GENETIC VARIATION AND MOLECULAR RELATIONSHIPS AMONG EIGHT TAXA OF DESMODIUM DESV . BASED ON RAPD MARKERS

Genetic variation and molecular relationships among eight taxa of Desmodium Desv. were assessed on the basis of random amplified polymorphic DNA (RAPD) markers. The banding patterns of eight taxa namely, Desmodium gangeticum (L.) DC., D. heterocarpon (L.) DC., D. heterophyllum (Willd.) DC., D. motorium (Houtt.) Merr., D. pulchellum (L.) Benth., D. triflorum (L.) DC., D. triquetrum (L.) DC. and D. triquetrum subsp. alatum (DC.) Prain were compared. A total of 81 DNA fragments were detected by 11 primers. Among the taxa studied D. triquetrum and D. triquetrum subsp. alatum were found to be most closely related followed by close proximity between D. gangeticum and D. motorium. The highest genetic distance was observed between D. triflorum and D. heterophyllum followed by D. heterocarpon and D. heterophyllum. UPGMA dendrogram was constructed to show the genetic relatedness among the taxa employed and the tree revealed a close proximity among D. pulchellum, D. gangeticum and D. motorium. In contrast, D. heterophyllum was found distantly related with rest of the taxa. Introduction Desmodium Desv. belongs to the family Fabaceae comprises about 280 species widespread in the tropical and subtropical regions (Puhua and Ohashi, 2010). In Bangladesh Desmodium is represented by 19 taxa (Ahmed et al., 2009). They are annual to perennial herbs, undershrubs or shrubs, and characterized by possessing unior tri-foliolate leaves, simple raceme or panicle inflorescence and distinctly jointed pods. The systematics of the genus Desmodium is confusing and not yet resolved completely (Ohashi and Mill, 2000). Several taxonomic studies on Desmodium were carried out based on morphology and anatomy (Pedley and Rudd, 1996; Shaheeen, 2008; Puhua and Ohashi, 2010). Recently, Rahman and Rahman (2012) conducted a morphometric study of Desmodium and showed interspecific relationships among 14 species of the genus. However, molecular studies employing different DNA markers on this genus are very scanty (Yue et al., 2010; Ahmad Haji et al., 2016). Recent progress in DNA marker technology have augmented the marker resources for genetic analyses of a wide variety of genomes. The development of random amplified polymorphic DNA (RAPD) markers generated by polymerase chain reaction (PCR) using arbitrary primers has resulted in alternative molecular markers for the detection of nuclear DNA polymorphism (Williams et al., 1990). RAPD markers have application in many fields including DNA fingerprinting (Elavazhagan et al., 2009), assessment of genetic diversity (Bodo Slotta and Porter, 2006), cultivar identification (Sipahi et al., 2010), estimation of population genetics (Sales et al., Corresponding author. Email: prof.oliurrahman@gmail.com Department of Botany, University of Barisal, Barisal 8200, Bangladesh


Introduction
Desmodium Desv.belongs to the family Fabaceae comprises about 280 species widespread in the tropical and subtropical regions (Puhua and Ohashi, 2010).In Bangladesh Desmodium is represented by 19 taxa (Ahmed et al., 2009).They are annual to perennial herbs, undershrubs or shrubs, and characterized by possessing uni-or tri-foliolate leaves, simple raceme or panicle inflorescence and distinctly jointed pods.The systematics of the genus Desmodium is confusing and not yet resolved completely (Ohashi and Mill, 2000).Several taxonomic studies on Desmodium were carried out based on morphology and anatomy (Pedley and Rudd, 1996;Shaheeen, 2008;Puhua and Ohashi, 2010).Recently, Rahman and Rahman (2012) conducted a morphometric study of Desmodium and showed interspecific relationships among 14 species of the genus.However, molecular studies employing different DNA markers on this genus are very scanty (Yue et al., 2010;Ahmad Haji et al., 2016).
Recent progress in DNA marker technology have augmented the marker resources for genetic analyses of a wide variety of genomes.The development of random amplified polymorphic DNA (RAPD) markers generated by polymerase chain reaction (PCR) using arbitrary primers has resulted in alternative molecular markers for the detection of nuclear DNA polymorphism (Williams et al., 1990).RAPD markers have application in many fields including DNA fingerprinting (Elavazhagan et al., 2009), assessment of genetic diversity (Bodo Slotta and Porter, 2006), cultivar identification (Sipahi et al., 2010), estimation of population genetics (Sales et al., 2001), hybridization (Caraway et al., 2001), systematics (Vilatersana et al., 2005), phylogeny reconstruction (Ahmed et al., 2005), and genome mapping (Krutovaskii et al., 1998).
In legume species, RAPD markers have proven to be a useful tool in studies analyzing genetic variation (Yamaguchi and Jabadi, 2004;Bisoyi et al., 2010).Previous studies on Desmodium using isozymes were conducted mainly with species which are important as forage (Smith and Schaal, 1979;Imrie and Blogg, 1983).Application of RAPD markers for detecting genetic variation and interspecific relationships of Desmodium is very limited.Bedolla-Garcia and Lara-Cabrera (2006) applied RAPD markers to detect genetic variation within and among five population of Desmodium sumichrastii from Mexico.Very recently, Singh et al. (2016) employed RAPD analysis for DNA fingerprinting of only two species of Desmodium, viz., D. gangeticum and D. laxiflorum.However, no detailed study based on RAPD markers for detecting genetic diversity and interspecific relationships in Desmodium was carried out so far.Therefore, the aims of the present study are two-fold: i) to detect the genetic diversity among eight Desmodium taxa, and ii) to infer the relationship among these taxa of Desmodium based on RAPD analysis.

