415 Advances in Environmental Biology, 7(2): 415-426, 2013 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Comparative study of three species of Malvatheca (Bombacoideae and Malvoideae (Malvaceae sensu lato)using Morphological, Anatomical and RAPD-PCR analyses. 1Wafaa M. Said, 2Nahla O.M. Ehsan, 3Noha S. Khalifa 1,2Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University. 3Botany Dept., Faculty of Science, Ain Shams University Wafaa M. Said, Nahla O.M. Ehsan, Noha S. Khalifa: Comparative study of three species of Malvatheca (Bombacoideae and Malvoideae (Malvaceae sensu lato)using Morphological, Anatomical and RAPD-PCR analyses. ABSTRACT Bombacoideae and Malvoideae are two subfamilies belong to family Malvaceae sensu lato (s. l.). These two subfamilies had long been problematic at the taxonomical level. Moreover, some genera have not been adequately sampled for sufficiently variable molecular markers. In this work, we selected three species (Bombax ceiba, Ceiba pentandra and Ceiba speciosa) that are related to Bombacoideae and Malvoideae. They were mainly selected because they are commonly present in Egypt, and exhibit high medicinal values and contain cotton Silk fiber in their fruits which is valuable in fabric industry. In this study, we compared these three plants from the taxonomical point of view as an initial step to determine their characteristic features, and investigated their genomic DNA banding patterns using randomly amplified bands (RAPD- PCR). This was done not only to determine the phylogenetic relationships but also to find new molecular markers that will help in their specific characterization. Key words: Bombacoideae- Malvoideae- Egypt -RAPD PCR- Bombax ceiba, Ceiba pentandra, Ceiba speciosa. Introduction was the reason to combine Malvaceae s.s and Bombacaceae into Malvaceae s.l. [2,10,19] to form The large family Malvaceae sensu lato the clade Malvatheca [7]. Baum et al [8] observed comprises of approximately 4300 species and 250 that within clade Malvatheca, there are two major genera [11. The recent circumscription recognized lineages: Bombacoideae and Malvoideae. this family on the basis of a number of Bombacoideae includes 14 genera and 160 species morphological and molecular data which divided it from the family Bombacaceae [10]. Bombacaceae is into four traditional families: Tiliaceae, predominately exhibit tropical trees [33,32] that are Bombacaceae, Sterculiaceae and Malvaceae sensu distributed in America, Africa, Asia and Australia stricto (s.s.) [1,2,7,8,10,54,43,41]. In addition, [38]. Malvoideae includes the traditional Malvaceae traditional circumscription dealt with Malvaceae (Mallows or Eumalvoideae) that comprises 78genera (s.s.) as a very homogenous and cladistically and 1700 species [8]. The relationships between the monophyletic group [13,14,15,47,48,51,52], whereas two major lineages: Bombacoideae and Malvoideae it considered the other three families as paraphyletic still largely unresolved and problematic at the [33,32]. Malvaceae s.l. exhibits stellate hairs and taxonomical level [1,2,7,8,11,41]. Thus, the objective palmately veined leaves with inflorescence structures of this study was to determine the interrelationships consisting of bicolor units, valvate sepals, mucilage among the three species from the two subfamilies of cavities within cortex & pith and cyclopropanoid oil Malvacea s.l. Bombax ceiba Linn. (Bombacoideae), seed [10,11,35]. On the other hand, Bombacaceae Ceiba pentandra (L.) Gaertn. and Ceiba speciosa exhibits tree habit, have polyandrous flowers with (A.St. HiL.) Ravenna (Malvoideae) they are unilocular anthers and smooth pollen [14,48,28]. commonly present in Egypt and are famous by their Although, there were a close relationships between medicinal, economical and nutritional values, were Malvaceae s.s. and Bombacaceae, most classification selected for this purpose. systems, [3,11], and references [26,49] have Here, the taxonomical characterization will maintained them as separate families. Recent depend on detailed morphological, anatomical phylogenetic analysis using atpB ,rbcL, and ndhF studies while the molecular characterization will genes from chloroplast and ITS gene from nucleus depend on DNA banding patterns resulted from their Coressponding Author Noha S. Khalifa, Botany Dept., Faculty of Women for Arts, Science, and Education Ain Shams University. E-mail: [email protected] 416 Adv. Environ. Biol., 7(2): 416-426, 2013 genomic DNA using RAPD- PCR primers. This HCl, 0.05 EDTA, 1.25% SDS, pH 8) then incubated study is mainly done to determine the (1) at 60 ºC for 3 h. The temperature of homogenate is phylogenitic relationships & their evolution (2) to brought to room temperature and then 250 µl of cold find new molecular markers that can be attributed in 6M ammonium acetate was added, shacked their specific characterization. vigorously and incubated for 30 min at 4ºC. Samples were centrifuged in (Sigma centrifuge, 2K15, USA) Material and Method at 5000 rpm for 30 minutes at 4 ºC to collect the precipitated proteins and plant tissue. 