GENETIC VARIABILITY AND RELATIONSHIP STUDY OF SIX BAMBOO SPECIES USING ISOZYME MARKERS

International Journal of Latest Research in Science and Technology Volume 4, Issue 2: Page No.82-86, March-April 2015 http://www.mnkjournals.com/ijlrst.htm ISSN (Online):2278-5299 GENETIC VARIABILITY AND RELATIONSHIP STUDY OF SIX BAMBOO SPECIES USING ISOZYME MARKERS 1 Thingujam Suson, 1 Heisnam Rameshwari, 1 Leimapokpam Tikendra, and 1 Potshangbam Nongdam 1 Department of Biotechnology, Manipur University, Manipur, India Corresponding Author Email: nongdammeetei@yahoo.com Abstract: Bamboos are economically important member of the grass family Poaceae, under the subfamily Bambusoideae. They are also commonly known as poor man s timber because of their ability to replace the expensive timber as cheap alternative for various construction purposes. Classical taxonomic studies of bamboos based on floral morphology and growth habit can cause problems in identification due to erratic flowering behavior and long sexual cycle. Limited studies have been carried out using molecular techniques to overcome this problem. In this study, investigation was conducted on identification and genetic relationship study of six different bamboos using acid phosphatase, esterase and phosphoenol pyruvate carboxylase (PEPCase) isozyme markers. Bambusa balcooa was found to be distantly related to Bambusa mizorameana and Bambusa nutans though they belonged to the same genus. Dendrogram profile and distance matrix analysis of the three isozyme markers revealed Dendrocalamus hamiltonii to be more closely related genetically to than to Dendrocalamus giganteus though the two were placed under the genus Dendrocalamus and having greater morphological similar characters. Melocanna baccifera exhibited greater affinity to Bambusa mizorameana as they were present together in the same major clusters of different dendrograms of isozyme markers under study. The present study indicated close similarity between Melocanna baccifera and ; and while Bambusa nutans exhibited the least genetic similarity to any of the other. Keywords - Bamboos; Isozymes; Acid phosphatase; Esterase; PEPCase; Dendrogram; Distance matrix I. INTRODUCTION Bamboos are the multiutility versatile forest tree-grasses belonging to the family Poaceae of subfamily Bambusoideae. They are considered to be one of the most useful forest trees greatly impacting the livelihood of rural people and getting the name tag of Poor man s timber and Green Gold [1]. Their uses are extensively found in paper, handicraft and furniture industry, house construction, making water pipes, storage vessels and other household items. The bamboos are regarded as natural resources for millions of people all over the world because of wide distribution across the globe [2]. The identification of bamboos mostly relies on vegetative characters like culm and culm sheath morphology due to their abnormally long sexual cycle and absence of any diagnostic tools [3]. However, the vegetative characters are considered less reliable for taxonomic and systematic identification of as they are easily influenced by several environmental factors [4,5]. Many of the economically important bamboos are not properly identified and taxonomic studies on bamboos are limited as compared to other grass family. The classification of bamboos at generic level as well as ranking of varieties at level are not consistent and highly fluctuating [6]. Molecular approach based on isozyme and DNA markers can be employed to establish phylogenetic relationship among different bamboos. Many researchers used DNA-RAPD markers to determine genetic relationship in different bamboos [7-10]. The genetic variation study of Phyllostachys bamboos and four bamboo genera within subtribe Bambusinae was performed using RFLP and AFLP markers respectively [11,12]. ISSR markers were applied to study genetic diversity and differentiation in different populations of Phyllostachys pubescens and Dendrocalamus membranaceous [13,14]. Inexpensive but fast and reliable isozyme markers were also successfully used for evaluating variability and establishing genetic relatedness in many plants [15-17]. The literatures on the use of isozyme markers for genetic variability studies on bamboos are very limited with few reports of peroxidase isozyme utilization for Dendrocalamus latiflorus clones identification and application of esterase, malate dehydrogenase and malic isozyme in accession characterization of Arundinaria falcata [18,19]. Bamboo germplasm collection is essential for conservation as bamboo natural resources at present face alarming level of genetic erosion and overexploitation for domestic and conservational purposes. There is a necessity to focus on effective classification and identification of bamboos. Molecular approach through isozyme markers can generate reliable markers which can be useful in identification and establishing genetic relationship between different bamboo. The state of Manipur located at North-East region of India harbours rich bamboo resources including the six important bamboo viz., Bambusa balcooa, Dendrocalamus hamiltonii,, ISSN:2278-5299 82

