ABSTRACT

Cowpea, Vigna Unguiculata plays an important role in human diets because of its good protein quality and high nutritional value. On hand are two cowpea lines sourced from a local Zimbabwean humanitarian organization, with an aim to diversify the local germplasm base of cowpea. Activities involved characterizing and evaluating the foreign sourced lines alongside local ones for morphological and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) diversity. Five seed characteristics were evaluated, namely seed size, color, uniformity, presence or absence of seed spots and hilum color using specified scales. For RAPD-PCR analysis, a set of five oligonucleotides was used. DNA extraction, quantification, amplification, electrophoresis and band analysis of presence or absence of bands was carried out. Data was collected and an ANOVA was carried out using genstat discovery software. After an ANOVA, principal components analysis (PCA) and hierarchical cluster analysis were used to examine distribution patterns in the germplasm. Significant differences were observed for the five variables (p˂0.001). Both morphological and molecular data for the cowpea lines show limited diversity from results of multivariate analysis. The dendrogram for morphological data shows an overall similarity index of 78%, whereas for molecular data shows a similarity index of 75%. Of the two foreign lines, the white seeded foreign line, NGO1, seems to be consistently diverse from the rest. It will be a valuable addition to the collection and will be useful for possible introgression in future. The genetic diversity for the collection is generally low and may require continued enrichment with introductions in future.

Key words: Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) characterization, variables, primers, electrophoresis, gel scoring.

Germplasm

 

Twelve lines of cowpea Vigna Anguiculata were used for morphological characterisation and Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. Two of the lines were obtained from a local Zimbabwean humanitarian organisation working in Mashonaland Central. These lines were named NGO 1 and NGO 2 in the study. They were meant for drought relief but however farmers now seem keen to perpetuate them along local landraces. Ten other lines were obtained from the national gene bank, six Zimbabwean landraces, Nhepetera, Chikoromo, Nyadahwa 1381, Zai Redahwa, Tsumbe and Nyadahwa 1444 and an additional four foreign lines also kept at the gene bank namely Red Teak, Tinyawa Kalula, Tinyawa Kongonzi and Indumba. For RAPD-PCR analysis, common bean was used as control. A complete list of the lines is shown in Table 1. A picture of seed taken for each accession in the same order as in the table from left to right is shown in Figure 1.

 

The experiment was carried out in the Biosciences Department, Bindura University of Science Education, Bindura, Zimbabwe. Seed characteristics were scored after which RAPD-PCR characterization was carried out in the laboratory at African institute of Biomedical Research.

 

 

 

Morphological characterisation

 

The lines show diversity in terms of seed characteristics e.g. colour, shape, size etc. Five seed variables namely seed size, seed colour, seed uniformity, hilum colour, presence and absence of seed spots were evaluated using scales specified in Table 2. Five seeds were scored for each entry in order to carry out ANOVA and also calculate means.

 

 

Genstat Discovery Version 4.0 software was used to analyse all the measured traits. The same software was used to carry out the hierarchical cluster analysis and also to establish the level of relatedness among the lines with respect to seed characteristics.

 

RAPD-PCR characterisation

 

The following stages were followed towards RAPD-PCR analysis, DNA extraction, quantification, PCR optimization, amplification, electrophoresis and band analysis.  For extraction, the cetyl trimethylammonium bromide (CTAB) protocol was used for the extraction of DNA from the plant tissues (Harisha, 2007; Hoelzel, 1998).

 

DNA extraction

 

Fresh leaf tissue was obtained separately from the seedlings of the twelve cowpea lines. They were placed in a mortar, liquid N₂ added to lyophilize the plant tissue before grinding to a fine powder with a pestle.  Two milligram of the fine powder was weighed and put into 2 ml Eppendorf tubes. One milliliter of CTAB buffer previously heated at 65°C for approximately 20 min was added. The contents were mixed gently and incubated in the water bath for 2 h at 65°C. The tubes were removed from the water bath cooled down for five   minutes at room temperature five hundred microlitres of chloroform/iso-amyl alcohol (24:1) were added. It was then mixed by gentle inversion or rocking approximately 100 times. The mixture was spinned in a table-top centrifuge for 10 min at 3600 round per minute (rpm). Six hundred microlitres of clear liquid was pipetted and incubated at room temperature for 15 min after which 400 ul of ice cold isopropanol was added.

