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

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

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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 b

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 Department of Biotechnology, Sri Ramachandra University, #1, Ramachandra Nagar, Porur, Chennai 600116, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 8 October 2014 Received in revised form 15 December 2014 Accepted 16 December 2014 Available online xxxx Edited by E Balázs Keywords: Waterleaf RAPD fingerprinting DNA barcoding Dendrogram

a b s t r a c t Talinum triangulare is a medicinal herb known to have originated from tropical Africa and is now widely cultivated in the humid tropical countries. The characterization of plant germplasm using molecular descriptors aids in describing the genotypic traits of the plant T. triangulare. Ten different accessions of T. triangulare were collected from nine districts of Tamil Nadu. High quality genomic DNA was isolated from the different samples, checked for purity and quantified. The DNA samples were subjected to PCR amplification using RAPD markers and by DNA barcoding technique. RAPD fingerprints 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 a total of 78 fragments, of which, 57 bands were determined as polymorphic loci. Phylogenetic relationship and intra-specific variation among the samples were established by constructing a dendrogram based on Jaccard's coefficient. DNA barcoding studies included the amplification of chloroplast large subunit of ribulosebisphosphate carboxylase (rbcL), trnH-psbA intergenic spacer (trnH-psbA), maturase K (matK) and nuclear 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 In the past century, rapid globalization leading to adverse environmental conditions has resulted in massive loss of valuable plant species. This has triggered the conservation of plant genetic resources. Accurate identification and characterization of plant materials is essential for their sustainable utilization. The dramatic advances in molecular genetics over the last few years have provided researchers with an extensive range of new techniques for answering many evolutionary and taxonomic questions. Moreover, a majority of these techniques have been effectively employed to study the extent and distribution of variation in species gene-pools (Karp et al., 1996). The advent of DNA-based markers has revolutionized the practice of DNA fingerprinting and diversity analysis of plant species. Recently, DNA barcoding, a new biological tool based on the analysis of short, standardized and universal DNA regions (barcodes), has been proposed as a universal tool for species discrimination and identification (Hebert et al., 2003). DNA sequences generated from such barcodes

Abbreviations: DMRT, Duncan's multiple range test; ITS, Internal transcribed spacer; RAPD, Random amplified polymorphic DNA; SPSS, Statistical package for social sciences; UPGMA, Unweighted pair group method analysis ⁎ Corresponding author. Tel.: +91 44 28178200x330. E-mail address: [email protected] (J. Swarna).

http://dx.doi.org/10.1016/j.sajb.2014.12.012 0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

can be compared and evaluated against reference sequences already existing in online databases, thus, providing rapid and reproducible taxonomic recognition (Hebert et al., 2003). This method opens new perspectives for the identification of medicinal herbs which is useful to clarify taxonomic uncertainties within the family Portulacaceae. The Purslane family (Portulacaceae), a group of edible plants with cosmopolitan distribution, is represented by about 20 genera and 500 species (Jones and Luchsinger, 1987). The genus Talinum comprises of 15 species, of which only five species have been reported in India. Talinum triangulare (Jacq.) Willd., popularly known as waterleaf, is a fleshy-leaved perennial herb grown widely in the humid tropical countries as a leaf vegetable (Swarna and Ravindhran, 2013). The plant known to have originated in Central Africa, is now traditionally valued for its remarkable antioxidant activities and has been implicated in the management of diabetes, jaundice, cancer, stroke, obesity and measles (Fontem and Schippers, 2004). Waterleaf is characterized as tolerant to various soil types, temperatures and moisture levels, and grows well under shade. The 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 vegetative tissues (Steglich and Strack, 1990). By nature, T. triangulare produces betalain in flowers and under extreme environmental conditions the pigment gets accumulated in leaves and stems (Swarna et al., 2013).

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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 2 3 4 5 6 7 8 9 10

Ariyalur Chennai Coimbatore Erode Madurai Thanjavur Tiruchirappalli Tirunelveli Tiruvannamalai Chennai (Wild)

Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu Tamil Nadu

N 11°8′14″ N 13°5′2″ N 11°1′6″ N 11°21′0″ N 9°55′10.78″ N 10°46′30″ N 10°48′18″ N 8°43′48″ N 12°13′12″ N 13°5′2″

E 79°4′40″ E 80°16′12″ E 76°58′21″ E 77°44′0″ E 78°7′9.82″ E 79°8′20″ E 78°41′8″ E 77°42′0″ E 79°4′12″ E 80°16′12″

76 m 6m 411.2 m 183 m 101 m 88 m 88 m 47 m 171 m 6m

Of the five different Talinum species reported in India, T. triangulare is closely related to Talinum paniculatum and Talinum portulacifolium. The former differs from the other species by certain morphological traits including paniculate inflorescence borne on a triangular peduncle and flower sepals being prominently veined. These minute differences often lead to confusion misjudgement of the T. triangulare species, which could lead to adulteration in herbal preparations. Hence, in the present study, molecular characterization of T. triangulare was carried out for its authentication at the DNA level. The current work was aimed at identifying morphological, biochemical and genetic variability among 10 accessions of T. triangulare collected from different districts within Tamil Nadu. An integrative approach was pursued by RAPD fingerprinting and DNA barcoding studies to provide better genetic clarity of the plant. Moreover, intra-specific phylogenetic relationship was assessed by plotting a dendrogram. The performance of four candidate barcoding loci (rbcL, trnH-psbA, matK and ITS) were examined by PCR amplification and DNA sequencing for the recognition and validation of T. triangulare to establish unambiguous identification of this valuable medicinal plant. 2. Materials and methods 2.1. Collection of plant material T. triangulare plants were collected during the period of January to April from cultivated farmer's fields or natural localities of nine different districts within the Tamil Nadu state. The districts from where the samples were obtained were Ariyalur, Chennai, Coimbatore, Erode, Madurai, Thanjavur, Tiruchirappalli, Tirunelveli and Tiruvannamalai, which included regions with varying soil and climatic conditions. T. triangulare plants from the wild were also acquired for comparison purposes. A minimum of at least 30 plants were gathered from each site for the analyses which represented the total population of that area. The plant material was identified and authenticated by Dr. D. Narasimhan, Centre for Floristic Research, Madras Christian College, Chennai. A herbarium voucher specimen (LCH 42) was preserved for future reference and has been deposited in the Loyola College Herbarium. The place of collection along with the latitude and longitude data and its altitude above sea level are tabulated in Table 1. The leaves of the different samples were freshly used for DNA extraction while epidermal peel of stem was used for betalain analysis. 2.2. Morphological and biochemical descriptors To analyze the morphological variation among the 10 accessions, a twig from each sample was collected and examined. Visual examination was performed to detect any difference among the samples. For biochemical characterization, betalains of the 10 samples were analyzed. For this, the betalain extraction from the epidermal peel (1.0 g) was performed by homogenizing the samples in a mortar with liquid nitrogen, to preclude enzymatic degradation during grinding. The resulting powder was extracted with 10 ml of 80% (v/v) aqueous methanol and continuously

stirred for 30 min. The samples were centrifuged at 10,000 rpm for 10 min at 4 °C in a 5810r Eppendorf refrigerated centrifuge (Hamburg, Germany). The supernatant was transferred and the pellet was reextracted twice with 80% methanol. All the supernatants were pooled and stored at −20 °C in dark until use. The betacyanin, betaxanthin and total betalain content were calculated based on the spectrophotometric multiple-component method of Nilsson (1970). Determination of betalain concentration, i.e., violet and yellow pigments, was expressed in terms of betanin and vulgaxanthin I, respectively. Total pigment content was calculated as the sum of betacyanin and betaxanthin components (mg/g FW of the plant sample). The experiments were performed with a minimum of ten plants per treatment and the data were analyzed statistically using IBM SPSS statistics 19 (SPSS Inc., Chicago, USA). The mean values were expressed as mean ± SE of three repeated experiments and the significance of differences among means was carried out at 5% probability level using Duncan's Multiple Range Test (DMRT). 2.3. DNA isolation from T. triangulare The total genomic DNA was isolated from different accessions of T. triangulare by using a modified SDS method (Dellaporta et al., 1983). The collected DNA pellet was suspended in appropriate quantity of nuclease-free Milli-Q water and stored at −20 °C until use. Quality and the concentration of the DNA samples were assessed by agarose gel electrophoresis and by using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA) respectively. The purity of DNA was assessed by the sample absorbance ratio of A260/A280 and the DNA samples were diluted according to use. 2.4. Random amplified polymorphic DNA (RAPD) fingerprinting To investigate the genetic variation among the 10 different T. triangulare samples, RAPD analysis was performed. The PCR-based RAPD technique was executed by employing 10 random decamer primers, namely, OPA-13, OPA-16, OPA-17, OPA-18, OPA-19, OPB-08, OPB-11, OPB-13, OPB-17 and OPB-18 (Operon Technologies Inc., CA, USA) (Table 2). The PCR reaction was carried out in a volume of 20 μl reaction mixture consisting of the following components: Taq Buffer A with 15 mM MgCl2 (1×), dNTPs (0.2 mM), RAPD primer (Operon Technologies, USA) (0.5 pM), Taq DNA Polymerase (1 U) and genomic DNA (50 ng). For every primer used, a negative control containing nuclease-free Milli-Q water was included to ensure there were no false-positives due to crosscontamination. The RAPD-PCR amplification was carried out in a DNA thermal cycler (Mastercycler gradient, Eppendorf, Germany) with the following conditions: Initial denaturation Cycling reaction o Denaturation o Annealing o Extension Final extension

94 °C for 5 min 94 °C for 1 min 36 °C for 1 min 72 °C for 2 min 72 °C for 7 min

9 = ;

