Research Journal of Agriculture and Biological Sciences, 5(1): 63-71, 2009 © 2009, INSInet Publication

Genetic Variation among glabra Cultivars () Detected by Rapd Markers and Isozymes Patterns

Ibtisam Hammad

Botany Dept., Faculty of Science, Helwan University, Egypt.

Abstract. This study was aimed at determining the genetic relationships among the most important Bougainvillea cultivars grown at most of the gardens in Egypt using random amplified Polymorphic DNA (RAPD) technology and isozymes patterns. Ten random decamer primers were screened and five were selected for final RAPD analyses. Each primer produced a unique set of amplification products ranging in size from. Isoenzyme patterns peroxidase, esterase, Poly phenyl-oxidase (PPO) and Alkohol- Dehydrogenase (Adh) were studied in five cultivars of Bougainvillea having different colors (purple, red, orange, white and dwarf ) commonly grown in Egypt. Isozymes revealed higher polymorphism among bougainvillea cultivars with all enzymes used. The cluster analysis based on isozymes data showed that the entire cultivars fell in one group but the purple and red is very close to each other and orange cultivar is the different.

Key words: Genetic variation, Bougainvillea glabra cultivars, RAPD markers and isozymes patterns.

INTRODUCTION characterization of 40 bougainvillea cultivars from eastern Sicily, Italy was carried out genetic variation Bougainvillea is a genus of flowering native among the experimental materials was high due to the to South America from Brazil The name comes from broad genetic base of the population. A dendrogram, Louis Antoine de Bougainville, an admiral in the constructed based on the length of the bracts and French Navy who discovered the plant in Brazil in of the plants, as well as on the yellow: blue 1768. This plant also growing quite well in most of ratio of chromaticity coordinates showed the genetic garden in Egypt with different bright bracts colors. distance among the cultivars examined. (Photo 1). Molecular markers have been shown to be useful The plants are thorny, woody, vines growing for genetic variation of plant [30]. These markers anywhere from 1-12 meters tall, scrambling over other based on the polymerase chain reaction (PCR) plants with their hooked thorns. The thorns are tipped technique which is the most commonly used for these with a black, waxy substance that is easily left in the purposes. Several different PCR- based techniques have flesh of an unsuspecting victim. They are evergreen been developed during the last decade, each with where rainfall occurs all year, or deciduous if there is specific advantages and disadvantages. The randomly a dry season. The are alternate, simple ovate- amplified polymorphic DNA (RAPD) markers acuminate, 4-13 cm long and 2-6 cm broad. The actual techniques is quick, easy and requires no prior of the plant is small and generally white, but sequence information; it detects nucleotide sequence each cluster of three flowers is surrounded by three or polymorphisms using single primer of arbitrary six bracts with the bright colors associated with the nucleotide sequence[31]. plant, including pink, magenta, purple, red, orange, RAPD marker have been extensively used for white, or yellow. Bougainvillea glabra is sometimes DNA fingerprinting[22] Gilbert,[13]. Gilbert et al.[14] and referred to as "paper flower" because the bracts are Gilbert et al.[15] . genetic diversity studies[17,11]. thin and papery. The fruit is a narrow five-lobed population genetic studies[33,23,35]. and Abdel- Tawab et achene. Bougainvillea is rapid growing and flower all al.[1]. Electrophoretic pattern of isozymes, allowing year in warm climates, especially when pinched or direct study of geneproducts, represent also a valid tool pruned. They grow best in moist fertile soil. Bloom in detecting genetic variations. Several studies using cycles are typically four to six weeks. Bougainvillea either starch or acrylamide gel electrophoresis led to grows best in very bright full sun and with frequent the ability to identify bougainvillea varieties by protein fertilization, but the plant requires little water to flower. and isozyme banding patterns[7,8]. Concilio and Leonardi and Romano[20]. reported that Morphological Giovannini, and Quiroga et al.[6,26].

