According to Arif et al. (2010), appropriate identification and characterization of plant materials are essential for the successful conservation of plant resources and to ensure their sustainable use. Hence, in other to conserve the trees species growing in the study area for posterity, attempts have been made by several researchers (Adekanmbi and Ogundipe, 2009; Shonubi and Okusanya, 2007; Orebamjo and Njoku, 1970) to list and highlight the existing species in the study area. Since effective conservation of plant genetic resources requires a complementary approach which makes use of both ex situ and in situ conservation methods to maximize the genetic diversity available for use, this study aims at conserving the tree genetic resources in northeastern Lagos using DNA banking techniques.

Description of study area

The study was conducted in the University of Lagos campus at Akoka, Yaba, Lagos, Nigeria. The area which is largely surrounded by the scenic view of the Lagos lagoon comprising a total of 802 acres (3.25 km²) of land. It is located on longitude 3° 24' E and latitude 6° 30' N and on elevation of 40-90 m, which makes flooding difficult. The vegetation in this area is half cleared and developed and the remainder is represented by mangrove vegetation and most of the species recorded by Orebamjo and Njoku (1970) have diminished in number and density. It has an undulating terrain, half of which represents buildings, with various fresh water channels and creeks passing across at different location of this area. A large area of mangrove swamps, roughly 50%, dominates the vegetation. In the north and south east lies the brackish water lagoon which supports a typical terrestrial habitat, and experiences less human disturbance while in the south and south west lies the fresh water, where the soil is highly rich and supports a rich flora which is highly favored by the climate type much disturbed by human activities (Figure 1).

Sample collection and identification

This study is based on extensive field surveys conducted in the North-eastern Lagos, Akoka Yaba, Lagos. A Global Position System (GPS) was used during the sampling period. For sample collection, the study area was divided into four sampling plots. Trees were enumerated in 50 x 20 m plots, whereas 0.5 x 2 m quadrat was used to study herbs and grasses. Samples were collected at random within each plot and identified. The assessment of native versus introduced status of the trees was done following Keay (1989) Keay et al. (1964) Hutchinson and Dalziel (1954) and Dalziel (1937). Voucher specimens of all plants have been collected and deposited at the University of Lagos Herbarium, Lagos, Nigeria. For DNA analysis, fresh young leaves, fruit, seed and flower samples were collected and silica-gel was added to the each sample in a zip lock bag and preserved in freezer for molecular analysis.

DNA extraction and purification

Genomic DNA was extracted from fresh leaf samples using the modified cetyltrimethyl ammonium bromide (CTAB) protocol (Doyle and Doyle, 1989). The phenolic compounds were removed by passing the extracted DNA through the vacuum cleaner.

Gel electrophoresis

This involved quality check of the DNA samples on 1% agarose gel. The gel was run on 0.5x tris Borate EDTA (TBE) buffer at 75 V for 1 h 30 min. The gel was visualized by staining with 10 mg/ml ethidium bromide under ultra violet (UV) light and photographed with the gel documentation system (UVitec).

Quantification of DNA samples

This involved the determination of the concentration and relative absorbance of each DNA samples using an Eppendorf biophotometer. It was achieved by mixing 55 μL of sterile water with 2 μL of the DNA sample in a cuvette. The cuvette was then placed in an Eppendorf Biophotometer Plus, and readings were documented at 260 and 280 nm, respectively.

Our sampling showed a total of 66 woody tree species capable of attaining a maximum height of 12 m and girth of 60 cm (Table 1). They are made up of 58 genera which can be grouped into 27 families. Of these, only 42 species are indigenous to the environment (Plate 1) while 24 species are categorized as introduced (Plate 2). Most of the species encountered belong to the legume family Fabaceae (Plate 3) representing 20.89% of total number of species recorded. The vegetation of the study area is undulating, as some species were found existing in higher elevated areas, whereas some were found to exist in low areas.

