Molecular delimitation and phylogeny of Basella alba L. varieties applying DNA barcoding and ISSR polymorphism

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(Basellaceae) is a popular leafy vegetable in the hot humid tropics of India, tropical Asia and Africa. The species is represented by two cultivated colour-morphs – green stemmed and red-stemmed and one crop-wild twining variant with habitual and morphological variability. Such variability has favoured the introduction of three varieties under Basella alba L. viz.- var. alba , var. rubra and var. scandens . Phylogenetic linkage among the varieties has been traced applying DNA barcoding of ITS and rbcL sequences as well as ISSR polymorphism. Resultant ITS sequences showed extreme polymorphism but rbcL sequences appeared with high level of sequence uniformity. Phylogenetic tree constructed on ITS sequences clearly reflected separate molecular identity of the varieties, inclusion of cultivated varieties with close ally Anredera cordifolia in a single clade and crop-wild Basella alba var. scandens as probable progenitor of two cultivated varieties in separate lineage. Phylogenetic tree computed on rbcL sequences showed conspecific nature of the varieties, there inclusion in a single clade, so as the allied species of Anredera . Dendrogram on ISSR polymorphism also consolidated separate identity of the varieties as well as close alliance of crop-wild variety with red-stemmed cultivated form as the immediate descendent. The study strongly favoured infraspecific classification of Basella alba comprising three varieties rather than treating them as separate species. Basella alba L. Infra-specific classification DNA barcoding ISSR polymorphism Phylogenetic tree Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Basella alba L. (Basellaceae) known as vine spinach or Indian spinach, is a fast-growing pantropical popular leafy vegetable (Sperling and Bittrich 1993 ) in the hot humid tropics of south and south-eastern Asia (Saroj et al. 2012 ). Tender succulent stem and mucilaginous leaves, enriched with dietary protein, vitamins, mineral components, antioxidants (Kadam et al. 2017 ) as well as novel health promoting bioactive elements (Das 2025a ) are used as leafy vegetable. The species is represented by two colour variant - green-stemmed and red-stemmed, named previously as B. alba L. and B. rubra L. respectively by Linnaeus. Later Basella alba was adopted as the correct name establishing the priority of B. alba over B. rubra (Roxburgh 1832 ). Studies on cytology, palynology, seed surface feature, protein profile favoured this merger (Bolaji et al. 2023 ; Deshmukh and Gaikwad 2014 ; Roy et al. 2010 ). Recently a crop-wild variety B. alba var. scandens has been described from lower Gangetic plain of West Bengal, India and an infraspecific classification has been proposed identifying three varieties under B. alba (var. alba , var. rubra and var. scandens ) to address the morphological variability (Das 2025b ). Molecular techniques like DNA barcoding, ISSR polymorphism analysis has emerged as effective tools to substantiate or redefine traditional morphology-based taxonomic and phylogenetic interpretations (Hebert et al. 2003 ; Jiang et al. 2022 ). These techniques are not replacement but complementary in application to traditional taxonomy. Internal Transcribed Spacer (ITS) regions of nuclear genome is a powerful barcode for species delimitation, phylogeny analysis due to universality and greater variability, evolutionary rate (Vijayan and Tsou 2010 ). Large subunit of Ribulose 1,5-bisphosphate Carboxylase/oxygenase encoding gene (rbcL gene) is suitable to study genetic diversity and phylogeny for to its high conservation level (Hapsari et al. 2019 ). ISSR markers have been used extensively for characterization of germplasm (Wolff et al. 1995 ; Charters and Wilkinson 2000 ), to estimate the extent of genetic variability at both inter and intra-specific level in a wide range of crop species ( Joshi et al. 2000 ; Ajibade et al. 2000 ). Among the PCR based marker techniques, ISSR is one of the simplest, quickest and widely used marker system with high reproducibility. ISSR markers are highly polymorphic and are useful in studies on genetic diversity, phylogeny, distinguishing even closely related germplasm (Reddy et al. 2002 ). In the present study, molecular approach like DNA barcoding of ITS and rbcL sequence and ISSR polymorphism analysis were employed to evaluate infraspecific classification of Basella alba L. as well as to outline phylogenetic relationship among the varieties. Materials and methods Materials Three varieties of Basella alba L., two cultivated ( B. alba L. var. alba and B. alba var. rubra (L.) Saubhik Das) and one crop-wild ( B. alba var. scandens Saubhik Das) were taken into consideration. The taxonomic identity was authenticated from the voucher specimens of Central National Herbarium (CAL), BSI, Shibpur, Howrah, West Bengal. Extraction of DNA The genomic DNA was extracted from fresh 30 mg of leaf samples by CTAB (Cetyltrimethylammonium bromide) buffer pH 8.0 incubating at 65⁰C for 45 min. The extracted DNA samples were stored at -20⁰C. Gene sequencing PCR analysis It was performed in 20 µl reaction mixture containing 100 ng of template DNA, 20 pmol /µl of both forward and reverse primer (Table 1 ), 10 µl Promega GoTaq Green master mix, and volume made up to 20 µl with nuclease-free water. Amplifications were carried out in MiniAmp plus thermal cyclar from Applied Biosystems programmed with initial denaturation at 94⁰C for 4 min, followed by 30 cycles including denaturation at 94⁰C for 30 sec, annealing at 52⁰C for 45 sec, extension at 72⁰C for 45 sec and final extension at 72⁰C for 7 min. In PCR amplification of rbcL gene similar thermal profile was used except annealing at 50⁰C for 45 sec. The PCR products were electrophoresed in 1.5% Agarose gel with 1 Kb DNA ladder (GeneRuler). The amplified DNA products were eluted from gel for sequencing by Sanger Cycle sequencing method. Table 1 Primers used for amplification of ITS and rbcL sequences Primers Sequences rbcL – Forward CACAAACAGAGAGACTAAAGC rbcL – Reverse GAAACGGTCTCTCCAACG ITS – Forward CCTTATCATTTAGAGGAAGGA ITS - Reverse TCCTCCGCTTATTGATATGC Sanger cycle sequencing method Sequencing was done in SeqStudio Genetic Analyzer from Applied Biosystems applying BigDye terminator kit version 3.1. After sequencing, chromatogram obtained as raw data in Bio Edit format were converted into FASTA formats, edited, resultant sequences compared with reference sequences available in NCBI (National Centre for Biotechnology Information) based on percent identity using BLAST (Basic Local Alignment Search Tool). Multiple sequences (resultant sequences and retrieved closely similar sequences from NCBI) were aligned using MEGA 12 and phylogenetic trees were constructed applying neighbour-joining method with 1001 bootstrap replications, applying Tamura 3-parameter model. GC contents of the sequences were derived using Genomics % GC content calculator. ISSR polymorphism analysis PCR was performed in 20 µl of reaction mixture containing 100 ng of template DNA, 20 pmol /µl of ISSR primer (Table 2), 10 µl Promega GoTaq Green master mix, volume up to 20 µl with nuclease-free water. Amplifications were carried out in MiniAmp plus thermal cyclar from Applied Biosystems programmed with an initial denaturation at 94⁰C for 4 min, followed by 30 cycles including denaturation at 94⁰C for 1 min, annealing at primer-specific temperature for 1 min, extension at 72⁰C for 2 min and final extension at 72⁰C for 7 min, cooled down to 4⁰C. The Amplicons were separated in 2% Agarose gel with 100 bp DNA ladder. Table 2 ISSR Primers used in DNA finger printing Primers Sequences Annealing temperature ISSR – 01 (CA) 8 GG 54⁰C ISSR − 02 (GA) 6 CC 50⁰C ISSR − 03 (AC) 8 YG 54⁰C ISSR – 04 (CT) 8 AC 50⁰C ISSR – 05 (CA) 6 AG 50⁰C ISSR – 06 (GT) 6 GG 55⁰C Statistical analysis A matrix was prepared for each ISSR primer scoring the presence or absence of ISSR bands in different varieties. Percentage-based pairing affinity values between different combinations of variety-pairs were calculated applying the following formula – Number of ISSR bands common to sample A and B PA = --------------------------------------------------------------------- x 100 Total number of ISSR bands in sample A and B A Dendrogram was computed based on Group average (Unweighted Pair Group), using software NCSS 2026, applying Euclidean distance method and standard deviation scaling. Results Sequencing of ITS and rbcL DNA fragment The amplicons of ITS and rbcL DNA segments obtained from three varieties were in the range of 500 bp and 750 bp respectively (Fig. 1 ). The size of the edited sequences ranged from 430 to 561 bp for ITS and 408 to 576 bp for rbcL gene. The amino acid sequences of the resultant rbcL gene sequenecs were derived. The