Odeigah and Osanyinpeju (1998) were not far from reality when they stated that many indigenous African crop plants which have been used for thousands of years have recently been neglected, due to the introduction of new exotic crops. These 'lost crops' as they are sometimes referred to, have the potential of improving the food security status of developing nations but require scientific attention in order to reintroduce them into farming system. Bambara groundnut (Vigna subterranean L. Verde) is an indigenous tropical African crop. Prior to the introduction of the American groundnut (Arachis hypogaea L.) into Africa, bambara groundnut was thriving as a major crop. Consequently, it is currently classified as one of the world's underutilised crop species. The likely centre of origin of bambara groundnut is in West Africa with North-Eastern Nigeria and Northern Cameroon believed to be the exact places of origin. The crop is also cultivated in Asia, Australia and Latin America.
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Bambara groundnut is herbaceous, intermediate, annual, self-pollinating plant of the family Leguminosae, subfamily Papilionoideae. The plant has bunched leaves arising from branched stems that form a crown on the soil surface. Its podding habit is similar to that of A. hypogaea in that the pale yellow flower stalk bends downwards after fertilization, pushing the young pod into the soil, where it develops and matures (Amadou et al. 2001). For optimal growth and yield, the plant requires a frost-free period of 3.5 months, bright sunshine; average daily temperatures of 20-280C and 750-900mm of evenly distributed rainfall (Khonga et al., 2003).
Bambara groundnut has many nutritional, medicinal and environmental advantages over other crops in the same family. This grain legume is generally cultivated by women subsistence farmers in tropical sub Saharan Africa using unimproved techniques. It is relatively resistant to biotic and abiotic stresses and is said to be the third most important legume after groundnut and cowpea (Vigna unguiculata) (FAO, 2001). Bambara groundnut contributes soil nitrogen for other crops by fixing atmospheric nitrogen through symbiosis with Rhizobium bacteria and is therefore beneficial in crop rotations and intercropping. Chemical analyses of bambara seed have shown that it contains 32.72% of total essential amino acids and 66.10% of total non-essential amino acids. Lysine is the major essential amino acid and represents 10.3% of the total essential amino acid. Bambara groundnut is also a good source of leucine and contains a reasonable amount of phenylalanine, histidine and valine (Ntundu et al. 2004).
Despite the importance of bambara groundnut as a food legume in traditional farming systems, limited breeding efforts have been made to improve this crop. Little information is available about the extent of genetic diversity among bambara landraces, for long term conservation and improvement. The groundnut is still cultivated using landraces with low and unpredictable yields. Drabo et al. (1995) reported that world bambara groundnut production was about 330 000 t with 45-50% of the total world production coming from West Africa. Under various agro-ecological zones, bambara groundnut landraces have been reported to exhibit different grain yield potentials depending on the genotype and heavily influenced by environmental conditions like temperature and rainfall. For example, Khonga et al. (2003) have reported that average yields of bambara groundnut landraces could range between 13.7 kg ha-1 to 177.3 kg ha-1 depending on the genotype.
Till date, bambara groundnut is cultivated from unimproved local landraces exhibiting low yield associated with poor germination percentage and poor crop establishment. Ouedraogo et al. (2008) report that research on bambara groundnut has been very limited compared with investigation made on sorghum, millet, maize, peanut and cowpea. Traditionally, morphological, ecological location and agronomic traits have been used for identification of different genotypes of the crop (Massawe et al., 2005). Efforts to improve the yield potential of bambara groundnut is plagued with limitations arising from inadequate data on the genetic variability and diversity of the crop. Recently, scientists have begun to accumulate agronomic, physiological and molecular knowledge about the crop aimed at reducing the gap between current on-farm yield and its genetic potential.
