(L.) C. Jeffrey from India using internal transcribed - Academic ...

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African Journal of Biotechnology Vol. 10(72), pp. 16145-16151, 16 November, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.2002 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Genetic diversity assessment of Diplocyclos palmatus (L.) C. Jeffrey from India using internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA M. Ajmal Ali 1 *, Fahad M. A. Al-Hemaid 1 , Joongku Lee 2 , R. K. Choudhary 2 , Naif A. Al-Harbi 1,3 and Soo-Yong Kim 2 1 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh- 11451, Saudi Arabia. 2 International Biological Material Research Centre, Korea Research Institute of Bioscience and Biotechnology, Daejeon- 305-806, South Korea. 3 Addiriyah Chair for Environmental Studies, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Accepted 24 October, 2011 Internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 20 populations of Diplocyclos palmatus (L.) C. Jeffrey belonging to five different geographical locations (Bihar, Jharkhand, Maharashtra, Madhya Pradesh, and Tamil Nadu) of India were sequenced. Analysis of nucleotide sequences reveals polymorphism among the populations [π = 0.01482, θw = 0.01180 (0.00236) and total variance 3.260]. AMOVA was used to partition the genetic diversity and tested whether there is any hierarchy of ITS sequence variation among individuals. The genetic differentiation between the populations is high (ΦST = 0.591). The maximum likelihood tree reveals the evolution of D. palmatus under reproductive isolation and under different environmental conditions which may be most probably due to long distance distribution, and possibility of genetic exchange among the populations of D. palmatus distributed in Bihar and Jharkhnad. Key words: Diplocyclos palmatus (L.) C. Jeffrey, genetic diversity, internal transcribed spacer (ITS), nrDNA. INTRODUCTION Diplocyclos (Endl.) Post and Kuntze [Family Cucurbitaceae, Tribe Benincaseae (Schaefer and Renner, 2011)] is a small genus of four species; three [Diplocyclos leiocarpus (Hook. f.) C. Jeffrey, Diplocyclos schliebenii (Harms) C. Jeffrey, Diplocyclos tenuis C. Jeffrey] confined to tropical Africa and one [Diplocyclos palmatus (L.) C. Jeffrey] extends from tropical Africa to *Corresponding author. E-mail: majmalali@rediffmail.com. Tel: 966-75834. Fax: 966-75833. Abbreviation: ITS, Internal transcribed spacer. Malaysia (www.tropicos.org). In India the genus Diplocyclos is only represented by D. palmatus (commonly known as Sivalingi or Pachguria) which is growing wild on bushes, trees and hedges. The plant is a weak stemmed, branched tendril climber; leaves simple, alternate, 5-lobed, hairy above, pale and smooth beneath; flowers yellow, small, unisexual, male in small fascicles and females solitary; fruits quite conspicuous in the field due to bunches of globose green with white striped (or become bright red with white striped when ripe), smooth with 1 to 2 small seeds. Medicinally it is useful for skin diseases, inflammations and general debility (Chakravarty, 1982; Kirtikar and Basu, 1975; Ali and Pandey, 2007).
