Genetic variation between Xylocarpus spp . ( Meliaceae ) as revealed by Random Amplified Polymorphic DNA ( RAPD ) markers

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.


Introduction
Mangroves are unique plant communities inhabiting the estuarine and inter-tidal regions of both tropical and subtropical coasts.The total number of exclusive or true mangrove species in the world is 68 and they belong to 27 genera (Duke, 1992).The genus, Xylocarpus, belonging to the family Meliaceae has three distinct species, viz., X. granatum Koen., X. moluccensis Lamk.and X. mekongensis Pierre.These species are distributed in tropical tidal forests of old world, typical mangrove habitats or in sandy or in coastal habitats spread from Africa to Australia including India and Malayan Archipelago (Tomlinson, 1986).In India, these species were recorded from Andaman Islands, coastal line of Orissa, Andhra Pradesh and Pichavaram mangrove forest of Tamil Nadu in the East coast of India.X. granatum is distributed in West coast mangroves e.g.Maharashtra of India.X. granatum and X. mekongensis are moderately sized trees with a well-developed woody trunk, whereas X. moluccensis is a medium-sized crooked, muchbranched evergreen tree up to 10 m tall.They are found on the fringes of backwater creeks.Usually this taxon is associated with Avicennia, Excoecaria, Acanthus, Rhizophora and Bruguiera (Raju, 2003).The bark of X. mekongensis is rich in tannin and is used for tanning heavy hides, toughening fishingnets and dying cloth.The wood is of good quality and used for boat building, nails, house-posts, small objects, furniture and firewood.The seeds have medicinal properties.Molecular markers like random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism are extensively used to quantify the inter-specific, intra-specific and inter-generic variability in different plant groups and crop varieties (Chalmers et al., 1994;Lin et al., 1996;Garcia Mas et al., 2000;Sharma et al., 2000;Mukharjee et al., 2003;Begum et al., 2013).Parani et al. (1997) identified the parentage of Rhizophora hybrid, by establishing its maternal status using molecular markers such as RAPD and RFLP.Jian et al. (2004) analyzed the variation in inter simple sequence repeats (ISSR) in mangrove and non-mangrove populations of Heritiera littoralis.In 2004, Mukherjee and his associates studied the genomic relationship among nine mangrove and two non-mangrove species of India belonging to the family Rhizophoraceae and proved that mangroves and non-mangroves are related distantly.Su et al. (2006) studied genetic variation in the mangrove, Lumnitzera racemosa.. Triest (2008) reviewed the uses of dominant markers such as RAPD and AFLP for identifying the mangroves and studying their relationship.Jugale et al. (2009) assessed genetic diversity in intra and inter-populations of X. granatum using ISSR markers and showed that variation exists at the phenotypic level but the genetic variability among the populations of X. granatum is very low.The molecular markers are phenotypically neutral having no epistatic and developmental effects.Moreover, they can detect the variation in both coding and non-coding regions of the genome.Among the several types of markers RAPD markers have been successfully proven to distinguish the variations.There are no attempts made to study the genetic diversity of three mangrove species of the genus, Xylocarpus in peninsular India.
In the present study the genus Xylocarpus, a mangrove species has been assessed for genetic diversity.Three species viz., X. granatum, X. moluccensis and X. mekongensis were collected from different localities Tamil Nadu and Andhra Pradesh in India.These three species showed higher degrees of phenotypic variation in the vegetative parts such as leaf, bark and root.To assess the variations at the molecular level within three species of Xylocarpus, the RAPD technique was used.RAPD markers may help to find out the difference at the molecular level between these species, and which in turn will prove useful to explore the adaptation of these three species in different habitats.

Plant material and DNA extraction
The seeds of X. granatum and X. moluccensis were collected from Kothapalem (11º 51´ N and 80º 54´ E) of Andhara Pradesh and X.mekongensis from Pichavaram (11º 27´ N and 79º 47´ E) of Tamil Nadu.The physical characteristics of sampled location given below in Table 1.The seeds were collected during the monsoon season in the month of November-January (2010-2011).The seedlings of the 3 species (X.granatum, X. mekongensis and X. moluccensis) were raised in the Botanical garden, Department of Botany, Annamalai University.The morphological features of these three species were recorded (Table 2 and Figure 1) Genomic DNA was isolated from freshly collected leaves using the CTAB (cetyl trimethyl ammonium bromide) method (Saghai-Maroof et al., 1984) with some modifications.0.5 g of young leaf tissue was ground in liquid Nitrogen to fine powder using sterile pestle and mortar and suspended in 750 µl of preheated 2% CTAB buffer (2% CTAB; 0.1 M Tris pH 8.0; 20 mM EDTA; 1.4 M NaCl; 2% Polyvinylpyrrolidine-40 and 1% β-mercapto ethanol).The suspension was incubated at 65°C for one hour with occasional inversion.An equal volume of chloroform: isoamyl alcohol (24:1) mixture was added to the suspension and centrifuged at 10,000 g for 15 minutes.The aqueous phase was transferred to a new microfuge tube and extracted with 0.2 volumes of 5% CTAB buffer and equal volume of chloroform: isoamyl alcohol (24:1) mixture at 10,000 g for 15 minutes.The Chloroform: IAA extraction step was repeated twice.The aqueous phase was transferred to a fresh tube containing 0.6 volumes of ice-cold isopropanol and incubated overnight at -20°C and subsequently, centrifuged at 10,000 rpm for 15 minutes, to recover the nucleic acids.The pellet was washed with 70% ethanol and air dried and dissolved in TE buffer (10 mM Tris; 1 mM EDTA, pH 8.0).The extracted DNA was quantified by using Nanophotometer (IMPLEN, GmbH, Munich, Germany) and diluted to 15 ng/ µl for PCR amplifications.