 

The mixture was mixed by gentle inversion for 15 min till a precipitation was formed and then spinned in a centrifuge at 12 000 rpm for 10 min. The isopropanol was decanted carefully and 1 ml of 70% ethanol added, then mixed by inversion. The mixture was centrifuged at 12000 rpm for 10 min. The supernatant was poured off carefully by inverting the tube to obtain the pellet of DNA. The pellet was dried by leaving the tubes open in the laminar air flow. Hundred microliters of TE buffer of pH 8.0 was added and rocked gently overnight in the cold room to dissolve the DNA.

 

DNA quantification and quality check

 

DNA quantification was carried out using the UV spectrophotometer. For each sample, 5 ul of the DNA were pipetted and mixed with 995 ul of TE buffer in 1 ml quartz microcuvette. The mixture was read at 260 nm against a TE-buffer blank. The actual quantity of the DNA was calculated by converting the optical density (OD) readings to ug/ml (a reading of 1.0 at OD260 is equivalent to 50ug of DNA/ml) (Weising et al., 1995). The conversion also took into account the dilution factor. Finally all the quantified samples were aliquoted into dilutions of 4 ng/ul as required by the PCR conditions used.

 

 

The Random Amplified Polymorphic DNA analysis was carried out using primers that have been successfully used in cowpea by other researchers in various studies predominantly molecular characterisation studies (Sharawy and El-Fiky, 2002; Zannou et al., 2008; Prasanthi et al., 2012). A gel run of the twelve lines plus a common bean control was carried out to determine the success of the extraction process.

 

 

List of primers

 

The list of primers in Table 3 were tried for use in the study. The primers were sourced from University of Cape Town South Africa. 

 

 

The RAPD-PCR procedure

 

The primers were used for both amplification and detection of polymorphisms among the cowpea lines. Temperature cycling was carried out on the Gene Amp 2400 thermocycler. A number of reaction contents and protocols were tried by performing permutations between 3 sets of different reaction contents against 3 sets of different reaction conditions (Nienhuis et al., 1995). Each set of reaction was performed in 10 ul volumes containing, 1X RAPD buffer, 1.1 mM deoxynucleotide triphosphates (dNTPs), 4 mM primer, 1.25 units Taq DNA polymerase and 20 ng of genomic DNA. The only differences among the 3 sets were on the magnesium concentrations.

 

Amplification was performed in 0.2 ml tubes using a thermocycler (Eppendorf, Germany) programmed for initial denaturation at 94°C for 3 min followed by 45 cycles of denaturation at 94°C for 1 min at 35°C for 1 min and 72°C for 2 min. The amplification was completed at 72°C for 7 min and holding temperature of 4°C (Zannou et al., 2008).

 

RAPD-PCR products were separated run on a 1.5% agarose gel (Weising et al., 1995). The molten gel was stained with a 10 mg/ml ethidium bromide stock to a final concentration of 0.5 ug/ml before it had set (Henry, 1997). To each well, 10 ml of the sample in 1X loading dye, were loaded. The samples were electrophoresed in 0.5X TBE buffer at 80 V (1-5 V/cm) for 2 h.

Morphological characterization

 

The lines show diversity in terms of various seed characteristics that were scored namely seed colour, hilum colour, seed size, presence and absence of seed spots and seed uniformity. ANOVA results were generated using two-way analysis of variance (ANOVA) using Genstat Discovery Edition 4 software. The lines showed significant differences with respect to all of the variables namely seed size, seed spots, seed uniformity, hilum colour and seed colour, with p<0.001. Table 4 shows the means, standard error and the p values.

