International Journal of Molecular Biology ISSN: 0976–0482 & E-ISSN: 0976–0490, Vol. 1, Issue 2, 2010, PP-01-14 Trichosanthes anguina L. is variety of Trichosanthese cucumerina L.- evidence based on molecular phylogenetic analysis of internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA Ajmal Ali M.* and Fahad M. A. Al-Hemaid *Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia, [email protected] Abstract- Phylogenetic relationship among some species of Trichosanthes L ( Cucurbitaceae ) was assessed using internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA to infer the taxonomic status of Trichosanthes anguina . The parsimony analysis of the entire ITS region resulted in 85 maximally parsimonious trees (MPTs) with a total length of 100 steps, a consistency index (CI) of 0.8800 (0. 8378 excluding uninformative characters), a homoplasy index (HI) of 0.1200 (0. 1622 excluding uninformative characters), rescaled consistency index (RC) of 0.7733 and a retention index (RI) of 0.8788. Our findings support the recognition of T. cucumerina var. anguina (L.) Haines as a variety of T. cucumerina . Keywords: Trichosanthes anguina , Trichosanthese cucumerina , Cucurbitaceae , ITS, nrDNA Introduction Genus Trichosanthes L. of tribe Trichosantheae, of plant material used in this study, along with subtribe Trichosanthinae, family Cucurbitaceae voucher information and GenBank accession include c. 100 species [1-5]. Principally it is an numbers, are listed in the Table 1. Based on Asiatic genus. The geographic distribution of the chloroplast DNA sequences [17] and pollen genus indicates either an Indo-Malayan or morphology [18] a close relationship between Chinese Centre of origin [6]. Tichosanthes tribe Trichosantheae and Luffeae have been anguina (snake gourd or serpent gourd) is an suggested to which the genus Trichosanthes and usual cucurbits with long, white spackled fruits Luffa belong. Therefore, the sequences of Luffa that actually in morphology resembles with snake were used as outgroup. Total DNA was extracted and is widely grown as a vegetable in India and using the DNeasy Plant Mini Kit (QIAGEN, in the Orient. Roots and seeds are used to expel Amsterdam, Netherlands). ITS sequences of worms and to treat diarrhea and syphilis [7-8]. nuclear ribosomal DNA were amplified using Haines, 1921-1924 [9] recognized T. cucumerina primers ITS1 (Forward 5’- L. var. cucumerina (L) Haines as a wild variant GTCCACTGAACCTTATCATTTAG-3’) and ITS4 with short fruits and T. cucumerina var. anguina (Reverse 5’-TCCTCCGCTTATTGATATGC-3’) (L.) Haines as cultivated variant with elongated, [19] via the polymerase chain reaction (PCR) snake-like fruits. Jeffrey, 1980 [6] also followed using the AccuPower HF PCR PreMix (Bioneer, Haines, 1921-1924 [9] treatment of T. anguina as Daejeon, South Korea) in 20 µL volumes a variety of T. cucumerina. Chakravarty, 1982 containing 2 µL of 10X buffer, 300 µM dNTPs, 1 [10], however, treated these two varieties as two µL of a 10 pM solution of each primer, 1 unit of different species. This treatment has been HF DNA polymerase. The initial denaturation at followed in most of the Indian floras, monographs 94°C for 5 min, and followed by 40 cycles of and research paper published so far. A perusal of 94°C for 1 min, 49°C for 1 min, and 72°C for 1 literature reveals that taxonomic status of T. min, with a final extension step of 72°C for 5 min. anguina is controversial [11-17]. Hence, this The PCR products were ligated into the pT7Blue study was undertaken to compare sequences of cloning vector using Perfectly Blunt Cloning Kit the internal transcribed spacer regions of nrDNA (Novagen, Inc.) according to the manufacturer’s in some species of Trichosanthes in order to infer instructions. Resulting recombinant plasmids taxonomic status of T. anguina . were used to transform competent cells included in the kit. The transformation mix was incubated Materials and Methods in 250 µl SOC medium for 1hour at 37°C on a Present study sampled 10 accessions of rotary shaker, then plated on LB agar with 50 Trichosanthes (ingroup) and Luffa (outgroup) µg/mL ampicillin. Colonies were randomly from the geographical origin of Bihar, West selected and were put into PCR buffer. The PCR Bengal and Sikkim (India) and South Korea. products were purified with the SolGent PCR Voucher specimens are deposited at the BHAG Purification Kit-Ultra (SolGent, Daejeon, South (Herbarium, University Department of Botany, Korea) prior to sequencing. The purified Tilka Manjhi Bhagalpur University, Bhagalpur, fragments were directly sequenced using dye Bihar, India) and KRIB (Herbarium, Korea terminator chemistry following the manufacturer’s Research Institute of Bioscience and protocol. Cycle sequencing was conducted using Biotechnology, Daejeon, South Korea). Sources same primers used in amplification and BigDye vers. 3 reagents and an ABI PRISM 3730XL DNA Copyright © 2010, Bioinfo Publications International Journal of Molecular Biology, ISSN: 0976–0482 & E-ISSN: 0976–0490, Vol. 1, Issue 2, 2010 Trichosanthes anguina L . is variety of Trichosanthese cucumerina L . Analyzer (Perkin-Elmer, Applied Biosystems) by generation, resulting in 50,000 trees. The first following the manufacturer’s instructions. Cycling 10,000 trees were considered as the burn-in conditions included an initial denaturing set at phase and discarded. 