The use of molecular marker to study genetic diversity of Bamabara groundnut has been reported. Techniques such as RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) have been reported (Massawe et al., 2003; Amadou et al., 2001). In this study microsatellite or simple sequence repeats (SSRs) were used to study the genetic diversity of bambara groundnut. SSRs are reported to be highly polymorphic and co-dominant by Matthews et al. (1999). Knowledge about the diversity of these landraces would provide breeders with specific information required in controlled breeding of genotypes that are adapted to defined environmental conditions. It is also hoped that the results of this study will facilitate the documentation of the genetic diversity available to bambara groundnut breeders and thus assist the reintroduction of the crop into farming systems.
2.0 AIMS AND HYPOTHESIS
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This study aimed at determining the genetic diversity of 24 landraces using microsatellites or simple sequence repeats (SSRs). The study was based on the hypothesis that bambara groundnut landraces cultivated in different agro-ecological zones are genetically unrelated. There is therefore the need to determine the heterogeneity or homogeneity of these landraces.
3.0 MATERIALS AND METHOD
3.1 Plant material
Twenty-four landraces of V. subterranea from different African and Asian countries were kindly provided by the Plant and Crop Science Department, University of Nottingham, Sutton Bonington Campus. These included 8 landraces from Indonesia, 3 landraces from Namibia, 2 each from Cameroon and Botswana, 1 each from Swaziland, Ghana, Zimbabwe, Zambia, Nigeria, Tanzania, Mali, South Africa and Malawi (Table 1). Leaf samples from each landrace were collected for DNA extraction and analysis.
Table 1. Bambara groundnut landraces used and their origin
Name of Landrace
NAME OF LANDRACE
3.2 DNA Extraction
Young leaf tissue (2-5 g) was collected, quickly frozen in liquid nitrogen, transferred to lyophilize (350 µl of lysis solution [Part A] and 50µl of lysis solution [Part B]), and ground with mortar and pestle. Of this material, about 0.3-0.4 g was used to extract DNA using the Sigma GenEluteTM Plant Genomic DNA Miniprep Kit procedure. DNA concentration and visualisation was determined by comparison with lambda DNA standards on 1% agarose gel stained with ethidium bromide (0.5 µg µL-1) as shown in figure 1.
Fig 1: Picture showing DNA present in extracted samples.
3.3 PCR Amplification and DNA Analysis
PCR was carried out in 20-μl volume in a mixture containing 14.4µl sterile water, 2 µl MgCl2 PCR buffer, 0.5µl forward primer, 0.5µl reverse prime, 0.4µl dNTPs, 0.2µl Taq polymerase and 2µl template DNA. DNA amplification was performed as follows: 180 s at 94°C followed by 35 cycles with (i) 60 s at 94°C, (ii) 60 s at 60°C, and (iii) 120 s at 72°C. After the final cycle, the samples were held for 600 s (10 minutes) at 72°C (modified Mayes's Lab Protocol). Amplification products were analyzed by electrophoresis in agarose (2% agarose gel for PCR products, 1% for genomic DNA) gel in a TBE buffer. Permanent records were obtained by photographing ethidium bromide-stained gels under UV light. The 2-Log DNA ladder was used as a standard molecular weight size marker. Figure 2 shows a picture of the PCR products.
Fig 2. Products of our polymerase chain reaction
Capillary electrophoresis was performed using Beckman CEQ 8000 capillary sequencer. Eight random microsatellites of 20-base pair sequences that yielded reproducible and polymorphic products were used (Table 2). These were selected based on microsatellites sizes and mixed such that polymorphic bands of specific sizes could be amplified by respective primers without any overlap.
3.4 DATA ANALYSIS
Following satisfactory reproducibility of the separate DNA extractions and amplification process, the data was analysed using multivariate statistical package MVSP version 3.13q. Based on a procedure described by Kloda et al. (2008) a matrix was constructed counting each individual landrace sample as a single case and each microsatellite allele as a variable, scored as present (1) or absent (0) (see appendix). The composite landraces were presented as a single row. A matrix of allele frequencies was also constructed, representing the frequency of each allele for each landrace. The data sets were explored using Principal Coordinates Analysis (PCO) implemented through the Multivariate Statistical Package (MVSP). This ordination method according to the authors makes no assumptions about the distribution of the variates. Moreover, Euclidian distance was chosen in preference to other distance measures, as it does not class common absence of an allele as a shared characteristic, and was therefore judged to be most appropriate in the present study, which included highly polymorphic microsatellite data characterising bambara groundnut landraces. A dendogram was generated based on Nei and Li's similarity coefficient using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) from a matrix of presence and absence of each allele.
