International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 2, Issue 2, 2010, pp-42-60 Molecular phylogenetic study of Luffa tuberosa Roxb. (Cucurbitaceae) based on internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA and its systematic implication Ajmal Ali M.1*, Karuppusamy S.2 and Fahad M. Al-Hemaid1 *1Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia, [email protected] 2Department of Botany, The Madura College, Madurai, Tamil Nadu, India Abstract- The phylogenetic position of long been debatable species Luffa tuberosa was inferred in the present study using ITS sequence of nuclear ribosomal DNA data. The study sampled a total number of 16 accessions which include five accessions of Luffa (under four species i.e. Luffa acutangula, L. cylindrica, L. aegyptiaca and L. tuberosa), nine accessions of Momordica (under eight species i.e. M. angustisepala, M. balsamina, M. cabraei, M. charantia, M. charantia subsp. macroloba, M. cissoides, M. cochinchinensis, M. dioica and M. foetida) and two accessions of Trichosanthes under two species (i.e. T. lepiniana and T. tricuspidata). The sequence data analysis clearly reveals nesting of Luffa tuberosa within the clade of Momordica, thus, we herein support the inclusion of Luffa tuberosa into the genus Momordica as M. tuberosa (Roxb.) Cogn. Key Words: Luffa tuberosa, Momordica, Cucurbitaceae, ITS, nuclear ribosomal DNA INTRODUCTION tendril-bearing vine grows up to 5 meter. It bears The genus Luffa Miller, belongs to Tribe Luffeae, simple, alternate leaves, 4-12 cm across, with 3-7 Subfamily Cucurbitioideae, Family Cucurbitaceae deeply separated lobes [1]. are distributed mainly in tropical regions of the The taxonomic position of Luffa aroused much world [1]. Chakravarty, 1982 [2] recognized a interest after Jeffrey (1962) created a new total number of nine species of Luffa out of which Subtribe Luffinae under Cucurbitaceae to provide seven species (L. acutangula, L. cylindrica, L. separate rank of Subtribe to the genus Luffa [4]. echinata, L. graveolens, L. hermaphrodita, L. Singh (1964) has justified the position of Luffa in tuberosa and L. umbellata) occur in India. Luffa Jeffrey’s classification on the basis of tuberosa is distributed in Peninsular India comparative study of the endosperm haustorium (Andhra Pradesh, Karnataka, Madhya Pradesh in the family [5]. The species Luffa tuberosa was and Maharashtra) and Tropical Africa. The established by Roxburgh in 1832 [6]. Clarke species, Luffa tuberosa has been characterized (1879) transferred Luffa tuberosa to the genus morphologically by perennial climbing herb, Momordica under Momordica cymbalaria Fenzl monoecious; stems: slender, scandent, striate, [7]. Congiaux (1881) recognized Momordica pubescent, arising from a small perennial tuber; tuberosa (Roxb.) Cogn., based on Roxburgh’s leaf-blade: reniform-orbicular or pentagonal in Luffa tuberose [8]. The phylogenetic position and outline, cordate, obscurely sinuate to distinctly taxonomic status of Luffa tuberosa within the sinuate-toothed, glabrous or sparsely hairy genus is debatable [9]. especially on the nerves beneath, obscurely 5- The aim of this study is to evaluate phylogenetic lobed; petiole pubescent; tendrils simple; male position of Luffa tuberosa by comparing flowers: subtended by a minute bract, petals sequences of the internal transcribed spacer yellow, stamens 3 or 2; female flowers: peduncle regions of the nuclear ribosomal DNA with cylinder, ebracteate, stigma 2 or 3, bipartitie; species of Momordica. ovary: fusiform, glabrous or pubescent; fruit: fleshy, fusiform, shortly beaked, pubescent, MATERIALS AND METHODS longitudinally ribbed; seeds: subglobose, rugose- Present study sampled a total number of 16 appendaged at one end, testa smooth. The accessions which include five accessions of tender fruits of L. tuberosa are used as diuretic Luffa, nine accessions of Momordica and two and laxative in the traditional system of medicine accessions of Trichosanthes (outgroup). The in India [2]. Ayyangar, 1976 [3] reported plant material of Luffa acutangula, L. cylindrica, chromosome No. 2n= 22 in Momordica tuberosa L. tuberosa, Momordica dioica, Trichosanthes (= L. tuberosa). lepiniana and T. tricuspidata were collected in The genus Momordica L. belongs to Tribe nature from the state Bihar, West Bengal and Joliffieae, Subtribe Thladianthinae, Family Andhra Pradesh of India. GenBank accession Cucurbitaceae [1] comprises c. 45 species of numbers along with voucher information of newly annual or perennial climbing herbaceous or generated sequence for this study are listed in shrubby plants, natives of tropical and subtropical the Table 1. The voucher specimens submitted in Africa, Asia and