International Journal of Botany

Year: 2007 | Volume: 3 | Issue: 1 | Page No.: 56-63
DOI: 10.3923/ijb.2007.56.63

DNA Fingerprinting in Utricularia L. Section Utricularia as Revealed by PCR Based Assay

Mohammad Oliur Rahman

Abstract: DNA fingerprinting from Polymerase Chain Reaction (PCR) based assay ISSR (inter simple sequence repeat) has been investigated in order to detect interspecific variation and molecular relationships among nine aquatic species of the section Utricularia. PCR was performed with ISSR primers following agarose gel electrophoresis with ethidium bromide staining. The primer UBC 889 based on (AC)_n repeat presented the highest number of fingerprints while the primer UBC 891 based on (TG)_n repeat produced the least. Utricularia bremii showed the highest number of DNA fingerprints followed by U. gibba while U. aurea showed the least number of bands. The highest genetic affinity was detected between U. gibba and U. bremii and the highest genetic distance was found between U. bremii and U. dimorphantha among the species analyzed. Phenetic analysis based on the ISSR fingerprints using the Neighbour Joining analysis resolved the aquatic species into two groups: i) Utricularia aurea, U. australis, U. dimorphantha, U. intermedia, U. macrorhiza, U. minor and U. vulgaris ii) U. bremii and U. gibba. Principal coordinates analysis in aquatic Utricularia resulted in three clusters and the genetic relationships obtained from this analysis were found consistent with that of Neighbour Joining analysis. Molecular relationships obtained from PCR-based DNA fingerprinting is an agreement of taxonomy of Utricularia based on non-morphological approaches.

Keywords: neighbor joining, ISSR-PCR, genetic relationships, DNA fingerprints, Aquatic Utricularia and principal coordinates analysis

INTRODUCTION

DNA fingerprinting is an important tool for molecular characterization of various groups of plants. It offers a faster and more precise way of determining relationships among closely related species than that of morphological investigation because morphological characteristics are subject to environmental influence and thorough and extensive studies of mature plants are often necessary for taxonomic classification. During planning DNA fingerprinting, one of the most important decision is the marker systems and techniques to be employed. Several DNA marker systems and associated techniques are available today for fingerprinting and many of them are based on Polymerase Chain Reaction (PCR). The commonly used PCR-based techniques include random amplified polymorphic DNA (RAPD; Williams et al., 1990), amplified fragment length polymorphism (AFLP; Vos et al., 1995) and inter simple sequence repeat (ISSR; Zietkiewicz et al., 1994). Among these techniques ISSR appears as an easy, reliable, speedy and highly reproducible. ISSR markers have the ability to detect variation without prior sequence information. They are more advantageous than other DNA markers and overcome many of the limitations of these markers (Pujar et al., 2002). In addition, they are found most economical when a cost comparison was made between RAPD, RFLP (Restriction fragment length polymorphism) and ISSR methods (Yang et al., 1996).

The ISSR assay has been successfully used for DNA fingerprinting (Vijayan, 2004; Pharmawati et al., 2005), genetic diversity (Huang and Sun, 2000; Ge et al., 2005), phylogenetic and species relationships (Joshi et al., 2000; Wolfe and Randle, 2001), germplasm characterization (Moreno et al., 1998; Lanham and Brennan, 1999), cultivar identification (Fang and Roose, 1997; Chowdhury et al., 2002), population studies (Camacho and Liston, 2001; Xie et al., 2005), gene tagging (Akagi et al., 1996; Ratnaparkhe et al., 1998), genome mapping (Sankar and Moore, 2001; Carlier et al., 2004) and biogeography (Smissen et al., 2003).

Utricularia belongs to the family Lentibulariaceae is a carnivorous group of plants and consists of 35 sections (Taylor, 1989). It is found world-wide except for the Antarctic, but the greatest species richness is in tropical regions with the largest number of species originating from Central and South America. They are unique because of the structural complexity of their traps and the rapid movement of the opening and closing of the trapdoors. Many important accounts were made in the genus Utricularia based on morphology, cytology and palynology (Taylor, 1989; Casper and Manitz, 1975; Sohma, 1975). Out of 35 sections reported the section Utricularia includes all aquatic species and the members of this section are found in different ponds, lakes, ditches, marshy areas and other aquatic bodies. There is limited knowledge about the taxa of this section (Meyers and Strickler, 1979; Sasago and Sibaoka, 1985). In comparison with morphological studies molecular works in Utricularia are very scanty (Müller et al., 2002; Rahman and Kondo, 2003). DNA fingerprinting pattern has not been detected in aquatic species to determine genetic variability and to characterize them. Therefore, the present study was undertaken to evaluate the potential of PCR-based ISSR fingerprinting for detecting genetic variation and inferring molecular relationships among aquatic species of the section Utricularia for the first time. The results elucidate that ISSR markers are valuable for determining interspecific variation and molecular characterization in Utricularia.

MATERIALS AND METHODS

Plant materials: The plant materials employed in this study have been listed in Table 1. Plants were grown both in vivo and in vitro culture (B5 medium-Gamborg et al., 1968) at the Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, Japan.