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Table 2: List of the 20-base pair nucleotide primers used and their sizes
Size Rnge (bp)
Results from the cluster analysis are summarised as a dendrogram (Figure 3), showing the relationships among landraces as determined by the similarity between markers. Cluster analysis revealed that genetic distances between all pairs of the 24 bambara groundnut landraces varied from 0.22 to 1.00, with a total average genetic distance of 0.75. Generally, there were two main clusters and cluster 2 was further divided into three sub-clusters depicting varying degrees of genetic diversity between the landraces. Cluster one was made up of 2 landraces namely TVSu 999 from South Africa and Uniswa Red from Swaziland. Cluster 2 was made up of the remaining 22 landraces. There were generally close relationships between landraces from the same location; the sub-clusters of cluster 2 were observed to be grouped according to their geographical origins with landraces from Asia, West and Southern Africa each generally grouped together. GC and BHC, both from Indonesia were seen to be the same landrace but cultivated in different parts of the country with different names. With exception of primer 30 which did not produce clear banding patterns, all the remaining seven primers confirmed this homogeneity (Refer to appendix). Similarly, BH, GH and GCL exhibited similarity coefficient of 1 indicating that these landraces are also the same with different names according to six microsatellites baring primer 4 and primer 30 which was not scored for. A similarity coefficient of 0.92 between the two sub clusters of landraces from Indonesia indicates that these are indeed very closely related. A very interesting observation was the realisation that AS17 from the Republic of South Africa and BCL form Indonesia were the same landraces as amplified by six primers but for primers 16 and 30. These two landraces were observed to be closely related to the Southern African sub-cluster of landraces with a similarity coefficient of over 0.92. This observation is made clear from the PCO scatter plot (figure 4) where BCL is clearly seen to be part of the Southern African group and distinctively away from the Asian group of landraces. Moreover, Dod Red of Tanzania (East Africa) happens to be closely related to Asian landraces than to the African ones whereas VSSP 6 of Cameroon and SB16 5A of Namibia also exhibited close relationship (refer to figure 3 and 4).
Fig 3: UPGMA dendogram of 24 bambara groundnut landraces with Nei' and Li's similarity coefficient
Fig. 4: Euclidian PCO analysis for 24 bambara groundnut landraces. Axes 1 and 2 represent 28.70% and 17.40% of the variation present, respectively, based on an analysis for all the landraces.
The objective of the present work was to examine the amount of genetic diversity and to establish genetic relationships among contrasting bambara groundnut landraces from different growing regions in Africa and Asia using microsatellite markers. Grouping of the bambara groundnut landraces according to geographic origin indicates considerable genetic divergence probably due to different growing environments. Landraces used in this study represented the best possible range of morphological variability and geographical origin available in the cultivated form of bambara groundnut.
In total, 150 different alleles were detected across the 7 microsatellites genotyped on 24 landraces from Africa and Asia. The PCO analysis in Figure 4 suggests three broad groups: they include landraces from West Africa, Southern Africa and Asia. Although some landraces overlapped, these were generally limited with 46.10% of the molecular variation explained by the first two axes. Although, the study demonstrates that there is considerable diversity in landraces of bambara groundnut, some landraces however, produced identical electropherograms suggesting that these landraces are in fact the same genotype which due to their geographical origin have been given different names. The considerable high levels of genetic polymorphism observed in this study indicate that most of these landraces are highly diverse from one other. Massawe et al., (2002) have reported that the major contributory factor to high levels of genetic diversity in many species is the nature of their breeding systems with out-crossing species having higher diversity than self fertilized species. Although bambara groundnut is largely Self-fertilising, the authors reported that cross pollination occurs and is aided by ants.