DNA sequences were submitted in the GenBank and assigned with accession numbers (Table 3). The GC content found to be consistently much higher in case of ITS sequence (61.88% − 63.95%) than rbcL sequence (42.76% − 44.36%) (Table 4). Table 3 Nucleotide sequence of ITS and rbcL segments submitted in the GenBank ITS sequences B. alba var. scandens (GenBank Accession No. PZ020092.1) GGTGGACCCTCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTCCGGCC GAGGGCACGTCTGCCTGGGCGTCACGCATCGCGTCTCCCTCACCCGCCGC GCGGGGGGGAAGGACGATGGCCTCCCGTGCTTGAACGGGCGCGGCTGGC CTAAAACGGGAGCTTGCGGCGACGAGCTGCGGCGGCGTTTGGTTGACGGC GGGCCATCGGCCCTCGTAATGCATCGCGCCTCGCACGCACGTCGTCGGCAT GGGCTCGTCGGACCCTCGGAAAACCTTTGCGACCCCAGGTCAGGCGGGGC TACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGAGAAGAAACTTACGA GGATTCCCCTAGTAACGGCGAGCGAACCGGGAAGAGCCCAGCTTTAAAAT CGGGCGGCGTCGTCGTCCGAATTGTAGTTA B. alba var. alba (GenBank Accession No. PZ020093.1) ACAAGGTTTCCGTAGGTGACCTGCGGAAGGATCATTGTCGAAACCTGCC CAGCAGAATGACCCGAGGATGAGTTTCAAGCATAAGACGCGCGGGGAGG TCGCCTCCCCCGCGACCGCACAGCGCCCCCCTTGGGGTGGCACGTTGCGA CAACAAAACCCGGCGCGGACTGCGCCAAGGAACACGAACAGCGAGAGCG CCTGCCCGCGCCCGGTCCCCGGTGCGAGGGGCAGCGTCCCAGGCTAGAAA ACGTAATGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAA CGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCG AGTCTTTGAACGCAAGTTGCGCCCAAAGCCTTTCGGCCGAGGGCACGTCT GCCTGGGCGTCACACAATGCGTCTCCCCCACCCGTCGTGCGTGGGGAAGG ATGATGGCCTCCCGCGCCTTAGCGGGCACGGCTGGCCTAAAATGGGGAGCTCG B. alba var. rubra (GenBank Accession No. PZ020094.1) CAAGCATAAGACGCGCGGGGAGGTCGCCTCCCCCCGCGACCGCACAGCG CCCCCCTTGGGGTGGCACGTTGCGACAACAAAACCCGGCGCGGACTGCG CCAAGGAACACGAACAGCGAGAGCGCCTGCCCGCGCCCGGTCCCCGGTG CGAGGGGCAGCGTCGCAGGCTAGAAAACGTAATGACTCTCGGCAACGGA TATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTG TGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCC CAAAGCCTTTCGGCCGAGGGCACGTCTGCCTGGGCGTCACACAATGCGTC TCCCCCACCCGTCGTGCGGGGGGAAGGATGATGGCCTCACGCGCCTTAAC GGGCACGGCTGGCCTAAAATGGGAGCTCGTGGCGATGAGCTGCGGCGGC GTTTGGTTGACGGAGGGCCATTGGCCCTCGTAATACATTGCGCCTCGCACG CACGTCGTCGGCAAGGGCTCGTCGGACCCTGGTAAAACCTTTGCGACCCC AGGTCAGGCGGGGC rbcL sequences B. alba var. scandens (GenBank Accession No. PZ194680) TTGAATTATTATACTCCTCAATATCAACCCCTGGATACTGATATCTTGGCAGC ATTCCGAGTAACTCCTCAACCTGGAGTTCCGTCAGAAGAAGCAGGGGCCG CAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGGACCGACG GACTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGATCCCG TTCCTGGAGAAGACAATCAATATATTTGTTATGTAGCTTACCCATTAGACCT TTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATTGTGGGTAATGTAT TTGGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCTG TTGCTTATATAAAAACTTTCCAAGGCCCGCCTCACGGTATCCAAGTTGAGA GAGATAAATTGAACAAGTATGGCCGTCCTCTATTGGGATGCACTATTAAAC CGAAATTGGGGTTATCTGCTAAAAACTATGGTCGAGCAGTTTATGAATGTC TTCGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGTGAACTCCCAACCATTTATG B. alba var. alba (GenBank Accession No. PZ194679) TTGAATTATTATACTCCTCAATATCAACCCCTGGATACTGATATCTTGGCAG CATTCCGAGTAACTCCTCAACCTGGAGTTCCGTCAGAAGAAGCAGGGGCC GCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGGACCGAC GGACTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGATCCC GTTCCTGGAGAAGACAATCAATATATTTGTTATGTAGCTTACCCATTAGACC TTTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATTGTGGGTAATGTA TTTGGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCT GTTGCTTATATAAAAACTTTCCAAGGCCCGCCTCACGGTATCCAAGTTGAG AGAGATAAATTGAACAAGTATGGCCGTCCTCTATTGGGATGCACTATTAAA CCGAAATTGGGGTTATCTGCTAAAAACTATGGTCGAGCAGTTTATGAATGT CTTCGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGTGAACTCCCAACCATTTATGCGT B. alba var. rubra (GenBank Accession No. PZ201000) ATTATTATACTCGCTCAATATCAACCCCTGGATACTGATATCTTGGCAGCAT TCCGAGTAACTCCTCAACCTGGAGTTCCGTCAGAAGAAGCAGGGGCCGC AGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGGACCGACG GACTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGATCCCG TTCCTGGAGAAGACAATCAATATATTTGTTATGTAGCTTACCCATTAGACCT TTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATTGTGGGTAATGTAT TTGGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCTG TTGCTTATATAAAAACTTTCCAAGGCCCGCCTCACGGGTATCCAAGTTGA Table 4 G+C content in ITS and rbcL sequences of Basella alba varieties Varieties ITS segment rbcL segment Accession No. G + C content Accession No. G + C content B. alba var. scandens PZ020092.1 63.95% PZ194680 42.76% B. alba var. alba PZ020093.1 61.88% PZ194679 42.88% B. alba var. rubra PZ020094.1 63.81% PZ201000 44.36% The derived sequences and the nearest identified sequences based on BLAST search were used to make phylogenetic analysis. The evolutionary distances were calculated using Tamura 3-parameter method (Tamura 1992 ) in the unit of the number of base substitutions per site. Phylogenetic trees were computed using Neighbour-joining algorithm and with 1001 bootstrap replications and the branch length in the same unit as those of the evolutionary distance used. In Phylogenetic tree on ITS sequences, the optimal tree with the sum of branch length was 0.143 and analytical procedures included 4 coding nucleotide sequences using 1st, 2nd, 3rd and non-coding positions. But in tree on rbcL sequences the optimal tree with the sum of branch length was 0.015 and analytical procedures contained 7 coding nucleotide sequences using 1st, 2nd, 3rd and no-coding position. In the phylogenetic trees computed on ITS and rbcL sequences, the three varieties of Basella alba clustered with allied species Anredera cordifolia (Basellaceae) as out group species. Anredera cordifolia is an evergreen semi-succulent vine-like plant with simple, ovate-cordate, acute, thin fleshy leaves and pendent axillary racemes (Rasingam and Lakshminarasimhan 2012 ). In phylogenetic tree on ITS sequences (Fig. 2 ), both the green-stemmed and red-stemmed cultivated variety of Basella alba clustered with Anredera cordifolia in a single clade where green-stemmed variety appeared as nearest relative of Anredera cordifolia . But crop-wild variety B. alba var. scandens showed wide divergence from both the cultivated form representing a separate lineage. In phylogenetic tree on rbcL sequence (Fig. 3), out-group species Anredera cordifolia included in a different cluster. All the accessions of Anredera cordifolia [GQ436516.1, JQ933240.1 and MH049965.1] along with Anredera diffusa [OQ391768.1] formed a single clade while all the varieties of Basella alba were nested in a single clade. The rbcL deduced amino acid sequences appeared slightly variable in the green stemmed, red-stemmed and crop-wild varieties. ISSR polymorphism analysis Band profile (Fig. 4 ) in each gel was scored as presence or absence of bands. Six ISSR primers yielded a total of 72 bands of which only 26 were monomorphic and 46 were polymorphic with an average polymorphism percentage of 63.88 (Table 5). Highest polymorphism was shown by ISSR − 5 (94.11%) and least by ISSR – 2 (38.88%). Amplicons mostly varying in size range from 1000 bp to 200 bp. Table 5 ISSR polymorphism analysis showing primer-wise generated monomorphic, polymorphic bands and percentage of polymorphism Sl. No. Primer Total no. of bands No. of monomorphic bands No. of polymorphic bands % of Polymorphism 1. (CA) 8 GG 9 3 6 66.66% 2. (GA) 6 CC 18 11 7 38.88% 3. (AC) 8 YG 11 4 7 63.63% 4. (CT) 8 AC 12 4 8 66.66% 5. (CA) 6 AG 17 1 16 94.11% 6. (GT) 6 GG 5 3 2 40.00% Highest pairing affinity (PA) value on ISSR polymorphism was shown by variety pair B. alba var. alba and B. alba var. rubra (75.47%) while the least PA value (62.22%) was found between crop-wild B. alba var. scandens and B. alba var. rubra (Table 6). Table 6 Pairing affinity values of different varietal pairs of Basella alba B(A) B(R) B(S) B(A) 100 B(R) 75.47 100 B(S) 70.58 62.22 100 Dendrogram computed on ISSR data (Fig. 5 ) showed relative closeness between crop-wild variety B. alba var. scandens with red-stemmed cultivated variety Basella alba var. rubra nested in a single cluster. Green cultivated form ( B. alba var. alba ) appeared quite diverging. Discussion Basella alba L. (Basellaceae), commonly known as vine spinach is one of the favorite leafy vegetables in the hot humid tropics of India as well as in tropical Asia and Africa (Tindall 1983 ). The variability in morphology and habit in Basella alba has recently been addressed through the introduction of three varieties – green stemmed var. alba , red stemmed var. rubra and var. scandens , of which first two are cultivated varieties and B. alba var. scandens is crop-wild variety, new for science (Das 2025b ) . Both the cultivated forms are procumbent with thick, mucilaginous, fleshy, ridged stem and large oval leaves while the crop-wild B. alba var. scandens is characterized with twining thin cord-like, reddish stem and smaller cordate leaves. DNA sequence analysis is considered as a new approach in studying evolutionary relationship and genetic diversity in crop plants. In present study phylogenetic linkage among the varieties has been resolved with molecular parameter like – DNA barcoding with ITS and rbcL