Genomic DNA extraction: Genomic DNA was extracted from the leaf tissue. 1.0-1.8 g of leaf was homogenized in a mortar using liquid nitrogen. The powdered tissue was transferred to a 20 mL capacity of capped, sterilized centrifuged tube containing 10 mL of wash buffer [0.1 M Tris-HCl at pH 8.0, 2% 2-Mercaptoethanol, 1% Polyvinylpyrolidone K-30, 0.05M L-Ascorbic acid, dissolved in distilled water]. After shaking gently for 10 min the tube was centrifuged at 10,000 rpm at 20°C for 10 min. The supernatant was discarded from the tube and this process was repeated until the solution becomes transparent. After removing the supernatant, 10 mL of CTAB buffer [2% Cetyltrimethylammonium bromide (CTAB), 1.4 M NaCl, 0.1% Tris-HCl at pH 8.0, 20 mM EDTA-Na₂, dissolved in distilled water] and 0.5 mL 2-Mercaptoethanol were added to the tube followed by an incubation at 55°C for 60-90 min in order to supply the stabilization of DNA. Following that 10 mL chloroform:isoamylalcohol (24:1) was added to the tube and was shaken gently for 10 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a new sterilized centrifuged tube and it was continued until there was no precipitation on the border of the supernatant layer and chloroform: isoamylalcohol layer. The final supernatant was transferred to another centrifuged tube and 10 mL of 2-propanol was added followed by a centrifugation at 10,000 rpm for 15 min at 4°C. After discarding the solution, 5 mL 70% chilled ethanol was added to wash the pellet and was centrifuged at 10,000 rpm for 5 min. DNA was dried after decanting the ethanol and the dried DNA pellet was dissolved in 450 μL TE solution (10 mM Tris-HCl and 1mM EDTA) with 0.1 mg mL^-1 RNAse (Sigma). After incubation for 1 h at 37°C the solution was transferred in a 1.5 mL sterilized ependorf tube. Two hundred and fifty microlitter of neutral equilibrated phenol and 250 μL of chloroform: isoamylalcohol (24:1) were added to the tube. The tube was centrifuged for 10 min after shaking for 10 min. The upper phase was transferred to a new tube. Five hundred microlitter chloroform:isoamyl alcohol was added and was centrifuged for 10 min. The upper aquous solution was transferred to a new tube. Then 50 μL of 3M sodium acetate and 500 μL of 99.5% chilled ethanol were added to the tube and they were kept at -80°C for 20 min. The DNA pellet was found by spinning in a microcentrifuge at 15,000 rpm for 15 min at 4°C. The supernatant was removed and the pellet was washed with 70% chilled ethanol using centrifugation at 15,000 rpm for 15 min at 4°C. The ethanol was discarded and the DNA was dried into a Halogen Vacuum Concentrator for 3-5 min. The isolated DNA was dissolved in TE buffer and stored at -20°C.

PCR amplification: Seventy-two ISSR primers were screened for PCR amplification of Utricularia DNA, out of which the primers UBC 864[(ATG)₆], UBC 888[(CA)₇BDB], UBC 889[(AC)₇DBD], UBC 890[(GT)₇VHV] and UBC 891[(TG)₇HVH] were finally used because of their ability to produce clear and reproducible patterns of bands (Table 2). The PCR amplification was performed in a 10 μL reaction volume containing 10 ng of template DNA, 1 μM of a single primer (UBC, Vancouver, Canada), 1 μL of X10 Taq buffer, 0.8 μL of dNTP mixture and 0.05 μL of Taq polymerase enzyme and the remaining was filled with deionized distilled water. The mixture was overlaid with 30 μL mineral oil and subjected to PCR. Amplifications were carried out using a PTC-100 thermocycler with an initial denaturation step of 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 45 sec at annealing temperature 50°C and 2 min extension at 72°C. A final extension step for 5 min at 72°C was included. PCR products were visualized using 1.5% agarose gel electrophoresis stained with ethidium bromide. The electrophoretic gels were photographed under ultraviolet radiation.

Data scoring and analysis: Each ISSR band was considered as a character and the presence or absence of the band was scored in a binary code (present = 1, absent = 0). A data matrix was generated using the binary code. Data analyses were performed using the NTSYS-pc (Numerical Taxonomy System, Rohlf, 2000) version 2.1 computer program package. Genetic similarity was measured with the SIMQUAL (Similarity for qualitative data) program that computes similarity coefficients for qualitative data using Simple Matching coefficient. Genetic distance was calculated employing Jukes and Cantor (1969) coefficient and a Neighbour Joining tree was constructed. Among the ordination approaches, the Principal Coordinates Analysis (PCO) was conducted to view the clustering pattern of the taxa more precisely. In PCO, the distance matrix based on Dist coefficient was transformed using the double-centering option, eigenvectors calculated and the multidimensional hyper ellipsoid viewed as a three-dimensional model.

