organisms \u003csup\u003e112, 113\u003c/sup\u003e. The RSCU value compares the observed frequency of a specific synonymous codon to the expected frequency (no codon usage bias). A value of 1.0 suggests no bias, with equal codon usage for that amino acid. Values above 1.0 indicate positive bias, while those below 1.0 indicate negative bias. RSCU values exceeding 1.6 or falling below 0.6 indicate overrepresented and underrepresented codons, respectively \u003csup\u003e38, 114\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExcluding the termination codon, we found 61 codons for 20 amino acids in the winged bean chloroplast genome, with RSCU values ranging from 0.47 to 4.38. Notably, CGC, encoding alanine, exhibited the lowest RSCU, while TTT, encoding phenylalanine, showed the highest RSCU. Phenylalanine, lysine, and asparagine were the most abundant amino acids encoded by the winged bean genome. Among the various synonymous codons for amino acids such as arginine, asparagine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, and tyrosine in the winged bean chloroplast genome, codons ending with either A or U were overrepresented. This bias towards codons ending with A or U may be attributed to the higher AT content of chloroplast genomes, resulting from mutation and natural selection processes \u003csup\u003e38, 115\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnderstanding repeat sequences within genomes is critical for deciphering evolutionary patterns and genetic diversity \u003csup\u003e116, 117\u003c/sup\u003e. In the present study, we identified 59 repeats, primarily concentrated in intergenic spacers, consistent with patterns in other legumes studied recently \u003csup\u003e82, 118, 119\u003c/sup\u003e. A large proportion of repeats were also found in the genes namely \u003cem\u003eycf2, ndhA, ndhF, rps12, rpl22, pafI and psaA\u003c/em\u003e, indicating potential functional implications. The prevalence of repeats in intergenic regions highlights their role in genomic rearrangements and evolutionary dynamics \u003csup\u003e120, 121\u003c/sup\u003e. Leveraging these conserved patterns as genetic markers can enhance phylogenetic and population studies in legumes, providing insights into chloroplast genome evolution and plant adaptation \u003csup\u003e120\u0026ndash;122\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe identified 84 perfect SSRs, two compound SSRs, and 15 VNTRs in the chloroplast genome of winged bean. Their distribution in the LSC, SSC, and IR regions was generally similar to that observed in other legumes \u003csup\u003e99, 82, 118, 123, 124,125\u003c/sup\u003e. Typically, a significant proportion of SSRs in genic regions consist of trinucleotide repeats, which help mitigate the detrimental effects of frame-shift mutations \u003csup\u003e126, 127\u003c/sup\u003e. However, in our study, we found only mono- and dinucleotide repeats, predominantly concentrated in intergenic spacers and introns rather than exons. This may help counteract the detrimental effects of frame-shift mutations caused by mono- and dinucleotide repeats in genic regions \u003csup\u003e128\u0026ndash;130\u003c/sup\u003e. The SSR markers identified in the chloroplast genome of the winged bean are of significant advantage in evolutionary and taxonomic research due to their maternal inheritance and lower mutation rates \u003csup\u003e126, 127\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, the study of the chloroplast genome of \u003cem\u003eP. tetragonolobus\u003c/em\u003e and its comparative analysis with other legumes has provided valuable insights into the evolutionary dynamics and phylogenetic relationships within the legume family. The findings highlight the Andrews, utility of chloroplast genome sequences in deep phylogenetic analysis and support the strength of the cladistic approach to biological classification based on the hypotheses of most recent common ancestry. The observed genomic homogeneity among the leguminous species under investigation and the uniformity in GC content across different regions underscores the conserved nature of chloroplast genomes within this plant family. These results contribute to our understanding of legume systematics and emphasise the importance of combining expertise in genetics and bioinformatics to capitalise on new approaches for studying plant evolutionary biology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the funding and logistic support provided by Director, ICAR-IIAB, Ranchi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKUT \u0026ndash; Sequencing, data analysis, NKS \u0026ndash; Data analysis, AG \u0026ndash; Data analysis, HS - Data analysis and submission of sequences to NCBI, AP- Data analysis, SK- Generated the plant material, VPB-Coordinated the project, SR-Coordinated the project and manuscript editing, APT- Coordinated the project, BKS- Planning the experiment, sequencing and writing the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the finding of this study have been deposited in NCBI BankIt with primary accession code Psophocarpus PP894786.1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBennett, M. 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Plant Sci.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 726084; 10.3389/fpls.2021.726084 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Winged bean, Plastome, SSR, AKWB-1, Phylogenetic tree, Codon Usage Bias","lastPublishedDoi":"10.21203/rs.3.rs-4615004/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4615004/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe winged bean (\u003cem\u003ePsophocarpus tetragonolobus\u003c/em\u003e) is a fast-growing, underutilized legume thriving in hot, humid regions. It forms symbiotic associations with a broad-spectrum cowpea rhizobial group, making it ideal for crop rotation or intercropping systems. Winged bean seeds are rich in protein, fiber, vitamins, minerals, fat, and carbohydrates, highlighting its potential as a valuable agricultural crop. In this study, we conducted whole-genome sequencing of the winged bean chloroplast using high-coverage short-read sequencing on the Illumina platform, generating over 1\u0026nbsp;billion paired-end raw reads. We utilized the GetOrganelle toolkit to assemble the chloroplast genome comprising 130 genes, including 85 protein-coding genes, 37 tRNAs, and eight rRNA genes. We also identified 84 perfect SSRs, two compound SSRs, and 15 VNTRs. Our analysis revealed the typical quadripartite structure of the chloroplast genome, along with insights into its functional classification and phylogenetic relationships with other legumes. Additionally, we identified possible genomic rearrangements through synteny analysis. Characterizing the winged bean chloroplast genome provides crucial resources for research and crop improvement. Comparative genomics of the chloroplast offers significant insights into the evolutionary and molecular biology of legumes.\u003c/p\u003e","manuscriptTitle":"Chloroplast genome sequencing in winged bean (Psophocarpus tetragonolobus L.) and comparative analysis with other legumes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 12:08:34","doi":"10.21203/rs.3.rs-4615004/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7beaa2d9-35be-4dfe-a6d6-052f51c89f7e","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34562103,"name":"Biological sciences/Biotechnology"},{"id":34562104,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":34562105,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-05-04T01:37:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-19 12:08:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4615004","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4615004","identity":"rs-4615004","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}