94°C for 5 min., followed by 30 cycles of 96°C for 10 sec., 50°C for 5 sec., and 60°C for 4 minutes. Results Each sample was sequenced in the sense and Sequence Characteristics- The combined antisense direction. The sequences were length of the entire ITS region (ITS1, 5.8S and analyzed with ABI Sequence Analysis and ABI ITS2) from taxa sampled in the present study Sequence Navigator software (Perkin- ranged from 608-616 bp. The length of ITS1 Elmer/Applied Biosystems). Nucleotide region and %GC ranged from 191-201 bp and sequences of both DNA strands were obtained 61-62% respectively, the 5.8S gene was 163 bp, and compared to ensure accuracy. Initially the the length of ITS2 region and %GC ranged from sequence alignments were performed using 235-260 bp and 65-67% respectively (Table 2). ClustalX version 1.81 [20] with gap opening Data matrix has a total number of 632 characters penalty = 10 and gap extension penalty = 3.0. of which 554 characters are constant, 25 Sequence alignments were subsequently characters are variable but parsimony- adjusted manually using BioEdit [21] and uninformative and 55 characters are parsimony- SeaView [22]. Insertion-deletions (Indels) were informative. Insertions and deletions (indels) scored as single characters when we had were necessary to align the sequences. Indels confidence in positional homology (Annexure). were ranged from 1 to 11 bp. The boundaries between the ITS1, 5.8S, and ITS2 were determined by comparisons with Phylogenetic analyses- The parsimony analysis earlier published sequences available at National (using PAUP) of the entire ITS region resulted in Center for Biotechnology Information (NCBI) 85 maximally parsimonious trees (MPTs) with a GenBank (www.ncbi.nlm.nih.gov). Gaps were total length of 100 steps, a consistency index (CI) treated as missing data in phylogenetic analyses. of 0.88 (0. 8378 excluding uninformative All sequences generated in the present study characters), a homoplasy index (HI) of 0.12 (0. were deposited in GenBank and GenBank 1622 excluding uninformative characters), accession number included in Table 1. rescaled consistency index (RC) of 0.7733 and a Parsimony analyses were performed with PAUP* retention index (RI) of 0.8788. The bootstrap 4.0b10 [23]. Heuristic searches were conducted values above the line in bootstrap strict using 10,000 random addition sequence consensus tree (Fig. 1) show the relative support replicates, holding 10 trees at each step, and with of each clade. The number of base substitutions tree-bisection-reconnection (TBR) branch per site from analysis between sequences swapping, characters equally weighted, and gaps (evolutionary divergence) is shown in Table 3. treated as missing data. Support for internal The number of base substitutions per site from nodes was assessed using bootstrap analysis averaging evolutionary divergence over all [24] of 1000 replicates with 100 random additions sequence pairs was found 0.048. Homogeneity per replicate and holding 10 trees at each step. test of substitution patterns between sequences: Phylogenetic and molecular evolutionary The probability of rejecting the null hypothesis analyses (evolutionary divergence between that sequences have evolved with the same sequences, the number of base substitutions per pattern of substitution, as judged from the extent site from averaging evolutionary divergence over of differences in base composition biases all sequence pairs, homogeneity test of between sequences. A Monte Carlo test (1000 substitution patterns between sequences, base replicates) was used to estimate the P-values, composition bias difference between sequences, which are shown in diagonal in the Table 4. P- maximum composite likelihood estimate of the values smaller than 0.05 are considered pattern of nucleotide substitution, codon-based significant. The estimates of the disparity index test of neutrality for analysis between sequences, per site are shown for each sequence pair above and Fisher's exact test of neutrality for sequence the diagonal. pairs) were conducted using MEGA version 4 [25-28]. The result was