Corresponding Author: Ibtisam Hammad, Botany Dept., Faculty of Science, Helwan University, Egypt. E.mail: [email protected]

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The objectives of this study were to see the and the electrode buffer was Sodium borate, pH 8.0 [25]. genetic relationships among these cultivars of The staining procedures were modified from for ACP, bougainvillea based on the banding patterns of them AKP and PER. Esterase and GOT staining was done using RAPD-PCR and isozymes techniques. according to the methods of Jonathan and Norman[19]. Isozyme patterns were scored visually. Levels of MATERIAL AND METHODS marker polymorphism according to isozyme banding patterens were calculated. Asimilarity dendrogram was Plant Materials: Bougainvillea glabra represented produced using NTSYS (Numerical Taxonomic and family , five cultivars were used in this Multivariate/analysis System) software package, version study with different bracts colors (purple, red, orange, 2.1, Applied Biostatistics Inc[27]. white and dwarf plant). Leave of the five plants were collected from Royal Hills garden in 6 of October city Phylogenetic relationships: The banding patterns of in Egypt, It were freshly harvested for DNA. five RAPD primers were scored and data were inserted in a computer as values of (1) and (0) for the Genomic DNA extraction: For each cultivar, five presence and absence of bands, respectively (Table 2) hundred milligrams of young leaves rinsed with tap The data were analyzed using UPGMA cluster analysis water; frozen in liquid nitrogen; ground to a fine program in order to estimate the similarity indices powder and kept at -80C until use. The genomic DNA among the five genotypes and to develop their was extracted using CTAB method of Doyle and Doyle consensus tree relationships. [9] . with some modifications. After purification, DNA yield and quality were assessed by DNA using a Gene RESULT AND DISCUSSION Quant (Amersham pharmacia Biotech). RAPD- PCR analysis RAPD-PCR analysis: Five decamer oligonucleotides Genotype-specific markers based on RAPD (Table 1), Operon Technologies Inc., Alabameda, CA) analysis:The results of total amplified fragments were used for PCR amplifications according to (TFA), amplified fragments analysis with the 10 W illiams et al.[3 1 ] . with some modification. random primers as shown in Table (1) and Fig. (1). Table. 1: Names, sequences and GC% for the ten random primers Amplification reactions were performed in 25µl used in RAPD- PCR analysis. containing 1X PCR buffer (500mM KCL and 200 mM Serial number Primer code Sequence GC% Tris-Hcl pH 8.4); 1.5mM MgCl2; 100µM of each 1 OPB-02 TGATCCCTGG 70% Dntp; 5 p moles of primer; 1 unit of Taq DNA ------polymerase and 25 ng genomic DNA. The 2 OPC-02 GTGAG GCGTC 70% ------amplifications were performed in a Biometer TI 3 OPC-05 GATGACCGCC 70% gradient thermalcycler programmed for one cycle of 5 ------min at 95°C; followed by 35 cycles of 1 min at 94°C; 4 OPA-03 CAGGCCCTTC 70% ------1 min at 36°C; 2 min at 72°C and a final 10 min 5 OPA-01 GAGGCCCTTC 70% extension at 72°C[4]. Amplification products were analyzed by electrophoresis in 1% agarose; stained with A total number of 255 amplified fragments were ethidium bromide and photographed. The DNA lengths obtained using the five RAPD primers, all primers of the amplified fragments were estimated using 100bp showed high polymorphism which agreed with Welsh ladders (V-gene Biotechnology Limited, Shiqao, P.R. and McClelland[29] . who found that simple and China) reproducible fingerprints of complex genomes can be generated using single 5-me primer and PCR. Hu and Isozyme analysis: Four enzymes systems were Quios [18].stated that Broccoli and cauliflower could be analysed: peroxidase (PER).esterase (EST), Poly distinguished by using only two and three arbitrary phenyl- oxidase (PPO) and Alkaohol- Dehydrogenase primers, respectively. Similarity, Wolf and Rijn[34]. (ADH). All enzymes were studied in , small pieces demonstrated that, only two primers were necessary to of leaf was homogenized in a cool mortar without distinguish 18 Chrysanthemum cultivars. Aruna et al. buffer. Strips of filter paper were used to absorb the [3] .presented a key of black bye genotypes based on 11 cell homogenate and were placed on the origin line of marks amplified by four primers. RAPD assay o the get plates. The plates were stored at 4 C for 30 permitted the genetic variation of five cultivars (purple, minutes, and the filter papers were removed before red, orange, white and dwarf one) by unique positive electrophoresis. Agar- starch- polyvinylpyrolidine gel and negative markers (Table 3,4). Nine positive unique electrophoresis was used[28]. For ACP,AKP, GOT and RAPD markers were identified to characterize the PER, the gel buffer was Tris citrate- NaOH, pH 8.6 selected cultivars.

