Characterization of Talinum Triangulare (Jacq.) Willd

South African Journal of Botany 97 (2015) 59–68

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South African Journal of Botany

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Characterization of Talinum triangulare (Jacq.) Willd. germplasm using molecular descriptors

J. Swarna a,⁎, R. Ravindhran a, T.S. Lokeswari b a T.A.L Samy Centre for Plant tissue Culture and Molecular Biology, Department of Plant Biology and Biotechnology, Loyola College, Nungambakkam, Chennai 600 034, Tamil Nadu, India b Department of Biotechnology, Sri Ramachandra University, #1, Ramachandra Nagar, Porur, Chennai 600116, Tamil Nadu, India article info abstract

Article history: Talinum triangulare is a medicinal herb known to have originated from tropical Africa and is now widely Received 8 October 2014 cultivated in the humid tropical countries. The characterization of plant germplasm using molecular descriptors Received in revised form 15 December 2014 aids in describing the genotypic traits of the plant T. triangulare. Ten different accessions of T. triangulare were Accepted 16 December 2014 collected from nine districts of Tamil Nadu. High quality genomic DNA was isolated from the different samples, Available online xxxx checked for purity and quantified. The DNA samples were subjected to PCR amplification using RAPD markers fi Edited by E Balázs and by DNA barcoding technique. RAPD ngerprints were generated for the 10 different accessions of T. triangulare for the first time. Ten random decamer primers resulted in 73.08% of polymorphism by producing Keywords: a total of 78 fragments, of which, 57 bands were determined as polymorphic loci. Phylogenetic relationship and Waterleaf intra-specific variation among the samples were established by constructing a dendrogram based on Jaccard's RAPD fingerprinting coefficient. DNA barcoding studies included the amplification of chloroplast large subunit of ribulose- DNA barcoding bisphosphate carboxylase (rbcL), trnH-psbA intergenic spacer (trnH-psbA), maturase K (matK) and nuclear Dendrogram internal transcribed spacer (ITS). The amplicons were gel eluted, sequenced and checked for homology by using the BLAST tool. However, no variation was detected among the samples after sequencing, proving that the accessions were identical at the genetic level. © 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction can be compared and evaluated against reference sequences already existing in online databases, thus, providing rapid and reproducible In the past century, rapid globalization leading to adverse taxonomic recognition (Hebert et al., 2003). This method opens new environmental conditions has resulted in massive loss of valuable plant perspectives for the identification of medicinal herbs which is useful species. This has triggered the conservation of plant genetic resources. Ac- to clarify taxonomic uncertainties within the family Portulacaceae. curate identification and characterization of plant materials is essential for The Purslane family (Portulacaceae), a group of edible plants with cos- their sustainable utilization. The dramatic advances in molecular genetics mopolitan distribution, is represented by about 20 genera and 500 species over the last few years have provided researchers with an extensive (Jones and Luchsinger, 1987). The genus Talinum comprises of 15 species, range of new techniques for answering many evolutionary and taxo- of which only five species have been reported in India. Talinum triangulare nomic questions. Moreover, a majority of these techniques have (Jacq.) Willd., popularly known as waterleaf, is a fleshy-leaved perennial been effectively employed to study the extent and distribution of herb grown widely in the humid tropical countries as a leaf vegetable variation in species gene-pools (Karp et al., 1996). (Swarna and Ravindhran, 2013). The plant known to have originated in The advent of DNA-based markers has revolutionized the practice of Central Africa, is now traditionally valued for its remarkable antioxidant DNA fingerprinting and diversity analysis of plant species. Recently, activities and has been implicated in the management of diabetes, DNA barcoding, a new biological tool based on the analysis of short, stan- jaundice, cancer, stroke, obesity and measles (Fontem and Schippers, dardized and universal DNA regions (barcodes), has been proposed 2004). Waterleaf is characterized as tolerant to various soil types, as a universal tool for species discrimination and identification temperatures and moisture levels, and grows well under shade. The (Hebert et al., 2003). DNA sequences generated from such barcodes Portulacaceae members belonging to the order Caryophyllales are characterized by the occurrence of betalains, a class of nitrogen-containing plant pigments which accumulate in flowers, fruits and occasionally in vegeta- Abbreviations: DMRT, Duncan's multiple range test; ITS, Internal transcribed spacer; tive tissues (Steglich and Strack, 1990). By nature, T. triangulare produces RAPD, Random amplified polymorphic DNA; SPSS, Statistical package for social sciences; betalain in flowers and under extreme environmental conditions UPGMA, Unweighted pair group method analysis ⁎ Corresponding author. Tel.: +91 44 28178200x330. the pigment gets accumulated in leaves and stems (Swarna et al., E-mail address: [email protected] (J. Swarna). 2013).

Table 1 Latitude, longitude and altitude details of the 10 different accessions of T. triangulare collected from various districts within Tamil Nadu.

S. No. Place/District State Latitude Longitude Altitude(meters above sea level)

1 Ariyalur Tamil Nadu N 11°8′14″ E 79°4′40″ 76 m 2 Chennai Tamil Nadu N 13°5′2″ E 80°16′12″ 6m 3 Coimbatore Tamil Nadu N 11°1′6″ E 76°58′21″ 411.2 m 4 Erode Tamil Nadu N 11°21′0″ E 77°44′0″ 183 m 5 Madurai Tamil Nadu N 9°55′10.78″ E 78°7′9.82″ 101 m 6 Thanjavur Tamil Nadu N 10°46′30″ E 79°8′20″ 88 m 7 Tiruchirappalli Tamil Nadu N 10°48′18″ E 78°41′8″ 88 m 8 Tirunelveli Tamil Nadu N 8°43′48″ E 77°42′0″ 47 m 9 Tiruvannamalai Tamil Nadu N 12°13′12″ E 79°4′12″ 171 m 10 Chennai (Wild) Tamil Nadu N 13°5′2″ E 80°16′12″ 6m

