Genetic Variation Between Xylocarpus Spp. (Meliaceae) As Revealed by Random Amplified Polymorphic DNA (RAPD) Markers

Emir. J. Food Agric. 2013. 25 (8): 597-604 doi:10.9755/ejfa.v25i8.15403 http://www.ejfa.info/ PLANT SCIENCE Genetic variation between Xylocarpus spp. (Meliaceae) as revealed by Random Amplified Polymorphic DNA (RAPD) markers Umesh R. Pawar, Jitha Baskaran, I. Paul Ajithkumar, Rajaram Panneerselvam* Department of Botany, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India Abstract The genetic diversity of Xylocarpus a tree mangrove species was assessed. Three species viz., X. granatum, X. moluccensis and X. mekongensis were collected from different localities of the states of Tamil Nadu and Andhra Pradesh in India. These three species showed high degrees of phenotypic variation in the vegetative parts such as leaf, bark and root. To assess the variations at the molecular level within the three species of Xylocarpus a RAPD technique was used. A total of 283 DNA fragments were generated by 25 random primers, with an average of 11.32 easily detectable fragments per primer. A higher similarity was observed between X. mekongensis and X. moluccensis, whereas X. granatum and X. mekongensis showed lower similarity. X. granatum and X. mekongensis had a variation of more than 79 %. On the other hand, X. mekongensis and X. moluccensis seem to be closely related with 35% variation. These variations may be due to its genotypic variation, isolated distribution and adaptation to dissimilar edaphic and environmental factors. All the three species of Xylocarpus are related at various degrees. These results will be helpful in future to assess the existing interspecific genetic polymorphism in Xylocarpus species and to design strategy for their conservation. Key words: Genetic diversity, Xylocarpus, RAPD Introduction Mangroves are unique plant communities granatum and X. mekongensis are moderately sized inhabiting the estuarine and inter-tidal regions of trees with a well-developed woody trunk, whereas both tropical and subtropical coasts. The total X. moluccensis is a medium-sized crooked, much- number of exclusive or true mangrove species in branched evergreen tree up to 10 m tall. They are the world is 68 and they belong to 27 genera (Duke, found on the fringes of backwater creeks. Usually 1992). The genus, Xylocarpus, belonging to the this taxon is associated with Avicennia, Excoecaria, family Meliaceae has three distinct species, viz., X. Acanthus, Rhizophora and Bruguiera (Raju, 2003). granatum Koen., X. moluccensis Lamk. and X. The bark of X. mekongensis is rich in tannin and is mekongensis Pierre. These species are distributed in used for tanning heavy hides, toughening fishing- tropical tidal forests of old world, typical mangrove nets and dying cloth. The wood is of good quality habitats or in sandy or in coastal habitats spread and used for boat building, nails, house-posts, small from Africa to Australia including India and objects, furniture and firewood. The seeds have Malayan Archipelago (Tomlinson, 1986). In India, medicinal properties. Molecular markers like these species were recorded from Andaman Islands, random amplified polymorphic DNA (RAPD) and coastal line of Orissa, Andhra Pradesh and amplified fragment length polymorphism are Pichavaram mangrove forest of Tamil Nadu in the extensively used to quantify the inter-specific, East coast of India. X. granatum is distributed in intra- specific and inter-generic variability in West coast mangroves e.g. Maharashtra of India. X. different plant groups and crop varieties (Chalmers et al., 1994; Lin et al., 1996; Garcia Mas et al., Received 27 November 2012; Revised 12 February 2013; 2000; Sharma et al., 2000; Mukharjee et al., 2003; Accepted 18 February 2013; Published Online 01 June 2013 Begum et al., 2013). Parani et al. (1997) identified *Corresponding Author the parentage of Rhizophora hybrid, by establishing Rajaram Panneerselvam its maternal status using molecular markers such as Department of Botany, Annamalai University, Annamalai RAPD and RFLP. Jian et al. (2004) analyzed the Nagar 608 002, Tamil Nadu, India variation in inter simple sequence repeats (ISSR) in Email: [email protected] mangrove and non-mangrove populations of Heritiera littoralis. In 2004, Mukherjee and his 597 Umesh R. Pawar et al. associates studied the genomic relationship among leaves using the CTAB (cetyl trimethyl ammonium nine mangrove and two non-mangrove species of bromide) method (Saghai-Maroof et al., 1984) with India belonging to the family Rhizophoraceae and some modifications. 