sequence as well as ISSR polymorphism analysis. DNA barcoding is a molecular diagnostic technology that uses standard, sufficiently variable DNA fragments for species identification and delimitation (Chen et al. 2010 ). Internal Transcribed Spacer (ITS) sequences, located between ribosomal RNA genes, are among the most widely sequenced molecular markers in plant systematics and DNA barcoding studies because of their rapid concerted evolution within and between constituent subunits, diverging at relatively high rate, fast evolution, and the availability of universal primers (Álvarez and Wendel 2003; Li et al. 2011 ; Schoch et al. 2012 ). ITS sequences are highly repeated, showing extensive sequence polymorphism making them reliable markers for reconstructing evolutionary history. The ribulose bisphosphate 1,5-carboxylase/oxygenase gene (rbcL) sequencing has been extensively utilized in various fields such as evolution, phylogeny, biogeography, population genetics, and systematics due to its ease of amplification, relatively conserved nature among related species, high level of universality and low mutation rate compared to other genes in chloroplast DNA (Sheng-Guo et al. 2008 , Candramila et al. 2023 ). ISSR polymorphism analysis is a PCR-based technique to generate multilocus marker through amplification of DNA segment present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction using microsatellite sequences as primer (Oliveira et al. 2010 ). The technique has emerged as a substitute system with the advantage and reliability of microsatellites (SSRs) due to their high polymorphism, rapidity, sensitivity, simplicity, reproducibility, and cost-effectiveness, not needing any prior information on DNA sequences. Highly polymorphic DNA fingerprints are generated which effectively distinguish between genetic individuals (Gemmill and Grierson 2021 ; Sevindik et al. 2023 ). Higher number of polymorphic bands generally indicative of higher level of genetic diversity within and among populations or cultivars. This marker is regarded as a reliable parameter for assessing the genetic diversity among closely related taxa and identifying genotypic similarities between accessions, populations, varieties, cultivars and species (Shaban et al. 2022 ). ISSR could be a suitable tool to assess the changes of diversity in agronomically important crops as well as underutilised crops with unknown sequences. Phylogenetic study on green-stemmed and red-stemmed Basella alba , tree spinach ( Talinum fruticosum ) applying rbcL and ITS barcoding recognized clear separation of two cultivated form in two well supported clade and close alliance of tree spinach (Portulacaceae) in a separate clade (Gayathree et al. 2020 ). In the present study, ITS sequence polymorphism has enabled clear delimitation of varieties as well as establishing the fact that ITS sequences are suitable for infraspecific delimitation. Phylogeny analysis also showed closer relationship between two cultivated domesticated varieties and wide divergence of crop-wild relative, representing the crop-wild variety B. alba var. scandens as probable ancestor of both the cultivated form. But due to high level of universality and relative conserved nature, the rbcL sequence analysis showed no varietal variability. Both the cultivated varieties and crop-wild variety of Basella alba were nested in a same clade so as the accessions of Anredera cordifolia and A. diffusa . Though amino acid sequence of rbcL gene in all the varieties showed little variability. The cluster pattern reflected in ISSR analysis is not concomitant with phylogenetic affinity derived from ITS sequencing. Crop-wild variety showed much closer affinity with B. alba var. rubra , suggesting the red-stemmed variety as the immediate descendant from crop-wild variety. Separate identity of three varieties was established by significant DNA polymorphism. Inclusion of all the varieties in a single clade with 100% area coverage signifies their high degree of genetic uniformity in rbcL sequences at the same time nullifies their consideration as separate species. Study on DNA barcoding of ITS sequences rather consolidate the perception of treating them as separate varieties only by virtue of ITS polymorphism and presenting crop-wild variety B. alba var. scandens as progenitor of both green-stemmed and red-stemmed cultivated variety. ISSR polymorphism also clearly distinguished three varieties as separate taxonomic entities. Close alliance between crop-wild variety and red-stemmed B. alba var. rubra is supported by their morphological similarities (Das 2025b ). Conclusion Morphological variability created ambiguity in identifying and delimiting different morphotypes of Basella alba . Such ambiguity was addressed segregating the population of Basella alba into three varieties – two cultivated (var. alba and var. rubra ) and one crop-wild variety (var. scandens) based on morphological variability. This infraspecific categorization was ratified with molecular identity and phylogenetic relationship among the varieties was also established. ITS sequencing conclusively indicated separate identity of the varieties, while uniformity of the rbcL sequences, as expected ignored their consideration as different species. Crop-wild variety Basella alba var. scandens projected as a probable progenitor of the cultivated varieties. ISSR polymorphism also clearly delimited all the varieties and reflected red-stemmed cultivated form as immediate descendant of the crop-wild variety. Declarations Authors have no relevant conflict of interest in the present work. Author Contribution S.D, conceptualized, designed the research plan, collected plant materials, investigated, analyzed data, prepared figures and manuscript, S. D. Jr. analysed and compiled data, reviewed the references, both the authors have read and agreed to the published version of the manuscript. Acknowledgement Authors express their sincere gratitude to Prof. Sankar Kumar Ghosh, Director of InBOL Healthcare Pvt.Ltd. 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Proc Natl Acad Sci USA 109(16): 6241–6246. https://doi.org/10.1073/pnas.1117018109 Sevindik E, Okan K, Sevindik M, Ercisli S (2023) Genetic diversity and phylogenetic analyses of Juglans regia L. (Juglandaceae) populations using RAPD, ISSR markers and nrDNA ITS regions. Erwerbs-Obstbau 65: 311–320. doi: 10.1007/s10341-023-00834-7 Shaban AS, Arab SA, Basuoni MM, Abozahra MS, Abdelkawy AM, Mohamed MM (2022) SCoT, ISSR, and SDS-PAGE Investigation of Genetic Diversity in Several Egyptian Wheat Genotypes under Normal and Drought Conditions. Int J Agron Article ID 7024028. https://doi.org/10.1155/2022/7024028 Sheng-Guo JI, Ke-Ke HUO, Jun WANG, Sheng-Li PAN (2008) A molecular phylogenetic study of Huperziaceae based on chloroplast rbcL and psbA trnH sequences. J Syst Evol 46(2): 213–219. DOI: 10.3724/SP.J.1002.2008.07036 Sperling CR, Bittrich V (1993) Basellaceae. In: Kubitzki K, Rohwer JG, Bittrich V (eds.) Flowering plants, Dicotyledons, the families and Genera of vascular plants. Berlin: Springer Verlag, p. 143–146. Tamura K (1992) Estimation of the number of nucleotide substitutions when there are strong transition- Transversion and G + C content biases. Mol Biol Evol 9: 678–687. Tindall HD (1983) Vegetables in the Tropics. London, Macmillan Press Ltd. https://doi.org/10.1007/978-1-349-17223-8 . Vijayan K, Tsou CH (2010) DNA barcoding in plants: taxonomy in a new perspective. Curr Sci 99(11), 1530–1541. Wolff K, Zietkiewicz E, Hofstra H (1995) Identification of Chrysantemum cultivars and stability of DNA fingerprint patterns. Theor Appl Genet 91:439–447. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 May, 2026 Reviews received at journal 14 May, 2026 Reviewers agreed at journal 09 May, 2026 Reviews received at journal 09 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 05 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 04 May, 2026 Editor assigned by journal 04 May, 2026 Submission checks completed at journal 04 May, 2026 First submitted to journal 29 Apr, 2026 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. 