Of the five different Talinum species reported in India, T. triangulare is stirred for 30 min. The samples were centrifuged at 10,000 rpm for closely related to Talinum paniculatum and Talinum portulacifolium.The 10 min at 4 °C in a 5810r Eppendorf refrigerated centrifuge (Hamburg, former differs from the other species by certain morphological traits Germany). The supernatant was transferred and the pellet was re- including paniculate inflorescence borne on a triangular peduncle and extracted twice with 80% methanol. All the supernatants were pooled flower sepals being prominently veined. These minute differences often andstoredat−20 °C in dark until use. The betacyanin, betaxanthin and lead to confusion misjudgement of the T. triangulare species, which total betalain content were calculated based on the spectrophotometric could lead to adulteration in herbal preparations. Hence, in the present multiple-component method of Nilsson (1970). Determination of study, molecular characterization of T. triangulare was carried out for its betalain concentration, i.e., violet and yellow pigments, was expressed authentication at the DNA level. in terms of betanin and vulgaxanthin I, respectively. Total pigment con- The current work was aimed at identifying morphological, biochemi- tent was calculated as the sum of betacyanin and betaxanthin compo- cal and genetic variability among 10 accessions of T. triangulare collected nents (mg/g FW of the plant sample). The experiments were performed from different districts within Tamil Nadu. An integrative approach was with a minimum of ten plants per treatment and the data were analyzed pursued by RAPD fingerprinting and DNA barcoding studies to provide statistically using IBM SPSS statistics 19 (SPSS Inc., Chicago, USA). The better genetic clarity of the plant. Moreover, intra-specific phylogenetic mean values were expressed as mean ± SE of three repeated experiments relationship was assessed by plotting a dendrogram. The performance and the significance of differences among means was carried out at 5% of four candidate barcoding loci (rbcL, trnH-psbA, matKandITS)wereex- probability level using Duncan's Multiple Range Test (DMRT). amined by PCR amplification and DNA sequencing for the recognition and validation of T. triangulare to establish unambiguous identification of this 2.3. DNA isolation from T. triangulare valuable medicinal plant. The total genomic DNA was isolated from different accessions of T. triangulare by using a modified SDS method (Dellaporta et al., 1983). 2. Materials and methods The collected DNA pellet was suspended in appropriate quantity of nuclease-free Milli-Q water and stored at −20 °C until use. Quality and 2.1. Collection of plant material the concentration of the DNA samples were assessed by agarose gel electrophoresis and by using NanoDrop ND-1000 Spectrophotometer T. triangulare plants were collected during the period of January to (NanoDrop Technologies, Wilmington, USA) respectively. The purity of April from cultivated farmer's fields or natural localities of nine different DNA was assessed by the sample absorbance ratio of A /A and the districts within the Tamil Nadu state. The districts from where the 260 280 DNA samples were diluted according to use. samples were obtained were Ariyalur, Chennai, Coimbatore, Erode, Madurai, Thanjavur, Tiruchirappalli, Tirunelveli and Tiruvannamalai, 2.4. Random amplified polymorphic DNA (RAPD) fingerprinting which included regions with varying soil and climatic conditions. T. triangulare plants from the wild were also acquired for comparison To investigate the genetic variation among the 10 different purposes. A minimum of at least 30 plants were gathered from T. triangulare samples, RAPD analysis was performed. The PCR-based each site for the analyses which represented the total population of RAPD technique was executed by employing 10 random decamer that area. The plant material was identified and authenticated by primers, namely, OPA-13, OPA-16, OPA-17, OPA-18, OPA-19, OPB-08, Dr. D. Narasimhan, Centre for Floristic Research, Madras Christian OPB-11, OPB-13, OPB-17 and OPB-18 (Operon Technologies Inc., CA, College, Chennai. A herbarium voucher specimen (LCH 42) was pre- USA) (Table 2). served for future reference and has been deposited in the Loyola College The PCR reaction was carried out in a volume of 20 μlreactionmixture Herbarium. The place of collection along with the latitude and longitude consisting of the following components: Taq Buffer A with 15 mM MgCl data and its altitude above sea level are tabulated in Table 1.Theleaves 2 (1×), dNTPs (0.2 mM), RAPD primer (Operon Technologies, USA) (0.5 of the different samples were freshly used for DNA extraction while pM), Taq DNA Polymerase (1 U) and genomic DNA (50 ng). For every epidermal peel of stem was used for betalain analysis. primer used, a negative control containing nuclease-free Milli-Q water was included to ensure there were no false-positives due to cross- 2.2. Morphological and biochemical descriptors contamination. The RAPD-PCR amplification was carried out in a DNA thermal cycler (Mastercycler gradient, Eppendorf, Germany) with the fol- To analyze the morphological variation among the 10 accessions, lowing conditions: a twig from each sample was collected and examined. Visual examination was performed to detect any difference among the samples. For biochem- Initial denaturation 94 °C for 5 min ical characterization, betalains of the 10 samples were analyzed. For this, Cycling reaction 9 the betalain extraction from the epidermal peel (1.0 g) was performed by o Denaturation 94 °C for 1 min = homogenizing the samples in a mortar with liquid nitrogen, to preclude o Annealing 36 °C for 1 min ; 35 cycles enzymatic degradation during grinding. The resulting powder was ex- o Extension 72 °C for 2 min Final extension 72 °C for 7 min tracted with 10 ml of 80% (v/v) aqueous methanol and continuously J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68 61

After PCR amplification, the RAPD-PCR products were resolved in Table 3 1.8% (w/v) agarose gel with 1× Tris–acetic acid–EDTA (TAE) buffer. Sequence information of the forward and reverse primers of the different barcodes used for the DNA barcoding studies. GeneRuler 1 kb Plus DNA ladder (Thermo Scientific, Mumbai, India) (75 bp to 20 kb) was used as DNA marker. The amplified fragments Name Primer code Sequence (5′–3′) Reference were visualized under UV light and documented using the Gel rbcL primers Documentation equipment (UVP, Ultra-Violet Products Ltd., UK). Forward rbcLa_f ATGTCACCACAAAC Levin et al. (2003) PCR reactions were repeated at least thrice to confirm the reproduc- AGAGACTAAAGC ibility of the results. Reverse rbcLa_r GTAAAATCAAGTCC Kress and Erickson ACCRCG (2007) trnH-psbA primers 2.4.1. RAPD data analysis Forward psbA3_f GTTATGCATGAACG Sang et al. (1997) After excluding markers that were monomorphic for the entire data TAATGCTC set, a vector of molecular marker phenotype was established for each Reverse trnHf_05 CGCGCATGGTGGAT Tate and Simpson TCACAATCC (2003) individual accession of the plant that was analyzed. Variability was matK primers expressed as percentage of polymorphism which was computed as Forward MatK-1R_KIM_f ACCCAGTCCATCTG Ki-Joong Kim the number of polymorphic bands over the total number of scored GAAATCTTGGTTC (unpublished) bands. In addition, fragment data were treated as two state qualitative Reverse MatK-3F_KIM_r CGTACAGTACTTTT Ki-Joong Kim data and the RAPD prints were converted into binary matrices. For GTGTTTACGAG (unpublished) ITS primers genetic similarity estimates, the fragment data were coded as one for Forward ITS1 TCCGTAGGTGAACC White et al. (1990) the presence of band and zero indicating absence of band. Pairwise TGCGG genetic comparison of the sample data was performed by entering the Reverse ITS4 TCCTCCGCTTATTG White et al. (1990) binary scores into Numerical Taxonomy and Multivariate Analysis ATATGC System (NTSYS-pc), version 2.02i (Exeter Software, NY, USA) using the NTedit program. Genetic similarity was estimated using Similarity for Qualitative Data (SIMQUAL) to generate Jaccard's similarity coef- For estimating the size of the amplicons, 1 kb DNA ladder (New England ficient in NTSYS-pc (Applied Biostatistics). Finally, diversity group Biolabs Inc.,UK) (500–10,000 bp) was used as the DNA marker. The clusters were analyzed using Sequential Agglomerative Hierarchical amplified fragments were visualized under UV light and document- Nested cluster analysis (SAHN) and Unweighted Pair Group Method ed using the Gel Documentation equipment (UVP, Ultra-Violet Products Analysis (UPGMA) using average linkages clustering routines within Ltd., UK). PCR reactions were repeated at least thrice to confirm the the NTSYS-pc software package to construct the dendrogram. reproducibility of the results.