0.5 g of young leaf tissue was proved that mangroves and non-mangroves are ground in liquid Nitrogen to fine powder using related distantly. Su et al. (2006) studied genetic sterile pestle and mortar and suspended in 750 µl of variation in the mangrove, Lumnitzera racemosa.. preheated 2% CTAB buffer (2% CTAB; 0.1 M Tris Triest (2008) reviewed the uses of dominant pH 8.0; 20 mM EDTA; 1.4 M NaCl; 2% markers such as RAPD and AFLP for identifying Polyvinylpyrrolidine-40 and 1% β-mercapto the mangroves and studying their relationship. ethanol). The suspension was incubated at 65°C for Jugale et al. (2009) assessed genetic diversity in one hour with occasional inversion. An equal intra and inter-populations of X. granatum using volume of chloroform: isoamyl alcohol (24:1) ISSR markers and showed that variation exists at mixture was added to the suspension and the phenotypic level but the genetic variability centrifuged at 10,000 g for 15 minutes. The among the populations of X. granatum is very low. aqueous phase was transferred to a new microfuge The molecular markers are phenotypically neutral tube and extracted with 0.2 volumes of 5% CTAB having no epistatic and developmental effects. buffer and equal volume of chloroform: isoamyl Moreover, they can detect the variation in both alcohol (24:1) mixture at 10,000 g for 15 minutes. coding and non-coding regions of the genome. The Chloroform: IAA extraction step was repeated Among the several types of markers RAPD markers twice. The aqueous phase was transferred to a fresh have been successfully proven to distinguish the tube containing 0.6 volumes of ice-cold isopropanol variations. There are no attempts made to study the and incubated overnight at -20°C and subsequently, genetic diversity of three mangrove species of the centrifuged at 10,000 rpm for 15 minutes, to genus, Xylocarpus in peninsular India. recover the nucleic acids. The pellet was washed In the present study the genus Xylocarpus, a with 70% ethanol and air dried and dissolved in TE mangrove species has been assessed for genetic buffer (10 mM Tris; 1 mM EDTA, pH 8.0). The diversity. Three species viz., X. granatum, X. extracted DNA was quantified by using moluccensis and X. mekongensis were collected Nanophotometer (IMPLEN, GmbH, Munich, from different localities Tamil Nadu and Andhra Germany) and diluted to 15 ng/ µl for PCR Pradesh in India. These three species showed amplifications. higher degrees of phenotypic variation in the Evaluation of genetic diversity vegetative parts such as leaf, bark and root. To The 25 RAPD 10-mers used (Table 3) were assess the variations at the molecular level within selected from among 134 RAPD primers (obtained three species of Xylocarpus, the RAPD technique from Operon Technologies USA) in a preliminary test was used. RAPD markers may help to find out the for oligos that amplified numerous discrete fragments. difference at the molecular level between these RAPD profile of five primers across the three species species, and which in turn will prove useful to is depicted in Figure 2. Every 15 µl reaction volume explore the adaptation of these three species in consisted of 15 ng of genomic DNA, 1.5 µl of 2mM different habitats. each dNTPs, 1.5 µl of 10x Taq DNA Polymerase Material and methods assay buffer, 1.8 µl of 15mM MgCl2, 5 µM RAPD Plant material and DNA extraction primers and 1 U Taq DNA polymerase (Fermentas). The seeds of X. granatum and X. moluccensis PCRs were performed using a in an Eppendorf were collected from Kothapalem (11º 51´ N and 80º Thermocycler and involved an initial denaturation 54´ E) of Andhara Pradesh and X.mekongensis from step (94°C, 3 min), 40 amplification cycles (each Pichavaram (11º 27´ N and 79º 47´ E) of Tamil 94°C, 30 s; 37°C, 30 s and 72°C, 60 s) and a final Nadu. The physical characteristics of sampled extension step (72°C, 15 min). location given below in Table 1. The seeds were collected during the monsoon season in the month Table 1. Physical characteristics of sampled locations. of November- January (2010-2011). The seedlings Location Physical Characteristics of the 3 species (X. granatum, X. mekongensis and Soil pH Salinity Soil type X. moluccensis) were raised in the Botanical (ppt) garden, Department of Botany, Annamalai Kothapalem 7.5 – Saline 6 – 14 University. The morphological features of these (Andhra Pradesh) 8.3 swampy Pichavaram Fine and three species were recorded (Table 2 and Figure 1) 7.8- 8.4 8 – 24 Genomic DNA was isolated from freshly collected (Tamil Nadu) coarse silt 598 Emir. J. Food Agric. 2013. 25 (8): 597-604 http://www.ejfa.info/ Table 2. General morphological characters to identify the 3 species of Xylocarpus. Morphological X. granatum X. mekongensis X. moluccensis characters Leaves Paripinnate leaves, light green Paripinnate leaves having Leaves paripinnate, long, glabrous; in colour with a rounded apex, 1, 2 or 3 pairs of leaflets leaflets upto 12 × 5 cm, elliptic, Elliptic /obovate leaflets with pointed apex oblong-elliptic or obovate-elliptic, apex usually rounded. Bark Trunk surface is pale, smooth Trunk surface is dark Bark smooth and yellowish or with its thin bark peelings in brown, fissured with the brown and green with flaking flakes or patches bark peelings in long thick narrow strips Root Horizontal cable roots develops Vertical conical laterally Surface roots laterally compressed into ribbon like plank roots compressed knee roots or and forming a spreading network pneumatophores present of ribbon like pneumatophores.

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