 

The means for the 12 lines are presented in Table 4 for the purposes of multivariate analysis. Principal component analysis (PCA) and hierarchical cluster analysis were used for the construction of a scatter plot and a dendrogram respectively.

 

 

Principal components analysis

 

PCA first converted the five variables to two vectors which can be plotted on a 2 dimensional scatter plot (Figure 2). The scatter plot produced after principal components analysis showed that most of the lines are clustered together in a large group, x-coordinates -2 to 1 and y coordinates -1.7 to 1.2 on the two dimentional plane. This group which is on the left hand plane includes entries, Nyadahwa 1444, Nyadahwa 1381, Tinyawa Kalula, Chikoromo, Red Teak, Zai Redahwa, NGO2, Tsumbe, Tinyawa Kongonzi and NGO2 with three outliers, Indumba, NGO1 and Nhepetera. This is an indication of similarity or uniformity within the group and the outliers may be significantly diverse from the rest. 

 

 

Cluster analysis

 

After hierarchical cluster analysis a dendrogram was constructed and presented in Figure 3. On a similarity scale of 0 to 1, total similarity index for the collection is 0.78. Zai redahwa and NGO2 are the most similar whereas Nyadahwa is the most dissimilar from the rest. Three main clusters are observed labelled C1, containing lines, Chikoromo, Tsumbe, Tinyawa Kongonzi, Zai redahwa and NGO2, C2 containing lines, Red Teak, indumba and Nhepetera and C3 containing lines, Nyadahwa 1444 and tinyawa Kalula. Each cluster has lines which are most related amongst themselves. Nyadahwa and NGO1 do not belong to any particular clusters. 

 

 

Molecular characterisation

 

When the DNA samples were run on the gel  to  ascertain

success of the extraction process, the following gel picture was captured (Figure 4). The DNA shows very faint bands showing some presence of the genetic material.

 

PCR analysis results

 

The banding patterns for primers AP5 and A07 are shown in Figures 5 and 6 respectively. Figure 5 shows DNA polymorphisms detected in the 12 accessions screened using primer AP5. While primer A07 was unable to clearly distinguish these accessions (Figure 6). Primer AP5 managed to detect polymorphic bands showing important variations among these accessions.

 

 

 

 

Principal components analysis

 

PCA converts the 8 alleles into two vectors which were again plotted on a 2 dimensional scatter plot. The scatter plot produced after principal components analysis showed the lines are distributed all over the plane some above   y=0  and  the other group below y=0. Figure 7 shows the scatter plot produced after principal component analysis was carried out.

 

The distribution is highly clustered together when the coordinates are examined which have small intervals although visually they seem widely distributed.

 

 

 

Cluster analysis

 

The dendrogram for RAPD-PCR data shows an overall similarity index of 75% or dissimilarity of 25%. It presents one main cluster containing eight lines (Tsumbe, NGO2, Nyadahwa, Tinyawa Kalula, Nhepetera, Zai Redahwa, Nyadahwa and Tinyawa Kongonzi. Red Teak and NGO 1  are the most dissimilar from the rest while Chikoromo and Indumba are the most similar (Figure 8).

 

 

The distribution pattern for morphological data are observed as different. Despite efforts to enrich a sample of cowpea lines from the gene bank, the genetic diversity is consistently low. It is comparable to literature reports that say genetic diversity in cowpea is generally low in spite of substantial variation seed color, seed proteins, plant type, pod type and seed size among cultivated cowpeas (Sharawy and El-Fiky, 2002). According to Huaqiang et al. (2012) the genetic diversity among cowpea cultivars they studied ranged from 0.1742 to 0.4054 on a scale of 0 to 1. Malviya et al. (2012) reported genetic diversity levels ranging 0.2 to 0.4 on diversity analysis of ten Indian cultivars.

 REFERENCES