2.5. Molecular characterization by DNA barcoding 2.5.2. DNA sequencing of the amplified product The double stranded PCR products were purified using a PCR For the DNA barcoding studies of the different accessions of purification kit and directly sequenced from both the ends using T. triangulare,amplification of the most frequently used barcodes of dye terminator technique. Forward and reverse cycle sequencing the chloroplast and nuclear genomes was performed. Two coding based on the Sanger's sequencing method was performed using the (rbcLandmatK) and one non-coding region (trnH-psbA) of the BigDye Terminator v3.1 Cycle Sequencing Kit (Perkin-Elmer, Applied chloroplast genome was used for the molecular identification of Biosystems) on ABI Prism 3730XL sequencer (Perkin-Elmer, Applied this plant. A portion of the internal transcribed spacer (ITS) region Biosystems, USA). The sequencing reaction mixture (10 μl) contained of the 18S–28S nuclear DNA was amplified in a PCR reaction using 0.5 μl BigDye v3.1 ready reaction mixture, 3 μl PCR product (10–50 ng), ITS1 and ITS4 primers. The sequences of the different primers and 2 μl sequencing buffer (5×), 1 μlprimer(10μM) and 3.5 μlautoclaved their references as recommended by CBOL (2009) are provided in Milli-Q water. The cycling regime was 30 cycles of 94 °C for 30 s, 50 °C Table 3.A20μl reaction mixture was prepared for each barcode as for 15 s and 60 °C for 4 min. Each sample was sequenced in the sense described in Table 4. The PCR amplification was carried out in a and antisense direction and analyzed with ABI sequence navigator soft- DNA thermal cycler as per the conditions detailed in Table 5. ware (Perkin-Elmer/Applied Biosystems). Nucleotide sequences of both DNA strands were obtained and compared to ensure accuracy. Sequence 2.5.1. Agarose gel electrophoresis of the amplified product comparison to set up the level of identification (species, genus, or family) After PCR amplification of the chloroplast and nuclear gene, the through BLASTn algorithms was performed. The nucleotide sequences products were checked in agarose gel with 1× Tris–acetic acid–EDTA obtained were used as queries in the BLASTn search for molecular- (TAE) buffer. For rbcL, matK and ITS amplicons, 1.2% (w/v) agarose gel based species identification matches with the available GenBank was used, while 2.0% gel was used to resolve trnH-psb Aproduct. sequences (Altschul et al., 1990).

3. Results

Table 2 3.1. Morphological variation Details of the RAPD primers used to evaluate genetic diversity among 10 accessions of T. triangulare. T. triangulare plants that were collected from 10 different districts Primer name Sequence Tm (°C) GC-content within Tamil Nadu were examined for morphological variation. A twig OPA-13 5′-CAGCACCCAC-3′ 34.0 70% collected from each locality was photographed and documented OPA-16 5′-AGCCAGCGAA-3′ 32.0 60% (Fig. 1). Morphologically, most of the plants showed distinct variation OPA-17 5′-GACCGCTTGT-3′ 32.0 60% with respect to the leaf color, size, betalain production in stems, etc. ′ ′ OPA-18 5 -AGGTGACCGT-3 32.0 60% The plants obtained from Ariyalur (Fig. 1a), Chennai (Fig. 1b) and OPA-19 5′-CAAACGTCGG-3′ 32.0 60% OPB-08 5′-GTCCACACGG-3′ 34.0 70% Coimbatore (Fig. 1c) appeared healthy with long leaves which were OPB-11 5′-GTAGACCCGT-3′ 32.0 60% green in color. The ﬂowers were normal sized with regular seed for- OPB-13 5′-TTCCCCCGCT-3′ 34.0 70% mation. The Erode accession (Fig. 1d) had smaller leaves which were OPB-17 5′-AGGGAACGAG-3′ 32.0 60% succulent in nature. The presence of betalains was noticed in the thin OPB-18 5′-CCACAGCAGT-3′ 32.0 60% stems of the Erode sample. Light green to yellow colored leaves with 62 J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

Table 4 PCR reaction mixture for the four different barcoding loci of T. triangulare.

S. Component rbcL, trnH-psbA and ITS barcodes matK barcode No. Working Volume required Working Volume required concentration per 20 μl reaction concentration per 20 μl reaction

1. Taq Buffer A with 15 mM MgCl2 (GeNei, Merck, India) 1× 2 μl1×2μl 2. dNTPs (GeNei, Merck, India) 0.4 mM 4 μl 0.2 mM 2 μl 3. Forward primer 50 nM 0.2 μl 50 nM 0.2 μl 4. Rreverse primer 50 nM 0.2 μl 50 nM 0.2 μl 5. Taq DNA Polymerase (GeNei, Merck, India) 1 unit 0.3 μl 1 unit 0.6 μl 6. Genomic DNA 50 ng 2 μl50ng2μl 7. Milli-Q water 11.3 μl13μl Total 20 μl20μl