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Fig. 1: Phylogenetic tree of DNA genome of Bougainvillea glebra have different colors (purple, red, orange, white and dwarf colors) based on RAPD-PCR analysis using 5 primers.

Twenty negative unique RAPD markers identified were detected with genotype (orange color) two to characterize the selected cultivars. Certain primers were positive 400 and 600 (bps) with primer OPB- were more informative than the others (Tables 2 and 3) 02and OPC-05 respectively and four were negative; .the The genotype (purple color) showed eleven 750, 450, 2000,650 (bps) with primers; OPC-02, OPA- specific markers; three positive bands 350, 1200 and 01 and OPC-05 respectively. A third set of 8 DNA 650 (bps) with primer OPB-02, OPA-01 and OPC-05 fragments were detected with the genotype ( white respectively and eight negative markers 800, color) ;one was positive 1200 with primer OPC-05 and 600,400,700,520,280, 500 and 300 (bps) with primers seven were negative 1700,1500,500 with primer OPB- OPB-02, OPC-02, OPA-01and OPA-03 respectively 02, 1500, 280 (bps) with primer OPC-02, 420 bp with which appear only with this cultivar and disappear with primerOPA-01. the others. The genotype (red color) was characterized When made comparison between the DNA by the presence of four unique fragments hat fragment of deeply red dwarf of bougainvillea with disappeared in the DNA extracted from other cultivars. other cultivars found that there were four negative Three positive fragments have approximate sizes of specific DNA fragments which characterized this 600, 300 and 200 (pb) with the primer (OPA-01) and cultivar and positive with other cultivars, these were one negative fragments 1500 bp with the primer OPC- 250 bp withOPC-02, 680 and 680 and 550 (bps)with 05. Another set of one DNA fragment (six fragments) primer OP-01 and 700bp with primer OPA-03. These

65 Res. J. Agric. & Biol. Sci., 5(1): 63-71, 2009 results confirmed the importance of using RAPD similar findings were obtained in a bougainvillea analysis to characterized each genotype with the growing in national park in Indian by Chattejee et al.[5] appearance of specific markers and produce informative and in other genera as Gaboreanu et al.[12] Hassan[16], bands that distinguished all the tested species, Momeni et al.[21].

Table. 2: Number of am plified fragm ents and specific markers of the five cultivars of Bougrainvillea species using RAPD analysis with five primers. Primer TAF 1 2 3 4 5 TSM ------AF SM AF SM AF SM AF SM AF SM OPB-02 14 11 1 8 0 12 1 7 0 8 2 0 ------OPA-03 15 11 0 13 0 13 0 12 0 12 0 0 ------OPC-02 14 10 0 12 0 12 0 9 0 11 0 0 ------OPC-05 13 7 1 8 0 9 0 8 2 6 3 0 ------OPA-01 17 11 1 14 2 12 0 10 0 9 3 0 Total 73 50 3 55 2 58 1 46 2 46 0 8 1 Bougainvillea (purple bract color), (2) B. (red bract color), (3)B (orange bract color), (4) B. (white bract color) and (5) dwarf plant). TAF: total amplified fragments. AF: amplified fragments, SM: specific marker, TSM: total specific markers.