35 cycles

J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

After PCR amplification, the RAPD-PCR products were resolved in 1.8% (w/v) agarose gel with 1 × Tris–acetic acid–EDTA (TAE) buffer. GeneRuler 1 kb Plus DNA ladder (Thermo Scientific, Mumbai, India) (75 bp to 20 kb) was used as DNA marker. The amplified fragments were visualized under UV light and documented using the Gel Documentation equipment (UVP, Ultra-Violet Products Ltd., UK). PCR reactions were repeated at least thrice to confirm the reproducibility of the results. 2.4.1. RAPD data analysis After excluding markers that were monomorphic for the entire data set, a vector of molecular marker phenotype was established for each individual accession of the plant that was analyzed. Variability was expressed as percentage of polymorphism which was computed as the number of polymorphic bands over the total number of scored bands. In addition, fragment data were treated as two state qualitative data and the RAPD prints were converted into binary matrices. For genetic similarity estimates, the fragment data were coded as one for the presence of band and zero indicating absence of band. Pairwise genetic comparison of the sample data was performed by entering the binary scores into Numerical Taxonomy and Multivariate Analysis 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 coefficient in NTSYS-pc (Applied Biostatistics). Finally, diversity group clusters were analyzed using Sequential Agglomerative Hierarchical Nested cluster analysis (SAHN) and Unweighted Pair Group Method Analysis (UPGMA) using average linkages clustering routines within the NTSYS-pc software package to construct the dendrogram. 2.5. Molecular characterization by DNA barcoding For the DNA barcoding studies of the different accessions of T. triangulare, amplification of the most frequently used barcodes of the chloroplast and nuclear genomes was performed. Two coding (rbcL and matK) and one non-coding region (trnH-psbA) of the chloroplast genome was used for the molecular identification of this plant. A portion of the internal transcribed spacer (ITS) region of the 18S–28S nuclear DNA was amplified in a PCR reaction using ITS1 and ITS4 primers. The sequences of the different primers and their references as recommended by CBOL (2009) are provided in Table 3. A 20 μl reaction mixture was prepared for each barcode as described in Table 4. The PCR amplification was carried out in a DNA thermal cycler as per the conditions detailed in Table 5. 2.5.1. Agarose gel electrophoresis of the amplified product After PCR amplification of the chloroplast and nuclear gene, the products were checked in agarose gel with 1 × Tris–acetic acid–EDTA (TAE) buffer. For rbcL, matK and ITS amplicons, 1.2% (w/v) agarose gel was used, while 2.0% gel was used to resolve trnH-psbA product.

61

Table 3 Sequence information of the forward and reverse primers of the different barcodes used for the DNA barcoding studies. Name

Primer code

Sequence (5′–3′)

Reference

rbcL primers Forward

rbcLa_f

Levin et al. (2003)

Reverse

rbcLa_r

ATGTCACCACAAAC AGAGACTAAAGC GTAAAATCAAGTCC ACCRCG GTTATGCATGAACG TAATGCTC CGCGCATGGTGGAT TCACAATCC

Sang et al. (1997)

ACCCAGTCCATCTG GAAATCTTGGTTC CGTACAGTACTTTT GTGTTTACGAG

Ki-Joong Kim (unpublished) Ki-Joong Kim (unpublished)

TCCGTAGGTGAACC TGCGG TCCTCCGCTTATTG ATATGC

White et al. (1990)

trnH-psbA primers Forward psbA3_f Reverse

trnHf_05

matK primers Forward MatK-1R_KIM_f Reverse

MatK-3F_KIM_r

ITS primers Forward

ITS1

Reverse

ITS4

Kress and Erickson (2007)

Tate and Simpson (2003)

White et al. (1990)

For estimating the size of the amplicons, 1 kb DNA ladder (New England Biolabs Inc.,UK) (500–10,000 bp) was used as the DNA marker. The amplified fragments were visualized under UV light and documented using the Gel Documentation equipment (UVP, Ultra-Violet Products Ltd., UK). PCR reactions were repeated at least thrice to confirm the reproducibility of the results. 2.5.2. DNA sequencing of the amplified product The double stranded PCR products were purified using a PCR purification kit and directly sequenced from both the ends using dye terminator technique. Forward and reverse cycle sequencing based on the Sanger's sequencing method was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Perkin-Elmer, Applied Biosystems) on ABI Prism 3730XL sequencer (Perkin-Elmer, Applied Biosystems, USA). The sequencing reaction mixture (10 μl) contained 0.5 μl BigDye v3.1 ready reaction mixture, 3 μl PCR product (10–50 ng), 2 μl sequencing buffer (5×), 1 μl primer (10 μM) and 3.5 μl autoclaved Milli-Q water. The cycling regime was 30 cycles of 94 °C for 30 s, 50 °C for 15 s and 60 °C for 4 min. Each sample was sequenced in the sense and antisense direction and analyzed with ABI sequence navigator software (Perkin-Elmer/Applied Biosystems). Nucleotide sequences of both DNA strands were obtained and compared to ensure accuracy. Sequence comparison to set up the level of identification (species, genus, or family) through BLASTn algorithms was performed. The nucleotide sequences obtained were used as queries in the BLASTn search for molecularbased species identification matches with the available GenBank sequences (Altschul et al., 1990). 3. Results

Table 2 Details of the RAPD primers used to evaluate genetic diversity among 10 accessions of T. triangulare. Primer name

Sequence

Tm (°C)

GC-content

OPA-13 OPA-16 OPA-17 OPA-18 OPA-19 OPB-08 OPB-11 OPB-13 OPB-17 OPB-18

5′-CAGCACCCAC-3′ 5′-AGCCAGCGAA-3′ 5′-GACCGCTTGT-3′ 5′-AGGTGACCGT-3′ 5′-CAAACGTCGG-3′ 5′-GTCCACACGG-3′ 5′-GTAGACCCGT-3′ 5′-TTCCCCCGCT-3′ 5′-AGGGAACGAG-3′ 5′-CCACAGCAGT-3′