Melocanna baccifera,, and B. nutans. In the present study, an attempt had been made to apply isozyme markers to evaluate and determine genetic variability and relationship among the selected commercially important bamboos of the region. II. MATERIAL AND METHODS Plant materials Fresh leaves of six bamboos selected for the present investigation were collected from different bamboo growing areas of Manipur and stored at -20 0 C. The plant materials were surface sterilized employing 70% ethanol before using them for isozyme extraction for genetic variability study. Extraction of isozymes Three isozyme systems viz., acid phosphatase, esterase and phosphoenol pyruvate carboxylase (PEPCase) were studied following the method of Weendel and Weeden [20]. Acid phosphatase enzyme was extracted by homogenizing 1gm leaves of bamboo with 5 ml ice-cold citrate buffer (ph 5.3) in a pre-chilled mortar and pestle. The leaf paste was centrifuged at 10000 rpm for 10 minutes at 4 C and the supernatant was collected for isozyme analysis. Esterase enzyme extraction was performed by grinding 1gm of leaf with 5ml of sodium phosphate extraction buffer (ph 9.5) in a pre-chilled mortar and pestle which gave a homogenized green paste. This was followed by centrifugation at 10000 rpm for 10 minutes at 4 C and the supernatant was preserved for isozyme study. For extraction of PEPCase enzyme, 1gm of leaves was grounded in extraction buffer composing of 100mM Tris-HCl (ph 7.8), 10mM MgCl 2, 10mM sodium bicarbonate, 2% PVP, 1mM EDTA and 15mM â- mercaptoethanol. The supernatant was collected after centrifugation of the grounded plant material at 10000 rpm for 10 minutes at 4 C. Data analysis and Scoring The image profiles of banding patterns were recorded and bands were scored based on the presence or absence of visible and reproducible bands (1 for present and 0 for absent). The binary data (1/0) was used to generate a similarity coefficient. Genetic similarity between genotypes was estimated by using Jaccard s coefficient. Pairwise comparisons based on the similarity matrix generated by this analysis were used to generate dendrogram of genetic relatedness by Unweighted pair group method with arithmetic averages (UPGMA). The whole data analysis process was carried out using SeqentiX GelQuest version 3.2.1 software downloaded from www.sequentix.de/gelquest/. III. RESULTS AND DISCUSSION Three isozymes viz., acid phosphatase, esterase and PEPCase were employed to determine the genetic variability and relationship between the six selected commercially important bamboos. Acid phosphatase profile showed colored bands in all the lanes of the six bamboo (Figure 1). The distance matrix derived from the acid phosphatase banding pattern indicated close phylogenetic relationship (0.882) between Melocanna baccifera and Bambusa mizorameana. Bambusa nutans was found to be distantly related not only with bamboos belonging to other genera but also with Bambusa balcooa (Table 1). The dendrogram showed two distinct clusters with, Dendrocalamus hamiltonii and forming one cluster, while and comprising the other cluster (Figure 2). B. nutans appeared to have evolved independently indicating least affinity with the rest of bamboo. Staining of isozymes The extracted isozymes were subjected to Native-PAGE and the gel was treated with corresponding isozyme staining solution to obtain the specific isozyme banding patterns. Acid phosphatase staining was performed by washing the gel in 0.1M acetate buffer (ph 5) about 3-4 times and incubating the gel in solution containing 1-napthyl phosphate (0.05gm), fast blue RR (0.05gm), sodium chloride (1.0gm), 10% magnesium chloride and 0.1M acetate buffer (ph 5). The esterase staining was done by incubating the gel in 100 ml solution consisting of sodium dihydrogen phosphate (2.8gm), disodium hydrogen phosphate (1.1gm), fast blue RR salt (0.2gm) and alpha-naphthyl acetate (0.03gm) for about 20-30 minutes at 37 0 C and keeping in dark till the reddish color bands appeared. For PEPCase enzyme, the gel was treated in staining solution of 100mM Tris-HCl (ph 8.0), 10mM magnesium chloride, 200mM calcium chloride, 10mM sodium bicarbonate and 5mM phosphoenol pyruvate for 30 min at 40 C. Bb Dh Dg Mb Bm Bn Fig.1 Acid phosphatase profile generated for the six bamboo :Bb: Bambusa balcooa; Dh:Dendrocalamus hamiltonii; Dg: Dendrocalamus giganteus; Mb: Melocanna baccifera; Bm:Bambusa mizorameana; Bn: Bambusa nutans Esterase bands were observed for five bamboos with the exception of Bambusa nutans signaling the absence of esterase enzyme in this (Figure 3). The distance matrix obtained from esterase profile showed closest genetic distance (0.840) between Melocanna baccifera and Bambusa mizorameana (Table 2). Dendrogram pattern produced two separate major clusters with and Dendrocalamus hamiltonii grouped in one cluster, while Melocanna baccifera, and constituting another cluster (Figure 4). ISSN:2278-5299 83