betalain pigmentation at the ends were observed in the Madurai and total betalains (4.520 ± 0.033 mg/g FW) were quantified in the sample (Fig. 1e). The leaves were also not strongly attached at the wild accession. This was closely followed by the Chennai sample, nodes and wilted on touching. This sample also showed characteris- which revealed the second highest total betalain content (3.276 ± tic rosette habit with shorter internode length having more number 0.065 mg/g FW) with 3.258 ± 0.071 mg/g FW betacyanins of leathery leaves (Fig. 1e). The flower stalk surpassed the shoot apex and 0.017 ± 0.006 mg/g FW betaxanthins. The betacyanins and which produced miniature flowers that were very small in size betaxanthins in the Tiruchirappalli accession was determined to (Fig. 1e). The Thanjavur (Fig. 1f) and Tiruchirappalli (Fig. 1g) sam- be 2.614 ± 0.091 mg/g FW and 0.110 ± 0.004 mg/g FW respectively, ples possessed maximum leaf length among all the samples studied yielding to a total betalain content of 2.724 ± 0.095 mg/g FW. The and the plants were dark green in color. The stem diameter was highest betaxanthin content was detected in the Coimbatore sample almost 1.5–2.5 cm and it was succulent in nature. Healthy flowers (0.246 ± 0.036 mg/g FW), whereas, the wild variety possessed maxi- with dark pink pigmentation were observed in the Thanjavur and mum betacyanins (4.365 ± 0.053 mg/g FW). Very low betalain content Tiruchirappalli accessions. The plant collected from Tirunelveli com- was calculated in Madurai (0.950 ± 0.036 mg/g FW) and Coimbatore prised of smaller leaves near the ground level and the size of the (1.155 ± 0.014 mg/g FW) samples (Fig. 2). leaves gradually increased towards the top (Fig. 1h). Thin stems with betalain pigmentation were detected in this plant sample. Peduncles greatly exceeding the length of the stem were noticed in 3.3. Molecular characterization the Tiruvannamalai (Fig. 1i) and the wild variety (Fig. 1j). The inflorescence at the apex consisted of diminutive flowers and numerous 3.3.1. RAPD fingerprinting fruits. In both the samples the stems were pink colored and showed A total of 78 amplicons were detected by examining 10 T. triangulare more branching. The size of the leaves was small and light green in accessions with 10 random RAPD primers (Table 6). Fig. 3a–j illustrates color (Fig. 1j). the RAPD fingerprints of the 10 samples produced with the primers OPA-13, OPA-16, OPA-17, OPA-18, OPA-19, OPB-08, OPB-11, OPB-13, 3.2. Biochemical variation OPB-17 and OPB-18 respectively. The total number of bands per primer ranged from 1 (OPA-16) to 14 (OPA-13, OPA-18) and varied in size The betalain content was assessed in each sample and the data between 551 bp and 6549 bp. Out of the 78 amplified bands, 57 bands have been represented graphically in Fig. 2. Maximum betacyanins were determined as polymorphic loci while the remaining 21 were (4.365 ± 0.053 mg/g FW), betaxanthins (0.154 ± 0.020 mg/g FW) monomorphic bands. The number of polymorphic loci detected per primer differed from 1 band (OPA-16) to 13 bands (OPA-18). The primer OPA-16 produced a single amplicon in all the accessions except Table 5 Tiruvannamalai sample resulting in 100% polymorphism. From this PCR amplification conditions of the barcodes of T. triangulare as carried out in a DNA data, an average of 5.7 polymorphic fragments per primer was calculat- thermal cycler. ed (Table 6). The percentage of polymorphism ranged from 44.44% for PCR conditions for rbcL and trnH-psbA barcodes primer OPB-08 to a maximum of 100.0% for the primer OPA-16. When Initial denaturation 95 °C for 4 min all primers were taken collectively, a mean polymorphism percentage Cycling reaction 9 of 73.08% was established. o Denaturation 94 °C for 30 s = The similarity indices among the individual samples were o Annealing 59.7 °C for 1 min; 35 cycles o Extension 72 °C for 1 min represented in terms of Jaccard's similarity coefficient (Table 7). Final extension 72 °C for 10 min The similarity coefficient for the 10 accessions ranged between 0.436 (Wild vs. Coimbatore) and 0.929 (Tirunelveli vs. Tiruchirappalli). PCR conditions for matK barcode fi Initial denaturation 94 °C for 1 min These ndings divulged the fact that the Tiruchirappalli and Tirunelveli Cycling reaction 9 samples were closely related. UPGMA-based cluster analysis grouped o Denaturation 94 °C for 30 s = the 10 accessions into three main cluster groups. Based on the dendro- o Annealing 55 °C for 20 s; 35 cycles gram (Fig. 4), three groups (I, II and III) were identified with the help of o Extension 72 °C for 30 s Final extension 72 °C for 5 min the pruning line at genetic distance of 0.60. The cluster I comprised of six samples including Tiruchirappalli, Tirunelveli, Chennai, Thanjavur, PCR conditions for ITS barcode Erode and Madurai. Three genotypes, Ariyalur, Tiruvannamalai and Initial denaturation 94 °C for 3 min Coimbatore were classified under the second cluster (group II). The Cycling reaction 9 o Denaturation 94 °C for 30 sec = wild variety was positioned the farthest from the other nine accessions o Annealing 55 °C for 30 sec; 35 cycles with a genetic distance of 0.54 and was categorized as group III. Group I o Extension 72 °C for 2 min and group II were further partitioned into subgroups which were Final extension 72 °C for 10 min connected at different genetic distances. J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68 63

abc

d e f

g h i j

Fig. 1. Morphological variation among 10 different samples of T. triangulare as observed with the naked eye. Plant samples were collected from the following districts of Tamil Nadu: a. Ariyalur,b. Chennai, c. Coimbatore, d. Erode, e. Madurai, f. Thanjavur, g. Tiruchirappalli, h. Tirunelveli, i. Tiruvannamalai and j. Chennai (Wild sample) (Bar: 2.0 cm).

3.3.2. Molecular characterization by DNA barcoding (ITS) region was amplified in all the 10 samples. The agarose gel separa- Three chloroplast barcodes and one nuclear gene were used for tion revealed an amplicon size of 630 bp for all the samples (Fig. 5d). the DNA barcoding studies among 10 T. triangulare accessions. Results These amplicons obtained for each barcode were further sequenced of our tests within the 10 samples with the four barcodes showed with both forward and reverse primers and the nucleotide sequence ob- prominent PCR amplification with 100% success rate. tained was aligned at both the ends. DNA sequence length of 531 bp for The rbcL gene which codes for the large subunit of ribulose- rbcL, 255 bp for trnH-psbA, 858 bp for matK and 649 bp for ITS barcodes 1,5-bisphosphate carboxylase/ oxygenase (RuBisCo) enzyme is com- were acquired for the T. triangulare accessions respectively. The monly used for molecular discrimination of plant species. This coding sequence information was uploaded into the GenBank database region was amplified for all the samples and amplicons of size 580 bp and the ids for the samples were KJ380905.1, KJ380906.1, KJ380907.1 was obtained in all the lanes (Fig. 5a). In the chloroplast genome and KJ380908.1. (cpDNA), trnH-psbA is an intergenic spacer. Amplicon size of 310 bp was recorded for the 10 samples when this non-coding region was 3.3.2.1. BLAST analysis of DNA sequence data. The DNA sequences amplified by PCR (Fig. 5b). The nucleotide sequence of the plastid obtained were checked for homology by using BLAST tool in the encoded gene matK which codes of maturase enzyme was also inves- NCBI webpage. Out of the top 100 BLAST results for rbcLgeneof tigated. PCR amplification for the 10 samples resulted in 980 bp size T. triangulare, only one hit belonged to the same genus, namely, amplicons (Fig. 5c). Nuclear ribosomal internal transcribed spacer T. paniculatum. Portulaca oleracea, Portulaca pilosa and Portulacaria 64 J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