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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-9567677","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":637435638,"identity":"e7ca33b9-c704-4dcd-8e9b-b476328e336a","order_by":0,"name":"Saubhik Das","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDACCR5mBgY2BgZ+MEmSFskGkrUYHCBWi/zs3sMGDGV29sY3kp89+FDBIM8vdgC/FoM755ITGM4lJ267kWZuOOMMg+HM2QkEtEjkGB9gbGNOMLuRYCbN28aQYHCbgBb5GWAt9fbGM9K/EaeF4UaOcQJj22HGDRI5RNpicOeMsQHDueOJM868KZOccUaCsF/kZ/cYSzCUVdvzt6dvk/hQYSPPL03IYUDA/AdECoBVShBWjgD8B0hRPQpGwSgYBSMJAABAMz3iL1L45AAAAABJRU5ErkJggg==","orcid":"","institution":"Bidhannagar College (Affiliated to West Bengal State University","correspondingAuthor":true,"prefix":"","firstName":"Saubhik","middleName":"","lastName":"Das","suffix":""},{"id":637435639,"identity":"8d2aa38c-7772-452b-8d32-c9f7e1013946","order_by":1,"name":"Sagnik Das","email":"","orcid":"","institution":"St. Xaviers College","correspondingAuthor":false,"prefix":"","firstName":"Sagnik","middleName":"","lastName":"Das","suffix":""}],"badges":[],"createdAt":"2026-04-29 15:11:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9567677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9567677/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109249137,"identity":"0a018d84-33d0-44b9-a40a-99e23f0ee3b0","added_by":"auto","created_at":"2026-05-14 08:42:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100903,"visible":true,"origin":"","legend":"\u003cp\u003eAmplified ITS and rbcL sequence of three varieties of \u003cem\u003eBasella alba\u003c/em\u003e –\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e. \u003cem\u003eBasella alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e, \u003cstrong\u003e2\u003c/strong\u003e. \u003cem\u003eBasella alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e and\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e. \u003cem\u003eBasella alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e, M = 1 kb DNA marker\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9567677/v1/1d358a1704368e25195d10c7.png"},{"id":109297828,"identity":"ec396ab1-fdf1-4bb0-9478-755583df5970","added_by":"auto","created_at":"2026-05-15 09:06:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120365,"visible":true,"origin":"","legend":"\u003cp\u003eEvolutionary relationship between three varieties of \u003cem\u003eBasella alba\u003c/em\u003e and allied \u003cem\u003eAnredera\u003c/em\u003e sp. based on ITS \u0026nbsp;sequence analysis using Neighbour-joining method\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9567677/v1/5113160e8db936b1ccb888f7.png"},{"id":109214414,"identity":"4dd2a85d-3646-4fa4-953c-0456895a6728","added_by":"auto","created_at":"2026-05-13 17:37:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48994,"visible":true,"origin":"","legend":"\u003cp\u003eEvolutionary relationship between three varieties of \u003cem\u003eBasella alba\u003c/em\u003e and allied \u003cem\u003eAnredera\u003c/em\u003e sp. based on rbcL sequence analysis using Neighbour-joining method\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9567677/v1/483d5f04e163100cf19b8d74.png"},{"id":109214415,"identity":"41ea905d-9097-40b2-8ce6-ad9d1904ebb0","added_by":"auto","created_at":"2026-05-13 17:37:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":224035,"visible":true,"origin":"","legend":"\u003cp\u003eISSR band profile in three different varieties of \u003cem\u003eB. alba\u003c/em\u003e with diagrammatic representation; M = \u0026nbsp;100 bp DNA ladder, \u003cstrong\u003e1\u003c/strong\u003e. \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e, \u003cstrong\u003e2\u003c/strong\u003e. \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e and \u003cstrong\u003e3\u003c/strong\u003e. \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9567677/v1/e9005144587196e9f910cf72.png"},{"id":109222258,"identity":"7409dccc-0a4c-4026-8442-ec5624b4dd86","added_by":"auto","created_at":"2026-05-13 21:06:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7525,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram on ISSR polymorphism analysis of \u003cem\u003eBasella alba\u003c/em\u003e varieties\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9567677/v1/8ca0c48571f44384f6adff77.png"},{"id":109252161,"identity":"049aab96-8096-47e5-99f0-4a23422d353f","added_by":"auto","created_at":"2026-05-14 09:21:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":658112,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9567677/v1/ff714877-79e7-408e-9cc2-a182863f2e83.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMolecular delimitation and phylogeny of \u003cem\u003eBasella alba\u003c/em\u003e L. varieties applying DNA barcoding and ISSR polymorphism\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eBasella alba\u003c/em\u003e L. (Basellaceae) known as vine spinach or Indian spinach, is a fast-growing pantropical popular leafy vegetable (Sperling and Bittrich \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) in the hot humid tropics of south and south-eastern Asia (Saroj et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Tender succulent stem and mucilaginous leaves, enriched with dietary protein, vitamins, mineral components, antioxidants (Kadam et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) as well as novel health promoting bioactive elements (Das \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e) are used as leafy vegetable. The species is represented by two colour variant - green-stemmed and red-stemmed, named previously as \u003cem\u003eB. alba\u003c/em\u003e L. and \u003cem\u003eB. rubra\u003c/em\u003e L. respectively by Linnaeus. Later \u003cem\u003eBasella alba\u003c/em\u003e was adopted as the correct name establishing the priority of \u003cem\u003eB. alba\u003c/em\u003e over \u003cem\u003eB. rubra\u003c/em\u003e (Roxburgh \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1832\u003c/span\u003e). Studies on cytology, palynology, seed surface feature, protein profile favoured this merger (Bolaji et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Deshmukh and Gaikwad \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Roy et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Recently a crop-wild variety \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e has been described from lower Gangetic plain of West Bengal, India and an infraspecific classification has been proposed identifying three varieties under \u003cem\u003eB. alba\u003c/em\u003e (var. \u003cem\u003ealba\u003c/em\u003e, var. \u003cem\u003erubra\u003c/em\u003e and var. \u003cem\u003escandens\u003c/em\u003e) to address the morphological variability (Das \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMolecular techniques like DNA barcoding, ISSR polymorphism analysis has emerged as effective tools to substantiate or redefine traditional morphology-based taxonomic and phylogenetic interpretations (Hebert et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These techniques are not replacement but complementary in application to traditional taxonomy. Internal Transcribed Spacer (ITS) regions of nuclear genome is a powerful barcode for species delimitation, phylogeny analysis due to universality and greater variability, evolutionary rate (Vijayan and Tsou \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Large subunit of Ribulose 1,5-bisphosphate Carboxylase/oxygenase encoding gene (rbcL gene) is suitable to study genetic diversity and phylogeny for to its high conservation level (Hapsari et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). ISSR markers have been used extensively for characterization of germplasm (Wolff et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Charters and Wilkinson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), to estimate the extent of genetic variability at both inter and intra-specific level in a wide range of crop species \u003cb\u003e(\u003c/b\u003eJoshi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Ajibade et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Among the PCR based marker techniques, ISSR is one of the simplest, quickest and widely used marker system with high reproducibility. ISSR markers are highly polymorphic and are useful in studies on genetic diversity, phylogeny, distinguishing even closely related germplasm (Reddy et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, molecular approach like DNA barcoding of ITS and rbcL sequence and ISSR polymorphism analysis were employed to evaluate infraspecific classification of \u003cem\u003eBasella alba\u003c/em\u003e L. as well as to outline phylogenetic relationship among the varieties.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMaterials\u003c/p\u003e\n\u003cp\u003eThree varieties of \u003cem\u003eBasella alba\u003c/em\u003e L., two cultivated (\u003cem\u003eB. alba\u003c/em\u003e L. var. \u003cem\u003ealba\u003c/em\u003e and \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e (L.) Saubhik Das) and one crop-wild (\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e Saubhik Das) were taken into consideration. The taxonomic identity was authenticated from the voucher specimens of Central National Herbarium (CAL), BSI, Shibpur, Howrah, West Bengal.\u003c/p\u003e\n\u003cp\u003eExtraction of DNA\u003c/p\u003e\n\u003cp\u003eThe genomic DNA was extracted from fresh 30 mg of leaf samples by CTAB (Cetyltrimethylammonium bromide) buffer pH 8.0 incubating at 65⁰C for 45 min. The extracted DNA samples were stored at -20⁰C.