Table 1. Distance matrix of six different of bamboo based on Acid phosphatase banding pattern B b D h D g M b B m B n B b 0.000 0.893 0.901 0.925 0.921 1.000 D h 0.893 0.000 0.916 0.920 1.006 0.958 Bb Dh Dg Mb Bm Fig.3 Esterase banding pattern for the five bamboo : Bb: Bambusa balcooa; Dh: Dendrocalamus hamiltonii; Dg: Dendrocalamus giganteus; Mb: Melocanna baccifera; Bm: Bambusa mizorameana D g 0.901 0.916 0.000 0.925 0.942 1.000 M b 0.923 0.920 0.925 0.000 0.882 1.000 43.333 B m 0.921 1.000 0.942 0.882 0.000 1.000 4.485 B n 1.000 0.958 1.000 1.000 1.000 0.000 43.333 42.000 Bm: Bambusa mizorameana; Bn: Bambusa nutans 1.915 3.903 42.000 2.6448 1.4729 2.8209 49.5833 0.7848 45.4657 44.1176 44.1176 44.6809 44.6809 B. nutans Fig.2 UPGMA dendrogram of acid phosphatase banding pattern depicting the genetic relationship of six of bamboos. Table 2. Distance matrix of six different of bamboos based on Esterase banding pattern B b D h D g M b B m B b 0.000 0.866 0.954 0.941 0.978 D h 0.866 0.000 0.940 0.948 0.981 D g 0.954 0.940 0.000 0.923 0.913 M b 0.941 0.948 0.923 0.000 0.840 B m 0.978 0.981 0.913 0.840 0.000 45.903 Fig. 4 UPGMA dendrogram of esterase banding pattern depicting the genetic relationship of five of bamboos. PEPCase banding profile exhibited distinct visible bands for five bamboos under study except for Bambusa mizorameana which showed lightly fainted band (Figure 5). The distance matrix analysis showed closest interspecific relationship (0.861) between B. nutans and while the least interspecific affinity (0.985) was observed between and though they belonged to the same genus (Table 3). The dendrogram study revealed Bambusa balcooa, Dendrocalamus hamiltonii and constituting one major cluster and Melocanna baccifera, Bambusa mizorameana and B. nutans forming the other (Figure 6). Bb Dh Dg Mb Bm Bn Fig.5 PEPCase banding profile for the six bamboo : Dg: D.giganteus; Mb: Melocanna baccifera; Bm: ; Bn: B. nutans Bm: Bambusa mizorameana ISSN:2278-5299 84

Table 3. Distance matrix of six different of bamboos based on PEPCase banding pattern B b D h D g M b B m B n B b 0.000 0.919 0.921 0.924 0.985 0.885 D h 0.919 0.000 0.892 0.965 0.942 0.955 D g 0.921 0.892 0.000 0.966 0.913 0.893 M b 0.924 0.965 0.966 0.000 0.913 0.929 B m 0.985 0.942 0.913 0.914 0.000 0.861 B n 0.885 0.955 0.893 0.929 0.861 0.000 behavior [22] whereas on the basis of biochemical parameters viz., chromatographic separation of phenolic compounds and isozyme patterns, the same genus was separated into four groups [23]. Encountering such taxonomic confusion, Wong [22] concluded that Dendrocalamus genus required further critical study using precise molecular biology methods as discussed above from the studies on Bambusa. Loh et al. [12] through manual AFLP analysis showed 2 of Dendrocalamus as distantly related. In our study Dendrocalamus hamiltonii and appeared in a single cluster in case of PEPCase and acid phosphatase analysis. There is a variation in the observation of Loh et al. [12] having the two Dendrocalamus as separate entities whereas the present report indicated the appearance of the two within one group. Bm: Bambusa mizorameana; Bn: Bambusa nutans 0.9400 0.8800 1.4200 3.0100 46.0900 46.0300 44.6200 44.6200 43.0800 43.0800 B. nutans Fig.6 UPGMA dendrogram of PEPCase banding pattern depicting the genetic relationship of six of bamboos. Identification and genetic relationships in bamboo is very difficult because of the lack of morphological differences and erratic flowering behavior. Authentic identification of taxa is necessary both for breeders to ensure protection of intellectual property right and also for propagators and consumers. The traditional method of identifying by morphological characters is now gradually being replaced by isozyme markers or DNA profiling methods largely because of several limitations associated with morphological data analysis. A comparison of isozyme derived dendrograms of the present investigation with classical taxonomy based plant grouping system showed bamboos belonging to same genus appearing in one cluster indicating enough closeness to warrant monophyletic origin while isozyme marker analysis revealed dissimilarity among the under same genera, thereby indicating polyphyletic origin. Polyphyletic origin of Bambusa genus was also suggested by Loh et al. [12] from AFLP studies with 7 of Bambusa distributing into three different clusters, widely separated from each other. It may be noted that the pattern of distribution in RAPD based dendogram showed 11 under Bambusa genus being distributed in different sub clusters reflecting polyphyletic origin [21]. Dendrocalamus genus was divided into two groups based on vegetative characters, inflorescence IV. CONCLUSION The six important bamboo were differentiated from each other through the study of banding patterns of the three isozyme markers. B.balcooa was distantly related to B.mizorameana and B.nutans as depicted in the dendrogram profiles of the three isozymes. The placing of these three bamboos under the same genus needs further investigation and deeper analysis using sophisticated DNA markers along with in-depth morphological studies. Isozyme analysis showed D.hamiltonii having closer genetic relationship with than though the two under Dendrocalamus have greater morphological similar characters. exhibited closer affinity to as indicated by their grouping in the same major cluster in dendrograms of isozymes under study. It is obvious that in recent years there has been an explosion in the number of different types of genetic markers available and in many cases; the new DNA-based markers provide the same type of information as that of isozymes. 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