Australia. The herbaceous, BHAG (Herbarium, Tilka Manjhi Bhagalpur Copyright © 2010, Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 2, Issue 2, 2010 Molecular phylogenetic study of Luffa tuberosa Roxb. (Cucurbitaceae) University, Bhagalpur, Bihar, India) and SKU following the manufacturer’s instructions. Cycling (Herbarium, Sri Krishnadevaraya University, conditions included an initial denaturing set at Anantapur, Andhra Pradesh, India). To compare 94°C for 5 min., followed by 30 cycles of 96°C for and analyse the ITS sequences of nuclear 10 sec., 50°C for 5 sec., and 60°C for 4 minutes. ribosomal DNA, eight accessions of Momordica Each sample was sequenced in the sense and under eight spices i.e. M. angustisepala antisense direction. The sequences were (AM981080), M. balsamina (AM981071), M. analyzed with ABI Sequence Analysis and ABI cabraei (AM981084), M. charantia (AM981062), Sequence Navigator software (Perkin- M. charantia subsp. macroloba (AM981061), M. Elmer/Applied Biosystems). Nucleotide cissoids (AM981079), M. cochinchinensis sequences of both DNA strands were obtained (AY606266) and M. foetida (AM981065) and two and compared to ensure accuracy. accessions of Luffa under two species i.e. L. Initially the sequence alignments were performed aegyptiaca (AM981167) and L. cylindrica using ClustalX version 1.81 [13] with gap opening (AF013324) were retrieved from the NCBI penalty = 10 and gap extension penalty = 3.0. GenBank database. The genus Luffa, Momordica Sequence alignments were subsequently and Trichosanthes belong to subfamily adjusted manually using BioEdit [14] and Cucurbitoideae [1]. Based on chloroplast DNA SeaView [15]. Insertion-deletions (Indels) were sequences [10] from two genes, one intron, two scored as single characters when we had spacers (rbcL, matK, trnL, trnL-trnF, rpl20- rps12) confidence in positional homology (Annexure I). and on ITS1 and ITS2 sequences of nrDNA [11] The boundaries between the ITS1, 5.8S, and suggested a close relationship between Tribe ITS2 were determined by comparisons with Luffeae and Trichosantheae to which Luffa and earlier published sequences available at National Trichosanthes belong (73% bootstrap support) Center for Biotechnology Information (NCBI) and in between Trichosantheae and Joliffieae to GenBank (www.ncbi.nlm.nih.gov). Gaps were which Trichosathes and Momordica belong. treated as missing data in phylogenetic analyses. Therefore, for the phylogenetic analysis, the All sequences generated in the present study species of Trichosanthes were selected as were deposited in GenBank and GenBank outgroup. accession number included in Table 1. Total DNA was extracted using the DNeasy Plant Parsimony analyses were performed with PAUP* Mini Kit (QIAGEN, Amsterdam, Netherlands). 4.0b10 [16]. Heuristic searches were conducted ITS sequences of nuclear ribosomal DNA were using 10,000 random addition sequence amplified using primers ITS1 (Forward 5’- replicates, holding 10 trees at each step, and with GTCCACTGAACCTTATCATTTAG-3’) and ITS4 tree-bisection-reconnection (TBR) branch (Reverse 5’-TCCTCCGCTTATTGATATGC-3’) swapping, characters equally weighted, and gaps [12] via the polymerase chain reaction (PCR) treated as missing data. Support for internal using the AccuPower HF PCR PreMix (Bioneer, nodes was assessed using bootstrap analysis Daejeon, South Korea) in 20 L volumes [17] of 1000 replicates with 100 random additions containing 2 L of 10X buffer, 300 M dNTPs, 1 per replicate and holding 10 trees at each step. L of a 10 pM solution of each primer, 1 unit of Phylogenetic and molecular evolutionary HF DNA polymerase. The initial denaturation at analyses (evolutionary divergence between 94°C for 5 min, and followed by 40 cycles of sequences, the number of base substitutions per 94°C for 1 min, 49°C for 1 min, and 72°C for 1 site from averaging evolutionary divergence over min, with a final extension step of 72°C for 5 min. all sequence pairs, homogeneity test of The PCR products were ligated into the pT7Blue substitution patterns between sequences, base cloning vector using Perfectly Blunt Cloning Kit composition bias difference between sequences, (Novagen, Inc.) according to the manufacturer’s maximum composite likelihood estimate of the instructions. Resulting recombinant plasmids pattern of nucleotide substitution, codon-based were used to transform competent cells included test of neutrality for analysis between sequences, in the kit. The transformation mix was incubated and Fisher's exact test of neutrality for sequence in 250 l SOC medium for 1hour at 37°C on a pairs) were conducted using MEGA version 4 rotary shaker, then plated on LB agar with 50 [18-21]. Parsimony analyses were also g/mL ampicillin.