Whereas some landraces were shown to be of the same genotypes, there was close relationship between some of the landraces in this study, presumably because they were collected from similar locations. From the SSR analyses these landraces may have been derived from the same location. For example TVSu 999 from Zambabwe and Uniswa red from Swaziland are closely related and may be indeed the same. Landraces have local names based on testa colour and their cultivation site. Such an informal taxonomy may lead to one landrace having more than a single name as a consequence of seed introductions from other places. This could be further attributed to the fact that these two countries are neighbours and there is informal movement of people across borders and free trade (Oucho and Crush, 2001). Generally some of the landraces from South African countries, West African countries and Indonesia, revealed close genetic affinities to members from their respective place of origin. An interesting point is that AS 17 and BCL, originally from South Africa and Indonesia respectively, were seen to be the same. Introduction of seeds from one location to the other by crop breeders could be the possible explanation of this occurrence. Also, Azam-Ali and Squire (2002) reported that bambara groundnut that is grown in Indonesia is a result of the slaves who took the crop to Surinam and was further dispersed to areas such as South and Central America, India, Indonesia, Malaysia, the Philippines, Sri Lanka and parts of northern Australia. Additionally, the world is now said to be a global village with free movement of people further enhancing the possibility of dispersion of crops across continents.
The grouping of SB-16 5A from Namibia with VSSP 6 from Cameroon and its subsequent grouping with the West African landraces probably indicates that SB-16 5A also originated from Cameroon or one of the West African states. This is probably because Cameroon forms a transition between West and South Africa and it is possible that genetic material was moved from one country to the other. The observation that, based on the UPGMA analyses, the only East African landrace examined (Dod Red from Tanzania) was not grouped with the African types according to its putative geographical origin but was rather closely related to the Indonesian landraces could also be attributed to the movement of seeds by farmers and plant breeders. Amadou et al. (2001) reported similar findings where it was suggested that genetic material might have been moved from one country to another.
Four landraces from West Africa, six from Asia and seven from South Africa were clustered together showing that SSRs are capable of revealing very close relationships. The genetic variability among groups of landraces suggests that landraces may comprise a mixture of genotypes (Massawe et al., 2002) and this will have implications on landrace stability across variable environmental conditions. In applied breeding, genetic distances have been said to provide predictors for heterosis (Ntundu et al., 2004). Our results for bambara groundnut landraces indicated short genetic distances between some landraces and relatively longer distances between others. This knowledge would provide breeders with specific information required in controlled breeding of genotypes in order to maximise hybrid vigour. For example, to maximize genetic diversity, artificial hybridization should be done between landraces that are genetically distinct (with low genetic similarity values), such as Uniswa Red and VSSP 6 with Nie and Li's similarity coefficient of just 0.22 as revealed by our PCO analyses.
Knowledge about the diversity of crops can be exploited by breeders to control the breeding of genotypes that are adapted to defined environmental conditions.
Molecular markers offer a powerful tool for identification purposes and in breeding programmes. Minor crops, such as bambara groundnut, could benefit from the application of such technologies in molecular breeding (Massawe et al. 2002). The DNA polymorphism detected using SSRs in bambara groundnut in this study further opens the possibility of developing a molecular genetic map that would be useful for genetic enhancement of Vigna subterranean. Using molecular markers could have general application for comparisons to unambiguously distinguish bambara landraces which could subsequently result in the characterisation and the release of bambara adapted to specific agro-ecological zones to maximise yield.
Primer 30 could not produce clear banding patterns and this may be attributable to the specificity of the primer or as a result of the presence of a secondary substance like bubble in the sample. Selection of parents for mapping studies could also greatly benefit from the present study Masawe et al. (2002); however, studies are still needed to compare results from different genetic marker systems. This present study could be enhanced with further research to assess the genetic diversity within the related bambara groundnut landraces using more than one molecular technique. Albeit, our objective of identifying the diversity between the 24 landraces being achieved, our hypothesis of non-relatedness between the landraces was disproved.