\u003c/p\u003e\n\u003cp\u003eGene sequencing\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePCR analysis\u003c/h2\u003e\n \u003cp\u003eIt was performed in 20 \u0026micro;l reaction mixture containing 100 ng of template DNA, 20 pmol /\u0026micro;l of both forward and reverse primer (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 10 \u0026micro;l Promega GoTaq Green master mix, and volume made up to 20 \u0026micro;l with nuclease-free water. Amplifications were carried out in MiniAmp plus thermal cyclar from Applied Biosystems programmed with initial denaturation at 94⁰C for 4 min, followed by 30 cycles including denaturation at 94⁰C for 30 sec, annealing at 52⁰C for 45 sec, extension at 72⁰C for 45 sec and final extension at 72⁰C for 7 min. In PCR amplification of rbcL gene similar thermal profile was used except annealing at 50⁰C for 45 sec. The PCR products were electrophoresed in 1.5% Agarose gel with 1 Kb DNA ladder (GeneRuler). The amplified DNA products were eluted from gel for sequencing by Sanger Cycle sequencing method.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Primers used for amplification of ITS and rbcL sequences\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePrimers\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eSequences\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003erbcL \u0026ndash; Forward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCACAAACAGAGAGACTAAAGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003erbcL \u0026ndash; Reverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eGAAACGGTCTCTCCAACG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eITS \u0026ndash; Forward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCCTTATCATTTAGAGGAAGGA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eITS - Reverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eTCCTCCGCTTATTGATATGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSanger cycle sequencing method\u003c/h3\u003e\n\u003cp\u003eSequencing was done in SeqStudio Genetic Analyzer from Applied Biosystems applying BigDye terminator kit version 3.1. After sequencing, chromatogram obtained as raw data in Bio Edit format were converted into FASTA formats, edited, resultant sequences compared with reference sequences available in NCBI (National Centre for Biotechnology Information) based on percent identity using BLAST (Basic Local Alignment Search Tool). Multiple sequences (resultant sequences and retrieved closely similar sequences from NCBI) were aligned using MEGA 12 and phylogenetic trees were constructed applying neighbour-joining method with 1001 bootstrap replications, applying Tamura 3-parameter model. GC contents of the sequences were derived using Genomics % GC content calculator.\u003c/p\u003e\n\u003cp\u003eISSR polymorphism analysis\u003c/p\u003e\n\u003cp\u003ePCR was performed in 20 \u0026micro;l of reaction mixture containing 100 ng of template DNA, 20 pmol /\u0026micro;l of ISSR primer (Table\u0026nbsp;2), 10 \u0026micro;l Promega GoTaq Green master mix, volume up to 20 \u0026micro;l with nuclease-free water. Amplifications were carried out in MiniAmp plus thermal cyclar from Applied Biosystems programmed with an initial denaturation at 94⁰C for 4 min, followed by 30 cycles including denaturation at 94⁰C for 1 min, annealing at primer-specific temperature for 1 min, extension at 72⁰C for 2 min and final extension at 72⁰C for 7 min, cooled down to 4⁰C. The Amplicons were separated in 2% Agarose gel with 100 bp DNA ladder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e ISSR Primers used in DNA finger printing\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePrimers\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eSequences\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAnnealing temperature\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eISSR \u0026ndash; 01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e(CA)\u003csub\u003e8\u003c/sub\u003eGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e54⁰C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eISSR\u0026thinsp;\u0026minus;\u0026thinsp;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e(GA)\u003csub\u003e6\u003c/sub\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e50⁰C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eISSR\u0026thinsp;\u0026minus;\u0026thinsp;03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e(AC)\u003csub\u003e8\u003c/sub\u003eYG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e54⁰C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eISSR \u0026ndash; 04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e(CT)\u003csub\u003e8\u003c/sub\u003eAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e50⁰C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eISSR \u0026ndash; 05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e(CA)\u003csub\u003e6\u003c/sub\u003eAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e50⁰C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eISSR \u0026ndash; 06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e(GT)\u003csub\u003e6\u003c/sub\u003eGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e55⁰C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eA matrix was prepared for each ISSR primer scoring the presence or absence of ISSR bands in different varieties. Percentage-based pairing affinity values between different combinations of variety-pairs were calculated applying the following formula \u0026ndash;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Number of ISSR bands common to sample A and B\u003c/p\u003e\n \u003cp\u003ePA = --------------------------------------------------------------------- x 100\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Total number of ISSR bands in sample A and B\u003c/p\u003e\n \u003cp\u003eA Dendrogram was computed based on Group average (Unweighted Pair Group), using software NCSS 2026, applying Euclidean distance method and standard deviation scaling.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSequencing of ITS and rbcL DNA fragment\u003c/p\u003e\n\u003cp\u003eThe amplicons of ITS and rbcL DNA segments obtained from three varieties were in the range of 500 bp and 750 bp respectively (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The size of the edited sequences ranged from 430 to 561 bp for ITS and 408 to 576 bp for rbcL gene. The amino acid sequences of the resultant rbcL gene sequenecs were derived. The DNA sequences were submitted in the GenBank and assigned with accession numbers (Table\u0026nbsp;3). The GC content found to be consistently much higher in case of ITS sequence (61.88% \u0026minus;\u0026thinsp;63.95%) than rbcL sequence (42.76% \u0026minus;\u0026thinsp;44.36%) (Table\u0026nbsp;4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eNucleotide sequence of ITS and rbcL segments submitted in the GenBank\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eITS sequences\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e (GenBank Accession No. PZ020092.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGGTGGACCCTCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTCCGGCC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGAGGGCACGTCTGCCTGGGCGTCACGCATCGCGTCTCCCTCACCCGCCGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGCGGGGGGGAAGGACGATGGCCTCCCGTGCTTGAACGGGCGCGGCTGGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCTAAAACGGGAGCTTGCGGCGACGAGCTGCGGCGGCGTTTGGTTGACGGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGGGCCATCGGCCCTCGTAATGCATCGCGCCTCGCACGCACGTCGTCGGCAT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGGGCTCGTCGGACCCTCGGAAAACCTTTGCGACCCCAGGTCAGGCGGGGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGAGAAGAAACTTACGA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGGATTCCCCTAGTAACGGCGAGCGAACCGGGAAGAGCCCAGCTTTAAAAT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCGGGCGGCGTCGTCGTCCGAATTGTAGTTA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e (GenBank Accession No. PZ020093.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eACAAGGTTTCCGTAGGTGACCTGCGGAAGGATCATTGTCGAAACCTGCC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCAGCAGAATGACCCGAGGATGAGTTTCAAGCATAAGACGCGCGGGGAGG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTCGCCTCCCCCGCGACCGCACAGCGCCCCCCTTGGGGTGGCACGTTGCGA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCAACAAAACCCGGCGCGGACTGCGCCAAGGAACACGAACAGCGAGAGCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCCTGCCCGCGCCCGGTCCCCGGTGCGAGGGGCAGCGTCCCAGGCTAGAAA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eACGTAATGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eAGTCTTTGAACGCAAGTTGCGCCCAAAGCCTTTCGGCCGAGGGCACGTCT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGCCTGGGCGTCACACAATGCGTCTCCCCCACCCGTCGTGCGTGGGGAAGG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eATGATGGCCTCCCGCGCCTTAGCGGGCACGGCTGGCCTAAAATGGGGAGCTCG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e (GenBank Accession No. PZ020094.