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Characterization of Talinum Triangulare (Jacq.) Willd

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Characterization of Talinum Triangulare (Jacq.) Willd South African Journal of Botany 97 (2015) 59–68 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb 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 charac- terized 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). http://dx.doi.org/10.1016/j.sajb.2014.12.012 0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved. 60 J. Swarna et al. / South African Journal of Botany 97 (2015) 59–68 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.
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