Evaluation of genetic diversity
The 25 RAPD 10-mers used (Table 3) were selected from among 134 RAPD primers (obtained from Operon Technologies USA) in a preliminary test for oligos that amplified numerous discrete fragments.RAPD profile of five primers across the three species is depicted in Figure 2. Every 15 µl reaction volume consisted of 15 ng of genomic DNA, 1.5 µl of 2mM each dNTPs, 1.5 µl of 10x Taq DNA Polymerase assay buffer, 1.8 µl of 15mM MgCl 2 , 5 µM RAPD primers and 1 U Taq DNA polymerase (Fermentas).PCRs were performed using a in an Eppendorf Thermocycler and involved an initial denaturation step (94°C, 3 min), 40 amplification cycles (each 94°C, 30 s; 37°C, 30 s and 72°C, 60 s) and a final extension step (72°C, 15 min).

Data analysis
The amplified products were separated on 1.5% (w/v) agarose gels by electrophoresis in 1X TBE buffer and visualized under UV light after ethidium bromide staining.To confirm the reproducible amplification of scored fragments, all amplifications were repeated twice.All the visible RAPD fragments were counted for each primer (Table 4) and robust polymorphic bands were scored as present (1) or absent (0) for each sample.For each primer, the number of polymorphic bands was calculated.The statistical analysis was carried out using the STATISTICA program (version 4.5 Stat soft Inc, USA).A phylogenetic dendrogram (Fig 3) was obtained by cluster analysis following the Unweighted Pair Group with Arithmetic Mean (UPGMA) method.

Results and Discussion
A total of 25 random primers were used for RAPD analysis with the 3 species of Xylocarpus.Majority of these primers showed polymorphic bands across the three species.PCR amplification using the genomic DNA of three species of Xylocarpus with 25 RAPD primers produced a total of 283 bands, of which, 242 bands were polymorphic.The size of the amplicons ranged from 240 to 6000 basepairs.Mangroves are constantly subjected to physiological stress caused by fluctuating growing conditions (Chapekar, 1994).Despite such extremes, they have successfully colonized suitable areas by developing morphological, physiological and reproductive adaptations (Clough et al., 1982;Clough et al., 1994;Saenger, 1982).Therefore depending on the genetic architecture of these species and their edaphic preferences and adaptations, different species are likely to display varying degree of polymorphism.Present observations on Xylocarpus granatum do support this presumption.In the three species, a maximum of 18 loci was obtained with the primer-OPW5, while the primer OPM11 resulted in a minimum of 3 loci.The total RAPD loci between the Xylocarpus species for individual primers differed according to the genomic characteristics of the species and the total number of amplicons resolved was 165 in X. moluccensis, followed by 159 in X. mekongensis and 145 in X. granatum.Ge and Sun (1999) studied genetic variation within and among populations of Aegiceras corniculatum.They recorded very low variation and low gene differentiation among populations.Huang et al. (2008) assessed interspecific and inter-population variation in three species of Ceriops and recorded low-genetic diversity at population level.
The average number of bands per primer varied from 5.8 to 6.6 in the species of Xylocarpus.The number of polymorphic loci ranged from a minimum of 102 in X. granatum to a maximum of 122 in X. moluccensis with an average ranging from 4.08 to 4.88 with the 25 random primers.It has been reported by Mukharjee et al. (2005) that nine members belonging to Rhizophoraceae formed a single cluster with RAPD analysis.The maximum average percent polymorphism of 79.62% was observed between the species of X. granatum and X. mekongensis, whereas the minimum DNA polymorphism of 35% existed between X. mekongensis and X. moluccensis and it was 72.76% with X. granatum and X. moluccensis.The cluster analysis of the RAPD profile with 25 primers following the method of Unweighted Pair Group with Arithmetic Mean (UPGMA) revealed that the 3 species of Xylocarpus clustered into two distinct branches of a single tree.X. mekongensis and X. moluccensis clustered together with a linkage distance of 9.2, whereas X. granatum is 4.8 linkage distance away from the above two species.Xylocarpus mekongensis and Xylocarpus moluccensis formed a single cluster while X. granatum formed different cluster.All the three species of Xylocarpus are related at various degrees, while X. granatum is distantly related with X. mekongensis and X. moluccensis.The X. mekongensis and X. moluccensips seem to be closely related and have 35% variation.This result coincides with the morphological and floristic variation exists among the species.Xylocarpus granatum is a critically endangered species of mangrove along the Indian coastline disappearing from many locations and represented by few individuals (Jugale et al., 2009).Loss of individuals or populations at certain locations may not cause immediate loss in genetic diversity, but more damage may occur in terms of long term genetic consequences due to the reduced numbers of populations and smaller population sizes.These variations may be due to isolated distribution and adaptation to dissimilar edaphic and environmental variation exists within the mangroves of Peninsular India.
In conclusion, our results demonstrate that molecular markers provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species and to design their conservation strategy.The Mangrove genus Xylocarpus having high genetic variability may be due to varied edaphic and climatic conditions of various mangroves localities.

Figure 3 .
Figure 3. UPGMA dendrogram showing the relationship among three species of Xylocarpus.

Table 1 .
Physical characteristics of sampled locations.

Table 2 .
General morphological characters to identify the 3 species of Xylocarpus.
Figure 1.Morphological variations among the three species of Xylocarpus.

Table 3 .
Details of the random primers used for the genetic relationship studies across three species of genus Xylocarpus.
Figure 2. RAPD profile of three species of Xylocarpus.

Table 4 .
Details of the RAPD analysis with the three Xylocarpus species.