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCAAGCATAAGACGCGCGGGGAGGTCGCCTCCCCCCGCGACCGCACAGCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCCCCCCTTGGGGTGGCACGTTGCGACAACAAAACCCGGCGCGGACTGCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCCAAGGAACACGAACAGCGAGAGCGCCTGCCCGCGCCCGGTCCCCGGTG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCGAGGGGCAGCGTCGCAGGCTAGAAAACGTAATGACTCTCGGCAACGGA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCAAAGCCTTTCGGCCGAGGGCACGTCTGCCTGGGCGTCACACAATGCGTC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTCCCCCACCCGTCGTGCGGGGGGAAGGATGATGGCCTCACGCGCCTTAAC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGGGCACGGCTGGCCTAAAATGGGAGCTCGTGGCGATGAGCTGCGGCGGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGTTTGGTTGACGGAGGGCCATTGGCCCTCGTAATACATTGCGCCTCGCACG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCACGTCGTCGGCAAGGGCTCGTCGGACCCTGGTAAAACCTTTGCGACCCC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eAGGTCAGGCGGGGC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003erbcL sequences\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e (GenBank Accession No. PZ194680)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTGAATTATTATACTCCTCAATATCAACCCCTGGATACTGATATCTTGGCAGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eATTCCGAGTAACTCCTCAACCTGGAGTTCCGTCAGAAGAAGCAGGGGCCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGGACCGACG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGACTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGATCCCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTCCTGGAGAAGACAATCAATATATTTGTTATGTAGCTTACCCATTAGACCT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATTGTGGGTAATGTAT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTGGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCTG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTGCTTATATAAAAACTTTCCAAGGCCCGCCTCACGGTATCCAAGTTGAGA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGAGATAAATTGAACAAGTATGGCCGTCCTCTATTGGGATGCACTATTAAAC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCGAAATTGGGGTTATCTGCTAAAAACTATGGTCGAGCAGTTTATGAATGTC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTCGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGTGAACTCCCAACCATTTATG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e (GenBank Accession No. PZ194679)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTGAATTATTATACTCCTCAATATCAACCCCTGGATACTGATATCTTGGCAG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCATTCCGAGTAACTCCTCAACCTGGAGTTCCGTCAGAAGAAGCAGGGGCC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGCAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGGACCGAC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGGACTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGATCCC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGTTCCTGGAGAAGACAATCAATATATTTGTTATGTAGCTTACCCATTAGACC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATTGTGGGTAATGTA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTTGGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGTTGCTTATATAAAAACTTTCCAAGGCCCGCCTCACGGTATCCAAGTTGAG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eAGAGATAAATTGAACAAGTATGGCCGTCCTCTATTGGGATGCACTATTAAA\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCCGAAATTGGGGTTATCTGCTAAAAACTATGGTCGAGCAGTTTATGAATGT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAAAACGTGAACTCCCAACCATTTATGCGT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e (GenBank Accession No. PZ201000)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eATTATTATACTCGCTCAATATCAACCCCTGGATACTGATATCTTGGCAGCAT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTCCGAGTAACTCCTCAACCTGGAGTTCCGTCAGAAGAAGCAGGGGCCGC\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eAGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGTATGGACCGACG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eGACTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGATCCCG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTCCTGGAGAAGACAATCAATATATTTGTTATGTAGCTTACCCATTAGACCT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTTTGAAGAAGGTTCTGTTACTAATATGTTTACTTCCATTGTGGGTAATGTAT\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTGGGTTCAAAGCCCTGCGTGCTCTACGTTTGGAGGATTTGCGAATCCCTG\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan fontcategory=\"NonProportional\" name=\"Emphasis\"\u003eTTGCTTATATAAAAACTTTCCAAGGCCCGCCTCACGGGTATCCAAGTTGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e G+C content in ITS and rbcL sequences of\u0026nbsp;\u003cem\u003eBasella alba\u003c/em\u003e varieties\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable float=\"No\" id=\"Tabd\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\n \u003cp\u003eVarieties\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\n \u003cp\u003eITS segment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\n \u003cp\u003erbcL segment\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAccession No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eG\u0026thinsp;+\u0026thinsp;C content\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eAccession No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eG\u0026thinsp;+\u0026thinsp;C content\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePZ020092.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e63.95%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003ePZ194680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e42.76%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePZ020093.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e61.88%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003ePZ194679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e42.88%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePZ020094.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e63.81%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003ePZ201000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e44.36%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe derived sequences and the nearest identified sequences based on BLAST search were used to make phylogenetic analysis. The evolutionary distances were calculated using Tamura 3-parameter method (Tamura \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) in the unit of the number of base substitutions per site. Phylogenetic trees were computed using Neighbour-joining algorithm and with 1001 bootstrap replications and the branch length in the same unit as those of the evolutionary distance used. In Phylogenetic tree on ITS sequences, the optimal tree with the sum of branch length was 0.143 and analytical procedures included 4 coding nucleotide sequences using 1st, 2nd, 3rd and non-coding positions. But in tree on rbcL sequences the optimal tree with the sum of branch length was 0.015 and analytical procedures contained 7 coding nucleotide sequences using 1st, 2nd, 3rd and no-coding position.\u003c/p\u003e\n\u003cp\u003eIn the phylogenetic trees computed on ITS and rbcL sequences, the three varieties of \u003cem\u003eBasella alba\u003c/em\u003e clustered with allied species \u003cem\u003eAnredera cordifolia\u003c/em\u003e (Basellaceae) as out group species. \u003cem\u003eAnredera cordifolia\u003c/em\u003e is an evergreen semi-succulent vine-like plant with simple, ovate-cordate, acute, thin fleshy leaves and pendent axillary racemes (Rasingam and Lakshminarasimhan \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In phylogenetic tree on ITS sequences (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), both the green-stemmed and red-stemmed cultivated variety of \u003cem\u003eBasella alba\u003c/em\u003e clustered with \u003cem\u003eAnredera cordifolia\u003c/em\u003e in a single clade where green-stemmed variety appeared as nearest relative of \u003cem\u003eAnredera cordifolia\u003c/em\u003e. But crop-wild variety \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e showed wide divergence from both the cultivated form representing a separate lineage.\u003c/p\u003e\n\u003cp\u003eIn phylogenetic tree on rbcL sequence (Fig. 3), out-group species \u003cem\u003eAnredera cordifolia\u003c/em\u003e included in a different cluster. All the accessions of \u003cem\u003eAnredera cordifolia\u003c/em\u003e [GQ436516.1, JQ933240.1 and MH049965.1] along with \u003cem\u003eAnredera diffusa\u003c/em\u003e [OQ391768.1] formed a single clade while all the varieties of \u003cem\u003eBasella alba\u003c/em\u003e were nested in a single clade. The rbcL deduced amino acid sequences appeared slightly variable in the green stemmed, red-stemmed and crop-wild varieties.