Table. 3: Unique markers characterizing the five genotype of Bougainvillea glebra as revealed by RAPDs. Primer Genotype(Purple) Genotype(red) Genotype(orange) Genotype(white) Dwarf(red) ------UPM UNM UPM UNM UPM UNM UPM UNM UPM UNM OPB-02 350 800 400 1700 1500 500 OPC-02 600 750 1500 250 400 280 OPA-01 1200 700 600 450 420 680 520 300 550 280 200 OPC-05 650 600 380 1200 OPA-03 500 1500 2000 700 300 650 Total 3 8 3 1 2 5 1 6 4 (UPM) Unique positive markers, (UNM) Unique negative markers.

Genetic similarity and cluster analysis based on and could identify a high level of interspecific RAPD markers: The RAPD data were used to polymorphism among the five genotypes. These result estimate the genetic similarities and phylogenetic agreed with Chattejee et al.[15]. The cluster of five relationships among the five cultivars of bougainvillea genotypes was separated within cluster (Fig. 4). In species. The highest similarity index (Table 5 and another words, the purple cultivar was grouped in one Fig.1) was observed between the five cultivars. Using group and all others in cultivars on other group. Also, scoring procedure, all DNA fragments were scored to white color genotype was grouped in subgroup. Also, generate genetic dissimilarity coefficients[24]. This data red color cultivars were grouped in other subdivion. matrix was used to generate the genetics distance matrix. The dendrogram was generated from the Isozymes analysis: In the present study, the genetic distance matrix according to the UPGMA electrophoretic spectra of 4 isozyme systems viz clustering method using the NTSYS program. The five peroxidase, esterase, Poly phenyl-oxidase (PPO) and genotypes were separated in one cluster, within cluster, Alkohol- Dehydrogenase (Adh) in five cultivars of two sub-clusters were obtained, the first one contained Bougainvillea having different bracts colors ( purple, purple color alone, while the second sub-cluster red, orange, white and dwarf) exhibited polymorphism comprised two sub-divisions; the first one obtained of major bands Figs. (5 to 12). A total of 82 bands included white genotype, while the red genotype and consisting of 7 isomorphs were resolved; 19 in purple dwarf genotype appeared in the other subdivision. bracts color cultivar, 18 in red bracts, 14 in orange The aforementioned data asserted that RAPD-PCR one, 13 white bracts cultivar and 17 in dwarf of analysis is good for phylogenetic relationships studies cultivar.

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Table. 5: Sim ilarities m atrix of DNA genom e of Bougainvillea Peroxidase isozymes: The electrophoretic pattern of glebra have different bract colors based on RAPD-PCR peroxidase isozymes of the plants for the five cultivars analysis using five primers. Bract colors Red Orange Dwarf White purple is shown in Fig. (5), their ideograms are shown in Fig. Red 1.000 (6). Twenty- five bands of peroxidase (PX) isozyme ------with Rm values ranging from 0.1 - 0.9 were observed, Orange 0.850 1.000 which were grouped into 7 PX isozyme phenotypes ------Dwarf 0.772 0.865 1.000 (PX1-PX7). A heavily stained monomorphic "fast" zone ------and a more moderately stained polymorphic "slow" White 0.752 0.712 0.739 1.000 zone were observed. Occasionally, lightly stained ------monomorphic zone near the original except the purple Purple 0.705 0.795 0.688 0.646 1.000 bracts co lour heavily stained. Px1 and Px 4 isozyme

Photo. 1: Bougainvillea glabra species with different bract colours.

phenotype viz EST-1 to EST 5 based on their mobility (Rm 0.2-0.7).