34.0 32.0 32.0 32.0 32.0 34.0 32.0 34.0 32.0 32.0

70% 60% 60% 60% 60% 70% 60% 70% 60% 60%

3.1. Morphological variation T. triangulare plants that were collected from 10 different districts within Tamil Nadu were examined for morphological variation. A twig collected from each locality was photographed and documented (Fig. 1). Morphologically, most of the plants showed distinct variation with respect to the leaf color, size, betalain production in stems, etc. The plants obtained from Ariyalur (Fig. 1a), Chennai (Fig. 1b) and Coimbatore (Fig. 1c) appeared healthy with long leaves which were green in color. The flowers were normal sized with regular seed formation. The Erode accession (Fig. 1d) had smaller leaves which were succulent in nature. The presence of betalains was noticed in the thin stems of the Erode sample. Light green to yellow colored leaves with

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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. No.

Component

rbcL, trnH-psbA and ITS barcodes

1. 2. 3. 4. 5. 6. 7.

Taq Buffer A with 15 mM MgCl2 (GeNei, Merck, India) dNTPs (GeNei, Merck, India) Forward primer Rreverse primer Taq DNA Polymerase (GeNei, Merck, India) Genomic DNA Milli-Q water Total

Volume required per 20 μl reaction

Working concentration

Volume required per 20 μl reaction

1× 0.4 mM 50 nM 50 nM 1 unit 50 ng

2 μl 4 μl 0.2 μl 0.2 μl 0.3 μl 2 μl 11.3 μl 20 μl

1× 0.2 mM 50 nM 50 nM 1 unit 50 ng

2 μl 2 μl 0.2 μl 0.2 μl 0.6 μl 2 μl 13 μl 20 μl

betalain pigmentation at the ends were observed in the Madurai sample (Fig. 1e). The leaves were also not strongly attached at the nodes and wilted on touching. This sample also showed characteristic rosette habit with shorter internode length having more number of leathery leaves (Fig. 1e). The flower stalk surpassed the shoot apex which produced miniature flowers that were very small in size (Fig. 1e). The Thanjavur (Fig. 1f) and Tiruchirappalli (Fig. 1g) samples possessed maximum leaf length among all the samples studied and the plants were dark green in color. The stem diameter was almost 1.5–2.5 cm and it was succulent in nature. Healthy flowers with dark pink pigmentation were observed in the Thanjavur and Tiruchirappalli accessions. The plant collected from Tirunelveli comprised of smaller leaves near the ground level and the size of the 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 the Tiruvannamalai (Fig. 1i) and the wild variety (Fig. 1j). The inflorescence at the apex consisted of diminutive flowers and numerous fruits. In both the samples the stems were pink colored and showed more branching. The size of the leaves was small and light green in color (Fig. 1j). 3.2. Biochemical variation The betalain content was assessed in each sample and the data have been represented graphically in Fig. 2. Maximum betacyanins (4.365 ± 0.053 mg/g FW), betaxanthins (0.154 ± 0.020 mg/g FW)

Table 5 PCR amplification conditions of the barcodes of T. triangulare as carried out in a DNA thermal cycler. PCR conditions for rbcL and trnH-psbA barcodes Initial denaturation 95 °C for 4 min Cycling reaction o Denaturation 94 °C for 30 s o Annealing 59.7 °C for 1 min o Extension 72 °C for 1 min Final extension 72 °C for 10 min

9 =

PCR conditions for matK barcode Initial denaturation 94 °C for 1 min Cycling reaction o Denaturation 94 °C for 30 s o Annealing 55 °C for 20 s o Extension 72 °C for 30 s Final extension 72 °C for 5 min

9 =

PCR conditions for ITS barcode Initial denaturation Cycling reaction o Denaturation o Annealing o Extension Final extension

9 =

;

;

35 cycles

35 cycles

94 °C for 3 min 94 °C for 30 sec 55 °C for 30 sec 72 °C for 2 min 72 °C for 10 min

;

matK barcode

Working concentration

35 cycles

and total betalains (4.520 ± 0.033 mg/g FW) were quantified in the wild accession. This was closely followed by the Chennai sample, which revealed the second highest total betalain content (3.276 ± 0.065 mg/g FW) with 3.258 ± 0.071 mg/g FW betacyanins and 0.017 ± 0.006 mg/g FW betaxanthins. The betacyanins and betaxanthins in the Tiruchirappalli accession was determined to be 2.614 ± 0.091 mg/g FW and 0.110 ± 0.004 mg/g FW respectively, yielding to a total betalain content of 2.724 ± 0.095 mg/g FW. The highest betaxanthin content was detected in the Coimbatore sample (0.246 ± 0.036 mg/g FW), whereas, the wild variety possessed maximum betacyanins (4.365 ± 0.053 mg/g FW). Very low betalain content was calculated in Madurai (0.950 ± 0.036 mg/g FW) and Coimbatore (1.155 ± 0.014 mg/g FW) samples (Fig. 2).