\u003c/p\u003e\n\u003cp\u003eISSR polymorphism analysis\u003c/p\u003e\n\u003cp\u003eBand profile (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) in each gel was scored as presence or absence of bands. Six ISSR primers yielded a total of 72 bands of which only 26 were monomorphic and 46 were polymorphic with an average polymorphism percentage of 63.88 (Table\u0026nbsp;5). Highest polymorphism was shown by ISSR\u0026thinsp;\u0026minus;\u0026thinsp;5 (94.11%) and least by ISSR \u0026ndash; 2 (38.88%). Amplicons mostly varying in size range from 1000 bp to 200 bp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e ISSR polymorphism analysis showing primer-wise generated monomorphic, polymorphic bands and percentage of polymorphism\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable float=\"No\" id=\"Tabe\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003eSl. No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003ePrimer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003eTotal no. of bands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003eNo. of monomorphic\u003c/p\u003e\n \u003cp\u003ebands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003eNo. of polymorphic bands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e% of Polymorphism\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003e1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e(CA)\u003csub\u003e8\u003c/sub\u003eGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e66.66%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003e2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e(GA)\u003csub\u003e6\u003c/sub\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e38.88%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003e3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e(AC)\u003csub\u003e8\u003c/sub\u003eYG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e63.63%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003e4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e(CT)\u003csub\u003e8\u003c/sub\u003eAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e66.66%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003e5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e(CA)\u003csub\u003e6\u003c/sub\u003eAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e94.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003e6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e(GT)\u003csub\u003e6\u003c/sub\u003eGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c8\" namest=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c10\" namest=\"c9\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" nameend=\"c12\" namest=\"c11\"\u003e\n \u003cp\u003e40.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eHighest pairing affinity (PA) value on ISSR polymorphism was shown by variety pair \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e and \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e (75.47%) while the least PA value (62.22%) was found between crop-wild \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e and \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e (Table 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u003c/strong\u003e Pairing affinity values of different varietal pairs of \u003cem\u003eBasella alba\u003c/em\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable float=\"No\" id=\"Tabf\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eB(A)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eB(R)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eB(S)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eB(A)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eB(R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e75.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eB(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e70.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e62.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDendrogram computed on ISSR data (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed relative closeness between crop-wild variety \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e with red-stemmed cultivated variety \u003cem\u003eBasella alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e nested in a single cluster. Green cultivated form (\u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003ealba\u003c/em\u003e) appeared quite diverging.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eBasella alba\u003c/em\u003e L. (Basellaceae), commonly known as vine spinach is one of the favorite leafy vegetables in the hot humid tropics of India as well as in tropical Asia and Africa (Tindall \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). The variability in morphology and habit in \u003cem\u003eBasella alba\u003c/em\u003e has recently been addressed through the introduction of three varieties \u0026ndash; green stemmed var. \u003cem\u003ealba\u003c/em\u003e, red stemmed var. \u003cem\u003erubra\u003c/em\u003e and var. \u003cem\u003escandens\u003c/em\u003e, of which first two are cultivated varieties and \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e is crop-wild variety, new for science (Das \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Both the cultivated forms are procumbent with thick, mucilaginous, fleshy, ridged stem and large oval leaves while the crop-wild \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e is characterized with twining thin cord-like, reddish stem and smaller cordate leaves.\u003c/p\u003e \u003cp\u003eDNA sequence analysis is considered as a new approach in studying evolutionary relationship and genetic diversity in crop plants. In present study phylogenetic linkage among the varieties has been resolved with molecular parameter like \u0026ndash; DNA barcoding with ITS and rbcL sequence as well as ISSR polymorphism analysis. DNA barcoding is a molecular diagnostic technology that uses standard, sufficiently variable DNA fragments for species identification and delimitation (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Internal Transcribed Spacer (ITS) sequences, located between ribosomal RNA genes, are among the most widely sequenced molecular markers in plant systematics and DNA barcoding studies because of their rapid concerted evolution within and between constituent subunits, diverging at relatively high rate, fast evolution, and the availability of universal primers (\u0026Aacute;lvarez and Wendel 2003; Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Schoch et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). ITS sequences are highly repeated, showing extensive sequence polymorphism making them reliable markers for reconstructing evolutionary history. The ribulose bisphosphate 1,5-carboxylase/oxygenase gene (rbcL) sequencing has been extensively utilized in various fields such as evolution, phylogeny, biogeography, population genetics, and systematics due to its ease of amplification, relatively conserved nature among related species, high level of universality and low mutation rate compared to other genes in chloroplast DNA (Sheng-Guo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Candramila et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eISSR polymorphism analysis is a PCR-based technique to generate multilocus marker through amplification of DNA segment present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction using microsatellite sequences as primer (Oliveira et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The technique has emerged as a substitute system with the advantage and reliability of microsatellites (SSRs) due to their high polymorphism, rapidity, sensitivity, simplicity, reproducibility, and cost-effectiveness, not needing any prior information on DNA sequences. Highly polymorphic DNA fingerprints are generated which effectively distinguish between genetic individuals (Gemmill and Grierson \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sevindik et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Higher number of polymorphic bands generally indicative of higher level of genetic diversity within and among populations or cultivars. This marker is regarded as a reliable parameter for assessing the genetic diversity among closely related taxa and identifying genotypic similarities between accessions, populations, varieties, cultivars and species (Shaban et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ISSR could be a suitable tool to assess the changes of diversity in agronomically important crops as well as underutilised crops with unknown sequences.