5 h i

h g

3 f g f f e e e d d 2 c c b b a a 1 a

d d c c c b c b a 0 a

Betaxanthin (mg/gm FW) Betacyanin (mg/gm FW) Total betalain (mg/gm FW)

Fig. 2. Biochemical variation among the 10 samples of T. triangulare as calculated based on the betaxanthin, betacyanin and total betalain content. (Values represent mean values ± SE of three replicates per sample. Mean bars with the same letter are not significantly different at 5% probability level using Duncan's Multiple Range Test (DMRT). FW: Fresh weight.) sp. (Didiereaceae) were also detected among the top hits. For these relationship with T. triangulare. Frequently, the size of the amplified sequences obtained, only partial conserved regions of the rbcL fragments of matK varied from 846 to 852 nucleotides depending gene have been reported. Other species belonging to various genera upon the plant species (Table 3). In the present study, the maturase and families were also attained in the homology pairing. The K(matK) gene of T. triangulare of size 858 bp, revealed 99% sequence sim- amplicon size of rbcL gene had earlier been reported as 654 bp when ilarity with Talinum fruticosum (gb|DQ855844.1) (Syn. T.triangulare). the primers rbcLa_f and rbcLa_r were used (Table 3). The rbcLgene Identity match of 98% with T. portulacifolium (gb|DQ855847.1) and showed 99% sequence identity with Portulacaria sp. (dbj|AB586510.1) 97% with T. paniculatum (gb|AY015274.1), Tspathulatum(gb| and T. paniculatum (gb|HM850388.1; gb|GQ436529.1) with 100% HQ620890.1), T polygaloides (gb|DQ855845.1) and Tlineare(gb| query coverage. EU834752.1) were found. Query coverage of 100% was detected in all The trnH-psbAgeneofT. triangulare was checked for homology with the species. A close relation to Talinella sp. matKgenehaving98%iden- different species.Out of the first 100 hits, just one sequence match with tity was also observed (gb|DQ855846.1). T. paniculatum was detected. Most of the sequence alignments were The nuclear ITS region of T. triangulare was amplified to produce a observed with Pereskia spp. (Cactaceae), followed by Opuntia spp. 680 bp amplicon. The partial ITS sequence was checked for homology (Cactaceae). The amplicon size of trnH-psbA ranged from 318 bp to match and three Talinum spp. were distinguished among the Talinella 820 bp when the psbA3_f and trnHf_05 primers were employed spp. (Portulacaceae) and Opuntia spp. (Cactaceae). The sequence of (Table 3) due to small scattered insertions or deletions without an internal transcribed spacer (ITS) region obtained (649 bp) revealed apparent taxonomic pattern. A partial sequence of the trnH-psbA 98% similarity with T paraguayense (gb|L78056.1) showing query cover- intergenic spacer was acquired with the forward and reverse primers. age of 89%. T. paniculatum (gb|EU410357.1) was 91% identical to our ITS An identity match of 97% with Pereskia zinniflora (gb|AY851571.1) sequence having 96% sequence cover. The ITS region also showed 93% (Cactaceae) was revealed in the BLAST results. Sequence similarity of match with T. portulacifolium (gb|L78057.1). Homology of the ITS region 98% with T. paniculatum (gb|AY851584.1) was detected with 77% of Talinella microphylla (gb|L78053.1) and Talinella pachypoda (gb| query coverage, but with low scores. L78054.1), another genus of the sample family, was detected at95% The BLAST results for matK gene sequence of T. triangulare revealed and 94% sequence similarity respectively. the closely related species. Among the various hits acquired, six sequences producing significant alignments belonged to the Talinum 4. Discussion genus. Talinella sp., a closely related genotype, also showed strong 4.1. Morphological and biochemical descriptors

Table 6 RAPD ﬁngerprinting data obtained from 10 random decamer primers for the different The 10 different accessions of T. triangulare were analyzed for accessions of T. triangulare. morphological and biochemical variation. Based on the external ap- pearance of the plant, samples collected from Ariyalur, Chennai, Primer Size of Total Number of Number of Percentage of fragments number monomorphic polymorphic polymorphism Coimbatore, Thanjavur and Tiruchirappalli were similar. All the (bp) of bands bands bands samples were healthy with long, dark green leaves. The Erode and Tirunelveli samples were comparable as they had smaller leaves OPA-13 826–4875 14 3 11 78.57% OPA-16 1227 1 0 1 100.0% and thinner stems. Tiruvannamalai and the wild sample collected OPA-17 763–2108 5 1 4 80.00% from Chennai were alike since their leaves were greenish yellow OPA-18 651–6549 14 1 13 92.86% in color and they had extended peduncles. The most diverse – OPA-19 584 1936 8 2 6 75.00% among the 10 samples was the Madurai accession. The morpholog- OPB-08 551–3003 9 5 4 44.44% OPB-11 816–4035 10 3 7 70.00% ical characteristics of this sample were completely different by OPB-13 594–5012 5 2 3 60.00% having short internodes and light green leathery leaves arranged OPB-17 627–3910 8 2 6 75.00% in a rosette. These distinctive features observed in the plants OPB-18 882–1837 4 2 2 50.00% could be due to varying environmental and soil conditions at the Total/average 551–6549 78 21 57 73.08% place of plant collection. .San ta./SuhArcnJunlo oay9 21)59 (2015) 97 Botany of Journal African South / al. et Swarna J.

ab c d e – 68

fgh i j

Fig. 3. DNA ﬁngerprinting patterns of 10 different samples of T. triangulare generated with RAPD primers. RAPD-PCR ampliﬁcation products obtained with primers:a. OPA-13, b. OPA-16, c. OPA 17, d. OPA 18, e. OPA 19, f. OPB 08, g. OPB 11, h. OPB 13, i. OPB 17 and j. OPB 18 as separated on 1.8% agarose gel [Lane M: GeneRuler 1 kb Plus DNA ladder (75 bp to 20 kb); Lanes 1–10: Ariyalur, Chennai, Coimbatore, Erode, Madurai, Thanjavur, Tiruchirappalli, Tirunelveli, Tiruvannamalai, Chennai (Wild sample); Lane NC: Negative control]. 65 66 J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

Table 7 Jaccard's similarity coefﬁcient calculated among the 10 different accessions of T. triangulare by NTSYS-pc software.