3.3. Molecular characterization 3.3.1. RAPD fingerprinting A total of 78 amplicons were detected by examining 10 T. triangulare accessions with 10 random RAPD primers (Table 6). Fig. 3a–j illustrates 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, 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 between 551 bp and 6549 bp. Out of the 78 amplified bands, 57 bands were determined as polymorphic loci while the remaining 21 were 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 Tiruvannamalai sample resulting in 100% polymorphism. From this data, an average of 5.7 polymorphic fragments per primer was calculated (Table 6). The percentage of polymorphism ranged from 44.44% for primer OPB-08 to a maximum of 100.0% for the primer OPA-16. When all primers were taken collectively, a mean polymorphism percentage of 73.08% was established. The similarity indices among the individual samples were represented in terms of Jaccard's similarity coefficient (Table 7). The similarity coefficient for the 10 accessions ranged between 0.436 (Wild vs. Coimbatore) and 0.929 (Tirunelveli vs. Tiruchirappalli). These findings divulged the fact that the Tiruchirappalli and Tirunelveli samples were closely related. UPGMA-based cluster analysis grouped the 10 accessions into three main cluster groups. Based on the dendrogram (Fig. 4), three groups (I, II and III) were identified with the help of the pruning line at genetic distance of 0.60. The cluster I comprised of six samples including Tiruchirappalli, Tirunelveli, Chennai, Thanjavur, Erode and Madurai. Three genotypes, Ariyalur, Tiruvannamalai and Coimbatore were classified under the second cluster (group II). The wild variety was positioned the farthest from the other nine accessions with a genetic distance of 0.54 and was categorized as group III. Group I and group II were further partitioned into subgroups which were connected at different genetic distances.

J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

63

a

b

c

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 Three chloroplast barcodes and one nuclear gene were used for the DNA barcoding studies among 10 T. triangulare accessions. Results of our tests within the 10 samples with the four barcodes showed prominent PCR amplification with 100% success rate. The rbcL gene which codes for the large subunit of ribulose1,5-bisphosphate carboxylase/ oxygenase (RuBisCo) enzyme is commonly used for molecular discrimination of plant species. This coding region was amplified for all the samples and amplicons of size 580 bp was obtained in all the lanes (Fig. 5a). In the chloroplast genome (cpDNA), trnH-psbA is an intergenic spacer. Amplicon size of 310 bp was recorded for the 10 samples when this non-coding region was amplified by PCR (Fig. 5b). The nucleotide sequence of the plastid encoded gene matK which codes of maturase enzyme was also investigated. PCR amplification for the 10 samples resulted in 980 bp size amplicons (Fig. 5c). Nuclear ribosomal internal transcribed spacer

(ITS) region was amplified in all the 10 samples. The agarose gel separation revealed an amplicon size of 630 bp for all the samples (Fig. 5d). These amplicons obtained for each barcode were further sequenced with both forward and reverse primers and the nucleotide sequence obtained was aligned at both the ends. DNA sequence length of 531 bp for rbcL, 255 bp for trnH-psbA, 858 bp for matK and 649 bp for ITS barcodes were acquired for the T. triangulare accessions respectively. The sequence information was uploaded into the GenBank database and the ids for the samples were KJ380905.1, KJ380906.1, KJ380907.1 and KJ380908.1. 3.3.2.1. BLAST analysis of DNA sequence data. The DNA sequences obtained were checked for homology by using BLAST tool in the NCBI webpage. Out of the top 100 BLAST results for rbcL gene of T. triangulare, only one hit belonged to the same genus, namely, T. paniculatum. Portulaca oleracea, Portulaca pilosa and Portulacaria

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J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

5 i

h

4 h g

3

f

g

f

f e e c

2

e

d

d

c b

b

a

1 d

a

d

c

b

a

0

Betaxanthin (mg/gm FW)

a

c

b

Betacyanin (mg/gm FW)

a

c

c

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 sequences obtained, only partial conserved regions of the rbcL gene have been reported. Other species belonging to various genera and families were also attained in the homology pairing. The amplicon size of rbcL gene had earlier been reported as 654 bp when the primers rbcLa_f and rbcLa_r were used (Table 3). The rbcL gene showed 99% sequence identity with Portulacaria sp. (dbj|AB586510.1) and T. paniculatum (gb|HM850388.1; gb|GQ436529.1) with 100% query coverage. The trnH-psbA gene of T. triangulare was checked for homology with different species.Out of the first 100 hits, just one sequence match with T. paniculatum was detected. Most of the sequence alignments were observed with Pereskia spp. (Cactaceae), followed by Opuntia spp. (Cactaceae). The amplicon size of trnH-psbA ranged from 318 bp to 820 bp when the psbA3_f and trnHf_05 primers were employed (Table 3) due to small scattered insertions or deletions without an apparent taxonomic pattern. A partial sequence of the trnH-psbA intergenic spacer was acquired with the forward and reverse primers. An identity match of 97% with Pereskia zinniflora (gb|AY851571.1) (Cactaceae) was revealed in the BLAST results. Sequence similarity of 98% with T. paniculatum (gb|AY851584.1) was detected with 77% query coverage, but with low scores. The BLAST results for matK gene sequence of T. triangulare revealed the closely related species. Among the various hits acquired, six sequences producing significant alignments belonged to the Talinum genus. Talinella sp., a closely related genotype, also showed strong