\u003c/p\u003e \u003cp\u003ePhylogenetic study on green-stemmed and red-stemmed \u003cem\u003eBasella alba\u003c/em\u003e, tree spinach (\u003cem\u003eTalinum fruticosum\u003c/em\u003e) applying rbcL and ITS barcoding recognized clear separation of two cultivated form in two well supported clade and close alliance of tree spinach (Portulacaceae) in a separate clade (Gayathree et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, ITS sequence polymorphism has enabled clear delimitation of varieties as well as establishing the fact that ITS sequences are suitable for infraspecific delimitation. Phylogeny analysis also showed closer relationship between two cultivated domesticated varieties and wide divergence of crop-wild relative, representing the crop-wild variety \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e as probable ancestor of both the cultivated form. But due to high level of universality and relative conserved nature, the rbcL sequence analysis showed no varietal variability. Both the cultivated varieties and crop-wild variety of \u003cem\u003eBasella alba\u003c/em\u003e were nested in a same clade so as the accessions of \u003cem\u003eAnredera cordifolia\u003c/em\u003e and \u003cem\u003eA. diffusa\u003c/em\u003e. Though amino acid sequence of rbcL gene in all the varieties showed little variability. The cluster pattern reflected in ISSR analysis is not concomitant with phylogenetic affinity derived from ITS sequencing. Crop-wild variety showed much closer affinity with \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e, suggesting the red-stemmed variety as the immediate descendant from crop-wild variety. Separate identity of three varieties was established by significant DNA polymorphism.\u003c/p\u003e \u003cp\u003eInclusion of all the varieties in a single clade with 100% area coverage signifies their high degree of genetic uniformity in rbcL sequences at the same time nullifies their consideration as separate species. Study on DNA barcoding of ITS sequences rather consolidate the perception of treating them as separate varieties only by virtue of ITS polymorphism and presenting crop-wild variety \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e as progenitor of both green-stemmed and red-stemmed cultivated variety. ISSR polymorphism also clearly distinguished three varieties as separate taxonomic entities. Close alliance between crop-wild variety and red-stemmed \u003cem\u003eB. alba\u003c/em\u003e var. \u003cem\u003erubra\u003c/em\u003e is supported by their morphological similarities (Das \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMorphological variability created ambiguity in identifying and delimiting different morphotypes of \u003cem\u003eBasella alba\u003c/em\u003e. Such ambiguity was addressed segregating the population of \u003cem\u003eBasella alba\u003c/em\u003e into three varieties \u0026ndash; two cultivated (var. \u003cem\u003ealba\u003c/em\u003e and var. \u003cem\u003erubra\u003c/em\u003e) and one crop-wild variety (var. scandens) based on morphological variability. This infraspecific categorization was ratified with molecular identity and phylogenetic relationship among the varieties was also established. ITS sequencing conclusively indicated separate identity of the varieties, while uniformity of the rbcL sequences, as expected ignored their consideration as different species. Crop-wild variety \u003cem\u003eBasella alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e projected as a probable progenitor of the cultivated varieties. ISSR polymorphism also clearly delimited all the varieties and reflected red-stemmed cultivated form as immediate descendant of the crop-wild variety.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eAuthors have no relevant conflict of interest in the present work.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.D, conceptualized, designed the research plan, collected plant materials, investigated, analyzed data, prepared figures and manuscript, S. D. Jr. analysed and compiled data, reviewed the references, both the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors express their sincere gratitude to Prof. Sankar Kumar Ghosh, Director of InBOL Healthcare Pvt.Ltd. Kolkata, West Bengal, India, for his support and cooperation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjibade SR, Weeden NF, Chite SM (2000) Inter-simple sequence repeat analysis of genetic relationships in the genus \u003cem\u003eViglla\u003c/em\u003e. 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Theor Appl Genet 91:439\u0026ndash;447.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Basella alba L., Infra-specific classification, DNA barcoding, ISSR polymorphism, Phylogenetic tree","lastPublishedDoi":"10.21203/rs.3.rs-9567677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9567677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBasella alba\u003c/em\u003e L. (Basellaceae) is a popular leafy vegetable in the hot humid tropics of India, tropical Asia and Africa. The species is represented by two cultivated colour-morphs \u0026ndash; green stemmed and red-stemmed and one crop-wild twining variant with habitual and morphological variability. Such variability has favoured the introduction of three varieties under \u003cem\u003eBasella alba\u003c/em\u003e L. viz.- var. \u003cem\u003ealba\u003c/em\u003e, var. \u003cem\u003erubra\u003c/em\u003e and var. \u003cem\u003escandens\u003c/em\u003e. Phylogenetic linkage among the varieties has been traced applying DNA barcoding of ITS and rbcL sequences as well as ISSR polymorphism. Resultant ITS sequences showed extreme polymorphism but rbcL sequences appeared with high level of sequence uniformity. Phylogenetic tree constructed on ITS sequences clearly reflected separate molecular identity of the varieties, inclusion of cultivated varieties with close ally \u003cem\u003eAnredera cordifolia\u003c/em\u003e in a single clade and crop-wild \u003cem\u003eBasella alba\u003c/em\u003e var. \u003cem\u003escandens\u003c/em\u003e as probable progenitor of two cultivated varieties in separate lineage. Phylogenetic tree computed on rbcL sequences showed conspecific nature of the varieties, there inclusion in a single clade, so as the allied species of \u003cem\u003eAnredera\u003c/em\u003e. Dendrogram on ISSR polymorphism also consolidated separate identity of the varieties as well as close alliance of crop-wild variety with red-stemmed cultivated form as the immediate descendent. The study strongly favoured infraspecific classification of \u003cem\u003eBasella alba\u003c/em\u003e comprising three varieties rather than treating them as separate species.\u003c/p\u003e","manuscriptTitle":"Molecular delimitation and phylogeny of Basella alba L. varieties applying DNA barcoding and ISSR polymorphism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 17:37:17","doi":"10.21203/rs.3.rs-9567677/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T00:43:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T22:34:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84531611446830243583075778265843728694","date":"2026-05-09T15:54:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T10:41:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189092477038623419294949739259012519951","date":"2026-05-08T13:07:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209889714589993150140668046592436002946","date":"2026-05-05T08:52:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54499636899656450609366574577311006832","date":"2026-05-04T15:54:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176495156647400746360737840201708674600","date":"2026-05-04T14:44:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-04T14:26:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T13:21:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-04T13:20:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genetic Resources and Crop Evolution","date":"2026-04-29T14:52:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a9bfd073-9b58-446e-9866-4711d69b23c6","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T00:43:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T22:34:42+00:00","index":23,"fulltext":""},{"type":"reviewerAgreed","content":"84531611446830243583075778265843728694","date":"2026-05-09T15:54:57+00:00","index":22,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T10:41:17+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"189092477038623419294949739259012519951","date":"2026-05-08T13:07:02+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"209889714589993150140668046592436002946","date":"2026-05-05T08:52:38+00:00","index":19,"fulltext":""},{"type":"reviewerAgreed","content":"54499636899656450609366574577311006832","date":"2026-05-04T15:54:44+00:00","index":17,"fulltext":""},{"type":"reviewerAgreed","content":"176495156647400746360737840201708674600","date":"2026-05-04T14:44:33+00:00","index":16,"fulltext":""},{"type":"reviewersInvited","content":"12","date":"2026-05-04T14:26:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-04T13:21:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-04T13:20:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genetic Resources and Crop Evolution","date":"2026-04-29T14:52:05+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T00:54:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 17:37:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9567677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9567677","identity":"rs-9567677","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}