Sample Ariyalur Chennai Coimbatore Erode Madurai Thanjavur Tiruchirappalli Tirunelveli Tiruvannamalai Wild

Ariyalur 1.0000000 Chennai 0.6617647 1.0000000 Coimbatore 0.6181818 0.6000000 1.0000000 Erode 0.5942029 0.7605634 0.5303030 1.0000000 Madurai 0.6406250 0.7142857 0.5737705 0.7205882 1.0000000 Thanjavur 0.6478873 0.8082192 0.5428571 0.8169014 0.8235294 1.0000000 Tiruchirappalli 0.5945946 0.8732394 0.5571429 0.7567568 0.7857143 0.8266667 1.0000000 Tirunelveli 0.6250000 0.9130435 0.5652174 0.7671233 0.7714286 0.8630137 0.9295775 1.0000000 Tiruvannamalai 0.6603774 0.6093750 0.6122449 0.5384615 0.6964286 0.5735294 0.5652174 0.5735294 1.0000000 Wild 0.5714286 0.5147059 0.4363636 0.6129032 0.5573770 0.5285714 0.5000000 0.5070423 0.5918367 1.0000000

The biochemical characters of the different plants were studied 4.2. Molecular characterization based on the quantity of betalain content. Betalains, which are charac- teristic pigments of the plants belonging to the order Caryophyllales, Molecular tools provide valuable data on genetic diversity based on were calculated in terms of betacyanins and betaxanthins. Maximum their ability to detect variation at the DNA level. Identification of any betalain content was determined in the wild accession followed by plant species is of fundamental importance in diversity studies. Thus, the Chennai sample, while, Madurai accession recorded the lowest for the evaluation of species diversity, it is essential that individuals be quantity of betalains. These divergent traits could be attributable to classified accurately. To establish effective management of plant genetic various reasons such as soil variation, excessive sunlight, low water diversity for sustenance and maintenance of vital plants, it is imperative content and nutrient depletion. to regard variation as richness and distribution at both inter and intra- Distinguishing characters among the different accessions can be specific levels. The genetic constitution of taxonomic units and endan- investigated using morphological and biochemical descriptors. gered species is distinct, so as a result their identification from their rel- For a long time, such traits have been the basis for characterization atives is important in the development of appropriate conservation of diversity (Endress, 2000). Morphological information play a strategies. Depending on the state of our heritable understanding of pivotal role in understanding life cycles, geographical distributions, taxon, genetic diversity may be considered at different organizational identification, conservation status, evolution, development, and spe- levels such as the genopool, population, individual genome, locus and cies delimitation (Buzgo et al., 2004; Kaplan, 2001). Nonetheless, DNA based sequence (Padmalatha and Prasad, 2007). there remains considerable debate about the precise role of morphology as they are inadequate for identifying the species, mainly 4.2.1. RAPD fingerprinting due to fluctuation in environmental conditions. Furthermore, these The isolation of plant DNA is the first step for any type of molecular descriptors are often restricted as the characters may not be obvious analysis. In the present study, crisp DNA bands without any shear were at all stages of the plant development and appearances may vary. observed in agarose gel for all the 10 samples. A search for Talinum gene Thus, additional classification and characterization at the molecular sequences showed lack of information for this species. Since RAPD is level are essential to identify and authenticate this particular plant commonly used for unknown genomes, we estimated the polymor- at the species level. phisms exhibited for a set of 10 randomly chosen decamer primers.

Ariyalur

Tiruvannamalai Group II

Coimbatore

Chennai

Tiruchirappalli

Tirunelveli Group I Thanjavur Clusters: 3; Genec distance: 0.60 Erode

Madurai

Wild variety Group III

0.53 0.63 0.73 0.83 0.93 Coeﬃcient

Fig. 4. Dendrogram generated by UPGMA cluster analysis based on Jaccard's coefﬁcient to reveal the phylogenetic relationship among 10 samples ofT. triangulare. The pruning line at a genetic distance of 0.60 clustered the samples into three major groups (I, II, III). J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68 67

a c

b d

Fig. 5. DNA barcoding proﬁles of 10 different samples of T. triangulare using:a.rbcL chloroplast gene, b.trnH-psbAintergenicspacer,c.matK chloroplast gene and d. nuclear ITS region. Amplicon size of:a. 580 bp for rbcL, b. 310 bp for trnH-psbA, c. 980 bp for matK and d. 630 bp for ITS were obtained on agarose gel [Lane M: 1 kb DNA ladder (500–1000 bp); Lanes 1–10: Ariyalur, Chennai, Coimbatore, Erode, Madurai, Thanjavur, Tiruchirappalli, Tirunelveli, Tiruvannamalai, Chennai (Wild sample); Lane NC: Negative control].