Fig. 2: RAPD- PCR analysis of genomic DNA of Bougainvillea glebra have different bract colors. Fig. 3: RAPD- PCR analysis of genomic DNA of Bougainvillea glebra have different bract Lane (M) represented the marker used (phi*174 Hae colors. III), lane (1)purple bract color, lane (2) red bract color, lane (3) orange bract color, lane (4) white bract color Lane (M) represented the marker used ( phi*174 Hae and lane (5) dwarf cultivar respectively, generated by III), lane (1)purple bract color, lane (2) red bract color, primer OPA-03. Lanes from (6- 10) are represented by lane (3) orange bract color, lane (4) white bract color primer OPB-02. and lane (5) dwarf cultivar respectively, generated by primer OPA-01. Lanes from (6- 10) are represented by groups had two specific negative bands in white and primer OPC-02. orange bracts cultivars respectively, where as PX7 present only in purple. Purple bract cultivar have two specific bands; one is negative specific band in EST 1 which detected with Esterase Isozyme: The electrophoretic pattern of all other cultivars, the second is positive specific band Esterase isozymes of the plants for the five cultivars is [10] shown in Fig. (7), their ideograms are shown in Fig. which absent with the rest. El–Demerdash . support (8). The number of isozyme bands range from one these results with on potatoes, showed variations of band in orange bract color cultivar to four bands in some bands between different cultivars of potato which purple bracts color cultivar. Generally thirteen bands of used to identified it i.e., Spunta plants generated in esterase isozymes were observed in five cultivars, culture show 0-2 esterase bands compared to field which were grouped into five esterase isozyme plants showing 2-4 band.

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Fig. 4: RAPD- PCR analysis of genomic DNA of Bougainvillea glebra have different bract colors.

Lane (M) represented the marker used (phi*174 Hae III), lane (1) purple bract color, lane (2) red bract color, lane (3) orange bract color, lane (4) white bract color and lane (5) dwarf cultivar respectively, generated by primer OPC-05.

Fig. 6: Ideogram of Peroxidase isozyme isozyme patterens in five cultivars of Bougainvillea glebra.

Fig. 5: Zymogram of peroxidase isozyme patterns in the bougainvillea cultivars having different bract colors, lane (1) purple bract, lane (2) red color, lane (3) orange bract, lane (4) white color and lane (5) dwarf cultivar.

Alkohol Dehydrogenase (ADH): The electrophoretic Fig. 7: Zymogram of isozyme Esterase patterns in patterns of ADH isozymes and the ideograms of the the bougainvillea cultivars having different leaves for five cultivars are shown in Figs. (9 and 10). bract colors, lane (1) purple bract, lane (2) red ADH isozymes banding patterns were similar in the color, lane (3) orange bract, lane (4) white extracts of five cultivars. The fifth cultivars (dwarf) color and lane (5) dwarf cultivar. was different, showing characterize positive band (ADH6) which was missing with others. Twenty bands of polyphenyl oxidase (PPO) were observed in five cultivars, which were grouped into 7 Poly phenyl oxidase (PPO): The electrophoretic poly phenyl oxidase (PPO) isozymes phenotypes via patterns of ADH isozymes and the ideograms of the PPO1-PPO7 ranging from Rm 0.1 to 0.9. Two distinct leaves for five cultivars are shown in Figs. (11 &12) zones of PPO activity were observed, first zone

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Fig. 8: Ideogram of Esterase isozyme isozyme patterens in five cultivars of Bougainvillea Fig. 10: Ideogram of Poly Phenyl Oxidase (PPO) glebra. isozyme isozyme patterens in five cultivars of Bougainvillea glebra.

Fig. 11: Z ym o gram o f isozyme A lko ho l Dehydrogenase (Adh) patterns in the bougainvillea cultivars having different bract colors, lane (1) purple bract, lane (2) red Fig. 9: Zymogram of isozyme Poly Phenyl Oxidase color, lane (3) orange bract, lane (4) white (PPO) patterns in the bougainvillea cultivars color and lane (5) dwarf cultivar. having different bract colors, lane (1) purple bract, lane (2) red color, lane (3) orange bract, orange bract color cultivar characterize by specific lane (4) white color and lane (5) dwarf positive band with PPO7. While the band PPO2 cultivar. missing in purple, red and dwarf cultivar. Most results published in this respect emphasize comprising of six bands of intermediate mobility (Rm the genetic nature of isozymes of potato. Several 0.2-0.9) were useful in distinguishing the cultivars. isozyme loci have been used repeatedly to detect Polymorphism of polyphenyl oxidase patterns for five genetic differences between several types of plants and cultivars was evident in 6 (PPO2, PPO3, PPO4, PPO5, cultivars as a well as linking such differences to PPO6 and PPO7 out of seven bands detected. The traits[32].

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