relationship with T. triangulare. Frequently, the size of the amplified fragments of matK varied from 846 to 852 nucleotides depending upon the plant species (Table 3). In the present study, the maturase K (matK) gene of T. triangulare of size 858 bp, revealed 99% sequence similarity with Talinum fruticosum (gb|DQ855844.1) (Syn. T.triangulare). Identity match of 98% with T. portulacifolium (gb|DQ855847.1) and 97% with T. paniculatum (gb|AY015274.1), T spathulatum (gb| HQ620890.1), T polygaloides (gb|DQ855845.1) and T lineare (gb| EU834752.1) were found. Query coverage of 100% was detected in all the species. A close relation to Talinella sp. matK gene having 98% identity was also observed (gb|DQ855846.1). The nuclear ITS region of T. triangulare was amplified to produce a 680 bp amplicon. The partial ITS sequence was checked for homology match and three Talinum spp. were distinguished among the Talinella spp. (Portulacaceae) and Opuntia spp. (Cactaceae). The sequence of internal transcribed spacer (ITS) region obtained (649 bp) revealed 98% similarity with T paraguayense (gb|L78056.1) showing query coverage of 89%. T. paniculatum (gb|EU410357.1) was 91% identical to our ITS sequence having 96% sequence cover. The ITS region also showed 93% match with T. portulacifolium (gb|L78057.1). Homology of the ITS region of Talinella microphylla (gb|L78053.1) and Talinella pachypoda (gb| L78054.1), another genus of the sample family, was detected at95% and 94% sequence similarity respectively. 4. Discussion 4.1. Morphological and biochemical descriptors

Table 6 RAPD fingerprinting data obtained from 10 random decamer primers for the different accessions of T. triangulare. Primer

Size of Total Number of Number of Percentage of fragments number monomorphic polymorphic polymorphism (bp) of bands bands bands

OPA-13 OPA-16 OPA-17 OPA-18 OPA-19 OPB-08 OPB-11 OPB-13 OPB-17 OPB-18 Total/average

826–4875 1227 763–2108 651–6549 584–1936 551–3003 816–4035 594–5012 627–3910 882–1837 551–6549

14 1 5 14 8 9 10 5 8 4 78

3 0 1 1 2 5 3 2 2 2 21

11 1 4 13 6 4 7 3 6 2 57

78.57% 100.0% 80.00% 92.86% 75.00% 44.44% 70.00% 60.00% 75.00% 50.00% 73.08%

The 10 different accessions of T. triangulare were analyzed for morphological and biochemical variation. Based on the external appearance of the plant, samples collected from Ariyalur, Chennai, Coimbatore, Thanjavur and Tiruchirappalli were similar. All the samples were healthy with long, dark green leaves. The Erode and Tirunelveli samples were comparable as they had smaller leaves and thinner stems. Tiruvannamalai and the wild sample collected from Chennai were alike since their leaves were greenish yellow in color and they had extended peduncles. The most diverse among the 10 samples was the Madurai accession. The morphological characteristics of this sample were completely different by having short internodes and light green leathery leaves arranged in a rosette. These distinctive features observed in the plants could be due to varying environmental and soil conditions at the place of plant collection.

b

c

d

e

f

g

h

i

j

J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68

a

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

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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 Chennai Coimbatore Erode Madurai Thanjavur Tiruchirappalli Tirunelveli Tiruvannamalai Wild

1.0000000 0.6617647 0.6181818 0.5942029 0.6406250 0.6478873 0.5945946 0.6250000 0.6603774 0.5714286

1.0000000 0.6000000 0.7605634 0.7142857 0.8082192 0.8732394 0.9130435 0.6093750 0.5147059

1.0000000 0.5303030 0.5737705 0.5428571 0.5571429 0.5652174 0.6122449 0.4363636

1.0000000 0.7205882 0.8169014 0.7567568 0.7671233 0.5384615 0.6129032

1.0000000 0.8235294 0.7857143 0.7714286 0.6964286 0.5573770

1.0000000 0.8266667 0.8630137 0.5735294 0.5285714

1.0000000 0.9295775 0.5652174 0.5000000

1.0000000 0.5735294 0.5070423

1.0000000 0.5918367

1.0000000

The biochemical characters of the different plants were studied based on the quantity of betalain content. Betalains, which are characteristic pigments of the plants belonging to the order Caryophyllales, were calculated in terms of betacyanins and betaxanthins. Maximum betalain content was determined in the wild accession followed by the Chennai sample, while, Madurai accession recorded the lowest quantity of betalains. These divergent traits could be attributable to various reasons such as soil variation, excessive sunlight, low water content and nutrient depletion. Distinguishing characters among the different accessions can be investigated using morphological and biochemical descriptors. For a long time, such traits have been the basis for characterization of diversity (Endress, 2000). Morphological information play a pivotal role in understanding life cycles, geographical distributions, identification, conservation status, evolution, development, and species delimitation (Buzgo et al., 2004; Kaplan, 2001). Nonetheless, there remains considerable debate about the precise role of morphology as they are inadequate for identifying the species, mainly due to fluctuation in environmental conditions. Furthermore, these descriptors are often restricted as the characters may not be obvious at all stages of the plant development and appearances may vary. Thus, additional classification and characterization at the molecular level are essential to identify and authenticate this particular plant at the species level.