For the first time, RAPD fingerprint data of different samples of to the families Araliaceae (Liuetal.,2012), Plumbaginaceae (Ding et al., T. triangulare were generated. The gel analysis of the samples revealed 2012) and Orchidaceae (Asahina et al., 2010). Conversely, the poor abil- a total of 78 bands, of which, 57 were determined as polymorphic loci. ity of rbcL to resolve phylogenetic relationships at the genus and species The amplification patterns demonstrated that the percentage of poly- level has been reported earlier (Doebley et al., 1990). Therefore, other morphism was 73.08%. UPGMA-based cluster analysis calculated in useful DNA regions that evolve faster than rbcL to facilitate lower-level terms of Jaccard's similarity coefficient grouped the 10 accessions into phylogenetic construction are explored. three main cluster groups (I, II, III). Similarly RAPD markers have been The trnH-psbA amplicon of T. triangulare (255 bp) exhibited upto used extensively to study the diversity among various crop plants and 97% sequence match with Pereskia zinniflora (gb|AY851571.1) belong- medicinal plants. Some of the reports for establishing genetic variation ing to the family Cactaceae. Molecular phylogenetic studies based on in medicinal plants using RAPD markers include Rauvolfia tetraphylla trnH-psbA DNA sequences have been reported for members belonging (Mahesh et al., 2008), Momordica charantia (Dey et al., 2006), Bacopa to Cucurbitaceae (Li et al., 2010), Myristicaceae (Newmaster et al., monnieri (Darokar et al., 2001)andLycoris ligutuba (Deng et al., 2006). 2008) and Asteraceae (Gao et al., 2010) families. However, in some The data obtained in the current research work substantiate the plant species, trnH-psbA does not undergo amplification or sometimes occurrence of genetic loss. The results summarized in the present results as multiple bands (Sass et al., 2007). Moreover, within certain study also elaborate that the grouping of these 10 accessions is indepen- plant lineages, trnH-psbA is not variable enough to discriminate dent of the geographical distance. Thus, the intra-specificdiversity among closely related species (Spooner, 2009)asinT. triangulare, could be due to individual species adaptation and their response to whereas, in some plant species high intra-specific variation is detected the change in environmental conditions. Thus, further analysis by DNA (Edwards et al., 2008). barcoding and sequencing was carried out to investigate the genetic In this regard, the matK gene was a promising barcode. Due to the fidelity of the different accessions. high nucleotide substitution rate of the matK gene of the chloroplast genome, it has been widely employed as a powerful tool to identify 4.2.2. DNA barcoding the botanical origin of medicinal plants and to examine inter and In the present study, four distinct barcodes namely, rbcL, intra-specific phylogenetic relationships (Ohsako and Ohnishi, 2000). trnH-psbA, matK and ITS were used to discriminate the 10 accessions In the current work, matKgeneofT. triangulare was amplified to acquire of T. triangulare. Literature survey divulged the fact that sequence infor- 980 bp sized amplicon. The fragment was further sequenced (829 bp) and mation for the complete coding region of matK gene of T. triangulare BLAST analysis revealed 99% sequence similarity with T. fruticosum was available (Nyffeler, 2007). On the other hand, the genomic data (gb|DQ855844.1) (Syn. T. triangulare) with 100% query coverage. for the other genes ofT. triangulare was totally absent. Hence, barcoding There have been several studies using the matKgenesequencein studies were carried out for the different accessions of T. triangulare. phylogenetic reconstruction which include the families Asparagaceae Sequencing the amplicons and BLAST analysis showed no variation (Boonsom et al., 2012), Fabaceae (Newmaster and Ragupathy, 2009) among the different samples. Of the four different barcodes tested, and Rosaceae (Pang et al., 2011). matK gene showed 99% sequence identity which was straightforward The internal transcribed spacer from nuclear ribosomal DNA (ITS) and unambiguous, making inferences possible at a broader scale. was recommended as an alternate supplementary barcode in groups In this work, the rbcL amplified product of T. triangulare (580 bp) where direct sequencing was achievable (Thomas, 2009). In this showed upto 99% identity with T. paniculatum (gb|HM850388.1) with study, the ITS gene of T. triangulare (602 bp) revealed 98% sequence 100% query coverage. The plastid-encoded rbcL gene which codes for match with T. paraguayense (gb|L78056.1). Hershkovitz and Zimmer the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (2000) carried out the phylogenetic analysis of ribosomal DNA ITS (RuBisCO) has been employed to discriminate among species belonging sequences from 35 members of western American Portulacaceae. For 68 J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68 their study, they used ITS1 and ITS2 genes, whose total length varied Tropical Africa/Ressources ve´ge´tales de l'Afrique tropicale), Wageningen, – Netherlands (http://database.prota.org/search.htm Accessed 20 Oct 2010). from 399 to 441 nucleotides. The length of ITS1 (187 226 bp) Francisco-Ortega, J., Fuertes-Aguilar, J., Gomez-Campo, C., Santos-Guerra, A., Jansen, R.K., wasreportedtobemorevariablethanITS2(207–221 bp). The ITS1 1999. Internal transcribed spacer sequence phylogeny of Crambe L. (Brassicaceae): sequences for Talinum and Talinella spp. were found to be the molecular data reveal two Old World disjunctions. Molecular Phylogenetics and – Evolution 11 (3), 361–380. shortest (187 208 bp) among the other Portulacaceae members Gao, T., Yao, H., Song, J., Zhu, Y., Liu, C., Chen, S., 2010. Evaluating the feasibility of using (Hershkovitz and Zimmer, 2000). Conversely, the nuclear ribosomal candidate DNA barcodes in discriminating species of the large Asteraceae family. primers used in the present study to analyze T. triangulare were ITS1 BMC Evolutionary Biology 10, 324. fi and ITS4, which yielded a larger amplicon of 630 bp. The sequence ob- Hebert, P.D.N., Cywinska, A., Ball, S.L., DeWaard, J.R., 2003. Biological identi cation through DNA barcodes. Proc. R. Soc. Lond. Ser. B 270, 313–321. tained for this barcode exhibited 98% homology with T. paraguayense Hershkovitz, M.A., Zimmer, E.A., 2000. Ribosomal DNA evidence and disjunctions of (584 bp) and 93% match with T. portulacifolium (578 bp) as reported western American Portulacaceae. Molecular Phylogenetics and Evolution 15 (3), – by Hershkovitz and Zimmer (2000). Genetic identification and classifi- 419 439. Jones Jr., S.B., Luchsinger, A.E., 1987. Plant Systematics. 