16146 Afr. J. Biotechnol. The nuclear ribosomal transcription unit (NRTU) is comprised of 18S, 5.8S and 28S genes, two internal transcribed spacers (ITS-1 and ITS-2), and an intergenic spacer (IGS). After transcription, the NRTU is processed to produce mature rRNAs that are key components of cytoplasmic ribosomes. NRTU are found in hundreds to thousands of tandem copies and usually several NRTU clusters are present within plant genomes. The conserved regions (18S and 28S genes) of NRTU are used to infer phylogenetic relationships at higher taxonomy levels, whereas the more rapidly evolving segments (ITS and IGS) are used for studies at the genic or population levels (Soltis and Soltis, 1998; Alvarez and Wendel, 2003). For over a decade, sequences of internal transcribed spacers (ITS) of NRTUs have been widely used to infer phylogenetic relationships, genetic diversity and to unravel evolution in a wide range of complexes in plants (Alvarez and Wendel, 2003; Baldwin and Markos, 1998; Baldwin et al., 1995; Hershkovitz et al., 1999; Kelch and Baldwin, 2003; Lee et al., 2002). Although, NRTUs are found in thousands of copies within a genome, intra-genomic diversity is generally low (Baldwin et al., 1995). This homogeneity among NRTUs is attributed to concerted evolution (Baldwin et al., 1995; Ainouche and Bayer, 1997), a process that acts through gene conversion and unequal crossing over. Despite the fact that homogenization is a norm among NRTUs in a genome, extensive intra-individual and intra-specific variation has been observed in various plant species (Campbell et al., 1997; Hughes et al., 2002). The accumulating evidence suggests that intra-individual variation of nuclear ribosomal ITS regions should not be considered as exceptional (Feliner et al., 2004). Because of the influence of concerted evolution, the occurrence of ancestral polymorphisms is not the most likely ultimate cause for intra-genomic variability in this marker. Instead, a more frequent origin is the merging of different ITS copies within the same genome as a consequence of gene flow. Once the two copies meet, the fate of the polymorphism depends on genetic, reproductive and population-level factors: Specifically, the number and location of ribosomal loci (on the same or different chromosomes), the occurrence of polyploidy and/or apomixes (Hershkovitz et al., 1999; Campbell et al., 1997; Buckler et al., 1997), and the relative abundance of different ITS copies in the breeding populations (Feliner et al., 2004). D. palmatus shows morphological intermediate among the population. Because of its medicinal properties the plant is being over explored by the local people from the wild. An important prerequisite for development of an effective conservation strategy is the proper evaluation of the distribution and study at the level of genetic variation (Milligan et al., 1994). A perusal of literature reveals that phylogeny and classification of the family Cucurbitaceae have been the focus of several studies (Schaefer and Renner, 2011; Ali et al., 2009; Kocyan et al., 2007; Zhang et al., 2006; Jeffrey, 2005; Decker-Walters et al., 2004; Chung et al., 2003; Jobst et al., 1998); however, the information on population structure and genetic variation of D. palmatus is lacking. Hence, the main objectives of the present study were to utilize the nucleotide data of the ITS region to evaluate the degree of differentiation among the populations of D. palmatus from India. MATERIALS AND METHODS Plant materials Leaf samples of D. palmatus were collected from different geographical region of India. All the collected voucher specimens have been deposited in the Tilka Manhji Bhagalpur University Herbarium (BHAG), Bihar, India. For comparison, the sequence of closely related species Coccinia grandis (L.) Viogt. from our earlier study (Ali et al., 2009) was included in the analysis. DNA extraction Leaves were dried in silica gel prior to DNA extraction. Total genomic DNA was extracted by following the 2X CTAB method (Doyle and Doyle, 1987) or using the DNeasy Plant Mini kit (QIAGEN Inc., Crawley, West Sussex, UK). Total genomic DNA was extracted from similar amounts of silica dried tissue (~10 to 50 mg dry mass) as well as from herbarium specimens following the cetyltrimethyl ammonium bromide (CTAB) procedure (Doyle and Doyle, 1987). After precipitation with isopropanol and subsequent centrifugation, the DNA pellet was washed with 70% ethanol, dried at 37°C, and resuspended in TRIS-EDTA (TE) buffer. Amplification of ITS region ITS sequences of nuclear ribosomal DNA were amplified using primers of White et al. (1990) ITS1F (5’-GTCCACTGAACCTT ATCATTTAG-3’) and ITS4R (5’-TCCTCCGCTTATTGATATGC-3’) via the polymerase chain reaction (PCR) using the AccuPower HF PCR PreMix (Bioneer, Daejeon, South Korea). One round of amplification consisting of denaturation at 94°C for 5 min followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 49°C for 1 min and extension at 72°C for 1 min, with a final extension step of 72°C for 5 min. The PCR products were purified using SolGent PCR Purification Kit-Ultra (SolGent, Daejeon, South Korea) prior to sequencing. DNA Sequencing The purified fragments were directly sequenced using dye terminator chemistry following the manufacturer’s protocol. The sequencing reaction was performed in a 10 µl final volume with the BigDye Terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems). Cycle sequencing was conducted using same primers used in amplification and BigDye vers. 3 reagents and an ABI PRISM 3100 DNA Analyzer (Perkin-Elmer, Applied Biosystems). Cycling conditions included an initial denaturing set at 94°C for 5 min, followed by 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Sequenced product was precipitated with 17 µl of
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