4.2. Molecular characterization Molecular tools provide valuable data on genetic diversity based on their ability to detect variation at the DNA level. Identification of any plant species is of fundamental importance in diversity studies. Thus, for the evaluation of species diversity, it is essential that individuals be classified accurately. To establish effective management of plant genetic diversity for sustenance and maintenance of vital plants, it is imperative to regard variation as richness and distribution at both inter and intraspecific levels. The genetic constitution of taxonomic units and endangered species is distinct, so as a result their identification from their relatives is important in the development of appropriate conservation strategies. Depending on the state of our heritable understanding of taxon, genetic diversity may be considered at different organizational levels such as the genopool, population, individual genome, locus and DNA based sequence (Padmalatha and Prasad, 2007). 4.2.1. RAPD fingerprinting The isolation of plant DNA is the first step for any type of molecular analysis. In the present study, crisp DNA bands without any shear were observed in agarose gel for all the 10 samples. A search for Talinum gene sequences showed lack of information for this species. Since RAPD is commonly used for unknown genomes, we estimated the polymorphisms exhibited for a set of 10 randomly chosen decamer primers.

Ariyalur Tiruvannamalai Group II

Clusters: 3; Genec distance: 0.60

Coimbatore Chennai Tiruchirappalli Tirunelveli Group I

Thanjavur Erode Madurai Wild variety

0.53

0.63

0.73

0.83

Group III

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-psbA intergenic spacer, 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 T. triangulare were generated. The gel analysis of the samples revealed a total of 78 bands, of which, 57 were determined as polymorphic loci. The amplification patterns demonstrated that the percentage of polymorphism was 73.08%. UPGMA-based cluster analysis calculated in terms of Jaccard's similarity coefficient grouped the 10 accessions into three main cluster groups (I, II, III). Similarly RAPD markers have been used extensively to study the diversity among various crop plants and medicinal plants. Some of the reports for establishing genetic variation in medicinal plants using RAPD markers include Rauvolfia tetraphylla (Mahesh et al., 2008), Momordica charantia (Dey et al., 2006), Bacopa monnieri (Darokar et al., 2001) and Lycoris ligutuba (Deng et al., 2006). The data obtained in the current research work substantiate the occurrence of genetic loss. The results summarized in the present study also elaborate that the grouping of these 10 accessions is independent of the geographical distance. Thus, the intra-specific diversity could be due to individual species adaptation and their response to the change in environmental conditions. Thus, further analysis by DNA barcoding and sequencing was carried out to investigate the genetic fidelity of the different accessions. 4.2.2. DNA barcoding In the present study, four distinct barcodes namely, rbcL, trnH-psbA, matK and ITS were used to discriminate the 10 accessions of T. triangulare. Literature survey divulged the fact that sequence information for the complete coding region of matK gene of T. triangulare was available (Nyffeler, 2007). On the other hand, the genomic data for the other genes ofT. triangulare was totally absent. Hence, barcoding studies were carried out for the different accessions of T. triangulare. Sequencing the amplicons and BLAST analysis showed no variation among the different samples. Of the four different barcodes tested, matK gene showed 99% sequence identity which was straightforward and unambiguous, making inferences possible at a broader scale. In this work, the rbcL amplified product of T. triangulare (580 bp) showed upto 99% identity with T. paniculatum (gb|HM850388.1) with 100% query coverage. The plastid-encoded rbcL gene which codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been employed to discriminate among species belonging

to the families Araliaceae (Liu et al., 2012), Plumbaginaceae (Ding et al., 2012) and Orchidaceae (Asahina et al., 2010). Conversely, the poor ability of rbcL to resolve phylogenetic relationships at the genus and species level has been reported earlier (Doebley et al., 1990). Therefore, other useful DNA regions that evolve faster than rbcL to facilitate lower-level phylogenetic construction are explored. The trnH-psbA amplicon of T. triangulare (255 bp) exhibited upto 97% sequence match with Pereskia zinniflora (gb|AY851571.1) belonging to the family Cactaceae. Molecular phylogenetic studies based on trnH-psbA DNA sequences have been reported for members belonging to Cucurbitaceae (Li et al., 2010), Myristicaceae (Newmaster et al., 2008) and Asteraceae (Gao et al., 2010) families. However, in some plant species, trnH-psbA does not undergo amplification or sometimes results as multiple bands (Sass et al., 2007). Moreover, within certain plant lineages, trnH-psbA is not variable enough to discriminate among closely related species (Spooner, 2009) as in T. triangulare, whereas, in some plant species high intra-specific variation is detected (Edwards et al., 2008). In this regard, the matK gene was a promising barcode. Due to the high nucleotide substitution rate of the matK gene of the chloroplast genome, it has been widely employed as a powerful tool to identify the botanical origin of medicinal plants and to examine inter and intra-specific phylogenetic relationships (Ohsako and Ohnishi, 2000). In the current work, matK gene of T. triangulare was amplified to acquire 980 bp sized amplicon. The fragment was further sequenced (829 bp) and BLAST analysis revealed 99% sequence similarity with T. fruticosum (gb|DQ855844.1) (Syn. T. triangulare) with 100% query coverage. There have been several studies using the matK gene sequence in phylogenetic reconstruction which include the families Asparagaceae (Boonsom et al., 2012), Fabaceae (Newmaster and Ragupathy, 2009) and Rosaceae (Pang et al., 2011). The internal transcribed spacer from nuclear ribosomal DNA (ITS) was recommended as an alternate supplementary barcode in groups where direct sequencing was achievable (Thomas, 2009). In this study, the ITS gene of T. triangulare (602 bp) revealed 98% sequence match with T. paraguayense (gb|L78056.1). Hershkovitz and Zimmer (2000) carried out the phylogenetic analysis of ribosomal DNA ITS sequences from 35 members of western American Portulacaceae. For