2nd edition. McGraw-Hill Book cation of plant species using ITS genes have been achieved in the follow- Company, New York. ing families; Brassicaceae (Francisco-Ortega et al., 1999), Lauraceae Kaplan, D.R., 2001. The science of plant morphology: definition, history, and role in (Lee et al., 2010) and Ulmaceae (Lee et al., 2011). modern biology. American Journal of Botany 88, 1711–1741. Karp, A., Seberg, O., Buiatti, M., 1996. Molecular techniques in the assessment of botanical Overall, in the present study, the distribution pattern of T. triangulare diversity. Annals of Botany 78, 143–149. populations using phenotypic (morphological, biochemical) and RAPD Kress, J., Erickson, D.L., 2007. A two-locus global DNA barcode for land plants: the coding markers elucidate substantial intra-specific variation. However, the rbcL gene complements the non-coding trnH-psbAspacerregion.PLoSOne6,1–10. Lee, S.C., Lee, C.H., Lin, M.Y., Ho, K.Y., 2010. Genetic identification of Cinnamomum species DNA barcoding results of three chloroplast genes (rbcL, trnH-psbA, based on partial internal transcribed spacer 2 of ribosomal DNA. Journal of Food and matK) and one nuclear gene (ITS) demonstrated that no variation Drug Analysis 18 (4), 225–231. was detected among the regionally collected plant samples proving Lee, S.C., Chang, C.F., Ho, K.Y., 2011. Genetic diversity and molecular discrimination of the closely related Taiwanese Ulmaceae species Celtis sinensis Persoon and Celtis that environmental changes and geographical divergence did not formosana Hayata based on ISSR and ITS markers. African Journal of Agricultural have any effect at the genetic level. Thus, molecular characterization Research 6 (20), 4760–4768. of T. triangulare played a promising role in species identification, Levin, R.A., Wagner, W.L., Hoch, P.C., Nepokroeff, M., Pires, J.C., Zimmer, E.A., Sytsma, K.J., molecular authentication and uncovering distinctiveness among 2003. Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data. American Journal of Botany 90, 107–115. closely related taxa. Additionally, elaborate investigations are necessary Li, H.T., Yang, J.B., Li, D.Z., Möller, M., Shah, A., 2010. A molecular phylogenetic study of to understand the patterns of gene flow within and between accessions Hemsleya (Cucurbitaceae) based on ITS, rpl16, trnH-psbA, and trnL DNA sequences. – for implementing effective conservation strategies. Plant Systematics and Evolution 285, 23 32. Liu, Z., Zeng, X., Yang, D., Chu, G., Yuan, Z., Chen, S., 2012. Applying DNA barcodes for identification of plant species in the family Araliaceae. Gene 499 (1), 76–80. Conflict of interest Mahesh, R., Kumar, N.N., Sujin, R.M., 2008. Molecular analysis in Rauvolfia tetraphylla L. using RAPD markers. Ethnobotanical Leaflets 12, 1129–1136. fl Newmaster, S.G., Ragupathy, S., 2009. Testing plant barcoding in a sister species complex The authors declare that they have no con ict of interest. of pantropical Acacia (Mimosoideae, Fabaceae). Molecular Ecology Resources 9 (1), 172–180. References Newmaster, S.G., Fazekas, A.J., Steeves, R.A.D., Janovec, J., 2008. Testing candidate plant barcode regions in the Myristicaceae. Molecular Ecology Resources 8 (3), – Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment 480 490. search tool. Journal of Molecular Biology 215, 403–410. Nilsson, T., 1970. Studies into the pigments in beetroot (Beta vulgaris L. ssp. vulgaris var. – Asahina, H., Shinozaki, J., Masuda, K., Morimitsu, Y., Satake, M., 2010. Identification of rubra L.). Lantbrukshögskolans Annaler 36, 179 219. medicinal Dendrobium species by phylogenetic analyses using matKandrbcL Nyffeler, R., 2007. The closest relatives of cacti: insights from phylogenetic sequences. Journal of Natural Medicines 64, 133–138. analyses of chloroplast and mitochondrial sequences with special emphasis Boonsom, T., Waranuch, N., Ingkaninan, K., Denduangboripant, J., Sukrong, S., 2012. on relationships in the tribe Anacampseroteae. American Journal of Botany – Molecular analysis of the genus Asparagus based on matK sequences and its 94 (1), 89 101. fi application to identify A. racemosus, a medicinally phytoestrogenic species. Ohsako, T., Ohnishi, O., 2000. Intra- and inter-speci c phylogeny of wild Fagopyrum Fitoterapia 83 (5), 947–953. (Polygonaceae) species based on nucleotide sequences of noncoding regions in – Buzgo, M., Soltis, D.E., Soltis, P.S., Ma, H., 2004. Towards a comprehensive integration of chloroplast DNA. American Journal of Botany 87, 573 582. fi morphological and genetic studies of floral development. Trends in Plant Science 9, Padmalatha, K., Prasad, M.N.V., 2007. Genetic diversity in Rauvol a tetraphylla L. using – 164–173. RAPD. Indian Journal of Biotechnology 40 (2), 18 22. CBOL Plant Working Group, 2009. A DNA barcode for land plants. Proceedings of the Pang, X., Song, J., Zhu, Y., Xu, H., Huang, L., Chen, S., 2011. Applying plant DNA barcodes for fi – National Academy of Sciences of the United States of America 106, 12794–12979. Rosaceae species identi cation. Cladistics 27 (2), 165 170. Darokar,M.P.,Khanuja,S.P.S.,Shasany,A.K.,Kumar,S.,2001.Low levels of genetic Sang, T., Crawford, D.J., Stuessy, T.F., 1997. Chloroplast DNA phylogeny, reticulate diversity detected by RAPD analysis in geographically distinct accessions of Bacopa evolution and biogeography of Paeonia (Paeoniaceae). American Journal of Botany – monnieri. Genetic Resources and Crop Evolution 48 (6), 555–558. 84, 1120 1136. Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA minipreparation. Version II. Plant Sass, C., Little, D.P., Stevenson, D.W., Specht, C.D., 2007. DNA barcoding in the Cycadales: fi Molecular Biology Reporter 1, 19–21. testing the potential of proposed barcoding markers for species identi cation of Deng, C.L., Zhou, J., Gao, W.J., Sun, F.C., Qin, R.Y., Lu, L.D., 2006. Assessment of genetic cycads. PLoS One 2, e1154. diversity of Lycoris longituba (Amaryllidaceae) detected by RAPDs. Journal of Genetics Steglich, W., Strack, D., 1990. Betalains. In: Brossi, A. (Ed.), The alkaloids - chemistry and – 85 (3), 205–207. pharmacology. Academic Press, London, pp. 1 62. Dey, S.S., Singh, A.K., Chandel, D., Behera, T.K., 2006. Genetic diversity of bitter gourd Spooner, D.M., 2009. DNA barcoding will frequently fail in complicated groups: an example – (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits. in wild potatoes. American Journal of Botany 96, 1177 1189. Scientia Horticulturae 109, 21–28. Swarna, J., Ravindhran, R., 2013. In vitro organogenesis from leaf and transverse thin cell Ding, G., Zhang, D., Yu, Y., Zhao, L., Zhang, B., 2012. Phylogenetic relationship among layer derived callus cultures of Talinum triangulare (Jacq.) Willd. Food Chemistry. 70, – related genera of Plumbaginaceae and preliminary genetic diversity of Limonium pp. 79 87. sinense in China. Gene 506 (2), 400–403. Swarna, J., Lokeswari, T.S., Smita, M., Ravindhran, R., 2013. Characterisation and determi- Doebley, J., Durbin, M.L., Goldenberg, E.M., Clegg, M.T., Ma, D.P., 1990. Evolutionary nation of in vitro antioxidant potential of Talinum triangulare (Jacq.) Willd. Food – analysis of the large subunit of carboxylase (rbcL) nucleotide sequence among the Chemistry. 141, pp. 4382 4390. grasses (Gramineae). Evolution 44, 1097–1108. Tate, J.A., Simpson, B.B., 2003. Paraphyly of Tarasa (Malvaceae) and diverse origins of the – Edwards, D., Horn, A., Taylor, D., Savolainen, V., Hawkins, J.A., 2008. DNA barcoding of a polyploid species. Systematic Botany 28, 723 737. large genus, Aspalathus L. (Fabaceae). Taxon 57, 1317–1327. Thomas, C., 2009. Plant barcode soon to become reality. Science 325, 526. fi Endress, P.K., 2000. Systematic plant morphology and anatomy – 50 years of progress. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Ampli cation and direct sequencing of fungal Taxon 49, 401– 434. ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., Fontem, D.A., Schippers, R.R., 2004. Talinum triangulare (Jacq.) Willd. [Internet] Record White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic – from Protabase. In: Grubben, G.J.H., Denton, O.A. (Eds.), PROTA (Plant Resources of Press, New York, USA, pp. 315 322.