600 µl of the Plant sample collection: supernatant were recovered into new collection micro tubes containing 360 µl of iso-propanol mix Bombax ceiba Linn. silk-cotton tree, Ceiba thoroughly and allow the DNA to precipitate pentandra (L.) Gaertn., kapok, Ceiba speciosa (A. overnight at -20ºC. Centrifuge for 15 minutes at 5000 ST. Hill.) Ravenna Silk Floss tree were collected of rpm to pellet DNA. The pellet was washed twice in year 2010 from different regions through out Cairo 500 µl of 70% ethanol and centrifuged for 15 city during the vegetative and flowering times minutes at 5000 rpm. The pellet was resuspended in January-March, May-October, July-November of the least amount of ddH2O and left overnight to each plant respectively . dissolve at 4°C. The purity and concentration of extracted genomic DNA were checked by Morphological and anatomical studies: spectrophotometer (PG Instrument Limited, T90+, and U.K.). DNA concentrations were calculated The morphological study of different plant using the formula [DNA = optical density (OD260) × organs was described either directly from the tree at dilution factor × constant (50 μg/ ml)]. DNA samples its location or from fresh specimens. Averages of were diluted to 50-100 ng/µl in sterile distilled water dimension and lengths of leaflet's petiole and and stored at -20 °C. The integrity and concentration petiolule for at least 20 specimens were measured in of the DNA was confirmed by running samples on cm. For anatomical study, cross microtome sections 1% (w/v) agarose gel electrophoresis for 45 min at of 10-20 µm were cut from stems, petioles and 80 V. DNA bands were visualized using UV leaves, stained with safranin - light green mixture transilluminator (Spectroline, TX 312,USA) after following the method described by Johanson [31] staining with ethidium bromide (Bioshop, Canada and photographed under light microscope. The Inc.) morphological and anatomical descriptions were conducted according to Willis (1973) and Metcalfe Optimization of RADP PCR analysis: and Chalk [39], respectively. A total of 20 ten mer RAPD primers were used Morphological and anatomical data analysis: in this study, of which 5 combinations were finally selected based on polymorphism, quality and Morphological and anatomical characters were reproducibility of the amplifications (Table 1). Due given the numerical code 0 or 1 according to their to the high sensitivity of the PCR- RAPD technology absence or presence, respectively. The similarity to changes in experimental parameters, primers were matrix was determined using NTSYS-PC software initially screened against the three plants (bulked (version 2.02, Rohlf, 1998). Phylogenetic leaves DNA). Optimization includes the adjustments dendrogram was generated from the similarity matrix of magnesium, template DNA concentrations, pH following the Unweighted Pair Group Method using values, length of the denaturation stage and primer Arithmetic Averages (UPGMA; Sneath and Sokal;, annealing temperature. The highest performance of 1973). primers is observed at 34 °C. Reactions were performed by volume of 20 μL in 5X Green GoTaq DNA extraction: Reaction Buffer pH 8.5 containing blue and yellow dyes, 7.5 mM magnesium. (M3008 promega, USA), DNA was extracted from fresh young leaves and 2.5 mM dNTPs (Promega, USA), 25 pmol of each was pooled from 20 different plants. Due to the high primers (Metabion International AG, Germany), 3 ng viscosity of the leaf material, commercially available DNA and 0.3 U of Taq polymerase and ddH2O. PCR kits such as DNazol, Trizole and plant DNA thermal cycler (Technie TC-4000, UK) was extraction kit failed to extract high quality DNA for programmed as follows, 94 °C for 5 min, followed PCR analysis. Thus, a new method was adopted here by 35 cycles of 40 sec at 94 °C, 40 sec at 34 °C, and for genomic DNA extraction. Fresh leaves were 3 min at 72 °C. The PCR tubes were kept at 72 °C ground in liquid N2 and 0.3 g from each sample was for 10 min as a final extension step and then stored at homogenized in 0.5 ml extraction buffer (0.1M Tris- 4 °C until analyzed using gel electrophoresis. 417 Adv. Environ. Biol., 7(2): 416-426, 2013 Table 1: List of RAPD primers combination that gave reproducible and polymorphic results with the three plants used in this study. # Primer pair Sequence '5-'3 GC content% A r34 GTCACCGGA 60% r1302.1 GGAAATCGTG 50% B r34 GTCACCGGA 60% OPA1 CAGGCCCTTC 70% C r34 GTCACCGGA 60% OPP2 TCGGCACGAC 70% D r34 GTCACCGGA 60% OPM13 GGTGGTCAAC 60% E r11 CCAAGCAGT 50% r1302.1 GGAAATCGTG 50% Analysis of DNA banding patterns: and needs further revision mainly due to the lack of more detailed systematic and phylogenetic studies. RAPD bands were separated electrophoretically This had greatly deprived these plants from the using 1% agarose gel in 1x TAE buffer, stained with discovery of an adequate molecular marker that can ethedium bromide and photographed on a UV- distinguish them apart.