All the samples yielded good quality DNA with high level of purity. The absorbance ratio of the extracted DNA samples as recorded from the spectrophotometric analysis ranging from 1.74 to 1.84 (Figure 2). Four of the samples however had absorbance ratio of > 1.9 Casuarina equisetifolia, Callophyllum innophylum, Spondias mombin and Tabebuia rosea) showing that impurities might be present in the samples. The concentration of the DNA samples obtained ranges from123 to 1670 ng/µl (Figure 3). All the samples have been deposited in the DNA bank at the University of Lagos, Akoka, Lagos, Nigeria for conservation purposes. 

The tree species encountered in this study have a maximum height of 12 m and girth of 60 cm and this conforms to the definition of trees as stated by Redhead (1971). However, continuous existence of these species is doubtful owing to the fact that nearly all the ancestral vegetation in the study area has been degraded mainly as a result of clearing secondary vegetation and Mangrove forest to build public houses or infrastructures. Also, the drainage patterns are drastically changed as streams are straightened, redirected and made into concretized canals and ditches. When compared with the report given by Orebamjo and Njoku (1970), many species were found to be missing (especially species such as Anogeissus leiocarpus, Triplochyton scleroxylon, Daniellia ogea, Celtis spp. and Entandrophragma spp., Daiella  olivieri, Pterocarpous  spp., Diosporos spp. and Pychanthus spp. Lovoa spp. and Vitex doniana,  among others) suggesting that these trees species are under the threat of anthropogenic human activities, as most of the trees species were probably replaced by buildings  after deforestation.

Although DNA extraction is the first step in every molecular studies research (Qi-Xing et al., 2013), the selection of suitable protocol for DNA extraction from  a specific plant species has always been  problematic, given that some plants are rich in cellulose, polysaccharides, polyphenols, proteins and lipids, which  are responsible for the complication of the nucleic acid separation and purification, and this is mostly associated  with tropical plants (Li et al., 2011; Mohammad et al., 2008; Tan and Yiap, 2009; Sharma et al., 2008; Wang et al., 2008; de la Cruz et al., 1997; Porebski et al., 1997; John, 1992). As a result, wide variety of DNA extraction techniques has been developed (Mohammad et al., 2008) however, this study adopted the CTAB protocol methods of Doyle and Doyle (1987) for the extraction of genomic DNA from the tree species, given that the samples studied are of tropical origin (Qi-Xing et al., 2013; Sahu et al., 2012; Oboh et al., 2009; Ogunkanmi et al., 2008). The study also highlights how rapid and reliable the CTAB protocol is specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident in the absorbance ratio recorded (1.74 to 1.84). This suggests that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds, with minimal contamination (Clark and Christopher, 2000). Hence, the purified DNA was dissolved in buffer and stored at -80°C in the established DNA Bank at the University of Lagos, Akoka, Lagos, Nigeria. The pure DNA extracted is a prerequisite to reliable molecular biology research (Maltas et al., 2011; Pagnotta, 2009; Savolainen et al., 2007; Mace, 2003) including molecular marker study such as AFLP, RAPD or any other PCR, based analysis or research. It could also be used in forensic research, understanding genetic and evolutionary relationships between taxa, functional analysis of genes, comparative genomics research, DNA barcoding and plant breeding. Furthermore, it can be used for studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hybridizations, and for cloning and sequencing; which provide additional option for conservation of biodiversity.

This study is probably the first attempt at using molecular techniques in conserving the flora of northeastern Lagos. Hence, this study has contributed to the genomic conservation of the tree species in Nigeria and the genomic DNA extracted would serve as a bench mark for further researches.

The author(s) have declared that there is no conflict of interests.

The authors thank the Head, Molecular Systematics Laboratory, Department of Botany, University of Lagos, for providing access to the laboratory as well as all the chemicals used in this work, and Prof. J.D. Olowokudejo for internal reviewed of the manuscript and suggestions.  Also, the support of Gashaka Primate Project is highly appreciated.