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their study, they used ITS1 and ITS2 genes, whose total length varied from 399 to 441 nucleotides. The length of ITS1 (187–226 bp) was reported to be more variable than ITS2 (207–221 bp). The ITS1 sequences for Talinum and Talinella spp. were found to be the shortest (187–208 bp) among the other Portulacaceae members (Hershkovitz and Zimmer, 2000). Conversely, the nuclear ribosomal primers used in the present study to analyze T. triangulare were ITS1 and ITS4, which yielded a larger amplicon of 630 bp. The sequence obtained for this barcode exhibited 98% homology with T. paraguayense (584 bp) and 93% match with T. portulacifolium (578 bp) as reported by Hershkovitz and Zimmer (2000). Genetic identification and classification of plant species using ITS genes have been achieved in the following families; Brassicaceae (Francisco-Ortega et al., 1999), Lauraceae (Lee et al., 2010) and Ulmaceae (Lee et al., 2011). Overall, in the present study, the distribution pattern of T. triangulare populations using phenotypic (morphological, biochemical) and RAPD markers elucidate substantial intra-specific variation. However, the DNA barcoding results of three chloroplast genes (rbcL, trnH-psbA, matK) and one nuclear gene (ITS) demonstrated that no variation was detected among the regionally collected plant samples proving that environmental changes and geographical divergence did not have any effect at the genetic level. Thus, molecular characterization of T. triangulare played a promising role in species identification, molecular authentication and uncovering distinctiveness among closely related taxa. Additionally, elaborate investigations are necessary to understand the patterns of gene flow within and between accessions for implementing effective conservation strategies. Conflict of interest The authors declare that they have no conflict of interest. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410. Asahina, H., Shinozaki, J., Masuda, K., Morimitsu, Y., Satake, M., 2010. Identification of medicinal Dendrobium species by phylogenetic analyses using matK and rbcL sequences. Journal of Natural Medicines 64, 133–138. Boonsom, T., Waranuch, N., Ingkaninan, K., Denduangboripant, J., Sukrong, S., 2012. Molecular analysis of the genus Asparagus based on matK sequences and its application to identify A. racemosus, a medicinally phytoestrogenic species. Fitoterapia 83 (5), 947–953. Buzgo, M., Soltis, D.E., Soltis, P.S., Ma, H., 2004. Towards a comprehensive integration of morphological and genetic studies of floral development. Trends in Plant Science 9, 164–173. CBOL Plant Working Group, 2009. A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America 106, 12794–12979. Darokar, M.P., Khanuja, S.P.S., Shasany, A.K., Kumar, S., 2001. Low levels of genetic diversity detected by RAPD analysis in geographically distinct accessions of Bacopa monnieri. Genetic Resources and Crop Evolution 48 (6), 555–558. Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA minipreparation. Version II. Plant Molecular Biology Reporter 1, 19–21. Deng, C.L., Zhou, J., Gao, W.J., Sun, F.C., Qin, R.Y., Lu, L.D., 2006. Assessment of genetic diversity of Lycoris longituba (Amaryllidaceae) detected by RAPDs. Journal of Genetics 85 (3), 205–207. Dey, S.S., Singh, A.K., Chandel, D., Behera, T.K., 2006. Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits. Scientia Horticulturae 109, 21–28. Ding, G., Zhang, D., Yu, Y., Zhao, L., Zhang, B., 2012. Phylogenetic relationship among related genera of Plumbaginaceae and preliminary genetic diversity of Limonium sinense in China. Gene 506 (2), 400–403. Doebley, J., Durbin, M.L., Goldenberg, E.M., Clegg, M.T., Ma, D.P., 1990. Evolutionary analysis of the large subunit of carboxylase (rbcL) nucleotide sequence among the grasses (Gramineae). Evolution 44, 1097–1108. Edwards, D., Horn, A., Taylor, D., Savolainen, V., Hawkins, J.A., 2008. DNA barcoding of a large genus, Aspalathus L. (Fabaceae). Taxon 57, 1317–1327. Endress, P.K., 2000. Systematic plant morphology and anatomy – 50 years of progress. Taxon 49, 401–434. Fontem, D.A., Schippers, R.R., 2004. Talinum triangulare (Jacq.) Willd. [Internet] Record from Protabase. In: Grubben, G.J.H., Denton, O.A. (Eds.), PROTA (Plant Resources of

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