RESULTS AND DISCUSSION

The potential of ISSR fingerprints has been observed for genetic analysis in the section Utricularia. Five ISSR primers employed generated a total of 250 ISSR fragments across nine aquatic species of the section Utricularia. Utricularia bremii presented the highest number of fingerprints followed by U. gibba. Utricularia aurea showed the least number of fragments and U. intermedia and U. vulgaris gave same number of fragments. Figure 1 represents the amplification patterns generated using the primer UBC 864 across nine aquatic species of Utricularia. The primer UBC 889 presented the highest number of bands (Fig. 2) followed by the primer UBC 891 (Fig. 3) in all species. The average number of fragments generated by all analyzed primers was 50.

The similarity index has been calculated in aquatic Utricularia species using Simple Matching coefficient based on ISSR fingerprints. The estimated genetic similarity index indicates that U. bremii was most closely allied to U. gibba among the aquatic species employed. Utricularia australis showed a high affinity with U. dimorphantha and U. vulgaris. U. macrorhiza is closely allied to U. vulgaris while U. intermedia is genetically close to U. minor (Table 3).

The genetic distance in the species studied has been calculated employing Jukes and Cantor coefficient. The highest genetic distance across the species was observed between U. bremii and U. dimorphantha showing that these two species were most far from each other among the species analyzed.

Utricularia bremii also exhibited a high genetic distance with U. macrorhiza. The same genetic distance was found between U. bremii and U. vulgaris and between U. dimorphantha and U. gibba. Among the species analyzed the lowest genetic distance was identified between U. bremii and U. gibba (Table 4).

Cluster analysis of the genetic distance values was carried out to generate a dendrogram indicating relationships between aquatic species studied. A Neighbour-joining (NJ) tree was constructed based on Jukes and Cantor coefficient. The NJ tree resolved that the aquatic species could be placed in two groups: One consisted of Utricularia aurea, U. australis, U. dimorphantha, U. intermedia, U. minor, U. macrorhiza and U. vulgaris and the other group constituted from U. bremii and U. gibba (Fig. 4). The first group is subdivided into two subgroups: one possessed U. aurea, U. australis, U. dimorphantha, U. macrorhiza and U. vulgaris. Utricularia dimorphantha has been found closest to U. australis in this cluster and the other subgroup contained U. intermedia and U. minor.

Principal Coordinates Analysis (PCO) was performed to more effectively view the clustering pattern of the taxa. The PCO analysis of ISSR data in aquatic species resulted in three clusters where Utricularia bremii and U. gibba grouped together, U. minor and U. intermedia formed another cluster and the third cluster consisted of U. aurea, U. australis, U. dimorphantha, U. macrorhiza and U. vulgaris (Fig. 5). The results obtained from the PCO analysis in the aquatic species studied were found coherent to the Neighbour Joining analysis.

DNA fingerprinting employing different marker systems is quite evident these days (Vanderpoorten et al., 2003; Roy et al., 2006). Recent studies revealed that ISSR markers are potential in molecular characterization of different groups of angiospermic taxa viz., Amaranthus (Xu and Sun, 2001), Gossypium (Liu and Wendel, 2001), Tolpis (Mort et al., 2003), Cicer (Sudupak, 2004), Phaseolus (Sicard et al., 2005), Juniperus (Meloni et al., 2006) and Morus (Vijayan et al., 2006). ISSR markers have also been found application in other groups of plant including phytoplankton (Bornet et al., 2004), fern (Thomson et al., 2005) and gymnosperm (Xiao and Gong, 2006).

Many authors paid acute attention on Utricularia and those studies were mainly based on morphological investigations. Taylor (1989) produced an excellent world monograph of Utricularia while Casper and Manitz (1975) did the pioneer chromosomal work of the genus. The pollen morphology was studied extensively by Huynh (1968). However, very little information is available on molecular studies of this genus. Although a previous study demonstrated the genetic variation among some terrestrial species of Utricularia, that study provided no information among the aquatic ones (Rahman and Kondo, 2003).

The present study shows that ISSR markers are very useful for genetic variability and species relationship in Utricularia. DNA fingerprinting from PCR-based ISSR analysis in aquatic Utricularia revealed that U. australis, U. dimorphantha, U. macrorhiza and U. vulgaris grouped together (Fig. 4 and 5).

The close relationship among these species obtained from this work support the previous intrageneric classification of the genus based on morphological characters (Taylor, 1989). Cytologically, Utricularia dimorphantha was found close to U. vulgaris showing the same chromosome numbers. Recently Rahman et al. (2001) counted the somatic chromosome number 2n = 44 in U. dimorphantha which was same as in U. vulgaris (Pogan et al., 1987). The close relationship between these two species from cytological studies has been found coherent with the present investigation. This work further demonstrates that Utricularia bremii is genetically closest to U. gibba among the species studied and morphologically, these species are closely allied. The highest genetic distance among the aquatic species has been detected between U. bremii and U. dimorphantha (Table 4), which is in accordance with the morphology and cytology of these species (Taylor, 1989; Rahman et al., 2001). Hence, the present study has been found corroborated with morphological and cytological investigations indicating the potential of ISSR fingerprints for molecular analysis of the aquatic species of the section Utricularia.

ACKNOWLEDGMENT

I am thankful to Prof. Katsuhiko Kondo, Director, Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, Japan for his cooperation while carrying out this research.

REFERENCES