RAPD amplification
The oligonucleotide primers tested for RAPD analysis were presented in Table 2.These primers were chosen by their number and consistency of amplified fragments for analyzing Desmodium taxa.The amplification reaction contained 50 ng of genomic DNA, 0.5 unit of Taq DNA polymerase, 0.5 µl of each dNTPs, 10 mM MgCl 2 , 1µl decamer random primers (Operon Biotechnology, Germany) and 2.5 µl 10X amplification buffer in a total volume of 25 µl.The amplifications were performed in triplicate using PCR thermal cycler (Biometra UNOII, Germany) with initial denaturation of 5 min at 94°C, followed by 42 cycles at 94°C for 5 sec, 33°C for 1 min and 72°C for 2 min with final extension of 5 min at 72°C.The amplified products were separated on 1% agarose gel containing ethidium bromide, and photographed under UV light.Data analysis RAPD bands were recorded in a binary data matrix scored as presence (1) or absence (0).The score obtained using all primers in the RAPD analysis were then combined to create a single data matrix.The size of amplification products were estimated by comparing the migration of each amplified fragments with that of a known size fragments of 1 kb molecular weight marker.Genetic linkage distance was determined using the data matrix.UPGMA (Unweighted pair group method with arithmetic means) dendrogram was constructed to show the genetic relationships among the species (Sneath and Sokal, 1973).All analyses were performed using the Statistica program.

Results and Discussion
A total of 81 RAPD bands were scored with eleven RAPD primers in eight Desmodium taxa.The highest number of fragments was detected in Desmodium heterophyllum (31) followed by D. triflorum (20) and D. heterocarpon (7), while the lowest band observed in D. motorium (1).The highest number of fingerprints were generated by the primer OPA-8 and least number in OPA-1.
The RAPD markers have been found efficient to detect genetic variation in Desmodium.The highest dissimilarity (41.0) was observed between D. triflorum and D. heterophyllum followed by D. pulchellum and D. heterophyllum (35.5) and D. heterophyllum and D. heterocarpon (35.0) (Table 3).The lowest genetic distance (1.0) was found between D. gangeticum and D. motorium indicating that these species are very closely related (Table 3).
Cluster analysis of the genetic similarity estimates from RAPD markers was performed to generate the UPGMA dendrogram for showing genetic relationship among the taxa of Desmodium (Fig. 1).The dendrogram revealed that D. pulchellum, D. gangeticum, D. motorium, D. triquetrum, D. triquetrum subsp.alatum, and D. heterocarpon grouped together and formed a cluster showing a close relationships among them.This cluster further consisted of two subclusters, the first one contained D. pulchellum, D. gangeticum and D. motorium showing a close affinity between these three species, while the second sub-cluster comprised D. triquetrum subsp.alatum, D. triquetrum, and D. heterocarpon.The highest relatedness was observed between D. gangeticum and D. motorium among all the taxa employed in this study.The RAPD analysisalso shown that D. heterophyllum and D. triflorum retained ungrouped and they are distantly related from other taxa of Desmodium.The RAPD method is popular because of its technical simplicity and speed.The present study reveals that RAPDs are useful markers in identifying Desmodium species.The RAPD data shows that D. gangeticum and D. motorium are genetically closely related.This result is congruent with the previous study based on morphological characters such as unifoliolate, lanceolate leaves and triangular stipules (Ahmed et al., 2009); and also supported by foliar anatomical investigation (data not shown).Bedolla-Garcia and Lara-Cabrera (2006) studied genetic variation within and among five populations of Desmodium sumichrastii from Mexico based on RAPD analysis.Singh et al. (2016) employed RAPD approach for the genetic fingerprinting of Desmodium gangeticum and D. laxiflorum and found 60-65% similarity between these two species.Irshad et al. (2009) studied three species of Desmodium, viz.D. gangeticum, D. triflorum and D. velutinum (Willd.)DC. and compared with commercial samples of various origin.Among these D. triflorum appears closer to D. gangeticum reflecting narrow genetic diversity.However, the present study shows that D. triflorum and D. gangeticum are distantly related.Very recently, Malgaonkar et al. (2016) determined the genetic relatedness and diversity among accessions of four Desmodium species using RAPD markers, namely D. dichotomum (Willd.)DC., D. laxiflorum DC., D. scorpiurus (SW.)Poir.and D. triflorum.A close affinity has been observed between D. laxiflorum and D. scorpiurus.In the present study D. pulchellum has been found close to D. gangeticum and D. motorium indicating that these three species are closely related.D. triflorum and D. heterophyllum are closely allied as evidenced by anatomical study (Data not shown), however, this affinity is not supported by RAPD analysis.In order to have better understanding about genetic relatedness and interspecific relationships inclusion of more taxa with additional markers is necessary.