Assessment of the genetic diversity of African yam bean ( Sphenostylis stenocarpa Hochst ex . A Rich . Harms ) accessions using amplified fragment length polymorphism ( AFLP ) markers

1 Department of Biotechnology, Bells University of Technology, Ota, Nigeria. Department of Crop Protection and Environmental Biology, University of Ibadan, Ibadan Nigeria. Department of Biological Sciences, Bells University of Technology, Ota, Nigeria. Molecular Virology and Diagnostic Laboratory, International Institute of Tropical Agriculture, Ibadan Nigeria. Department of Biotechnology, Institute of Agricultural Research and Training, Moor Plantation, Ibadan, Nigeria.


INTRODUCTION
African yam bean (AYB) Sphenostylis stenocarpa, (Hochst.Ex. A. Rich) Harms is an important underutilized food crop in tropical Africa, the most economically important species in the genus Sphenostylis (Potter, 1992).It is an inexpensive source of protein (Apata and Ologhobo, 1990), high in sulphur containing amino acid (Ezueh, 1977).The amino acid content in AYB seeds is higher than those in pigeon pea, cowpea, and bambara groundnut (Uguru andMadukaife, 2001, Adewale andDumet, 2009).Despite the nutritional benefits of AYB, it is faced with several production constraints.The presence of high antinutritional factors such as tannins, saponins, oxalate, phytate, trypsin inhibitors and lectin, long cooking time, (Fasoyiro et al., 2006) and low seed yield (Saka et al., 2004) are the major constraints to its cultivation and utilization.The current germplasm base available to breeders for genetic improvement is also narrow.AYB is among the 400 under exploited legumenous crops of the lowland tropics (Rachie and Robert, 1974) and is currently susceptible to genetic erosion, which would undoubtedly reduce the genetic bases and ultimately in the future, it is in danger of extinction (Klu et al., 2001).Utilization of underutilized crops such as AYB could be one of the best strategies to buffer nutritional, environmental and financial vulnerability (Jaenicke and Pasiecznik, 2009).
In order to improve any crop species with valuable genetic attributes by either plant breeders or scientists, germplasm characterization and assessment of genetic diversity are essential prerequisites.The degree of success in the genetic improvement of any crop species to a large extent depends on the amount of genetic diversity existing among cultivated accessions as well as their wild relatives.Such variations would make up a valuable source of potential parents for hybridization and subsequent development of improved cultivars.Cultivars with a great amount of diversity, if collected and appropriately evaluated would provide a great chance for breeders to select and disseminate best performing genotypes (Bainiwal and Jatasra, 1980).Most AYB accessions are in the hands of farmers and little activity has been carried out so far to genetically characterize these accessions.Only a very small sector of the farmers appreciates its cultivation, hence, they are the holder of the crop's genetic resources (Adewale et al., 2012).Explorations to collect more landraces from farmers are necessary to increase the gene pool for crop improvement.The Genetic Resources Centre of the International Institute of Tropical Agriculture only conserves a few accessions.Research on understanding the genetic diversity of AYB accessions through morphological characterization has been reported (Akande, 2008;Popoola et al., 2011;Adewale et al., 2012;Aremu and Ibirinde, 2012).
Accessions are clustered into distinct groups based on the morphological data.Characterizations of morphological traits are however, affected by environmental factors and need to be assessed at particular stages of growth of the crop.It also requires expertise on crop and/or species.
AYB is one of the underutilized crops with a wide genetic base.So far only few studies have been carried out to assess the genetic diversity of AYB using molecular markers.Moyib et al. (2008) used random amplified polymorphic DNA (RAPD) markers to understand the pattern of genetic variability in a few Nigerian AYB accessions.Eleven RAPD primers were used for PCR amplification, but only nine of them produced scorable bands.The results of the study revealed high genetic diversity among accessions of AYB.Adewale (2011) assessed the genetic diversity in 80 AYB accessions using amplified fragment length polymorphism (AFLP) markers with five primer combinations.Very few bands were generated (227) of which only 59 were polymorphic.He obtained an average percentage polymorphism of 25.6% which is extremely low because majority of the bands were monomorphic.The potential of AFLP marker to detect high level of polymorphisms by single reaction has made AFLP as one of the popular molecular tools in understanding the pattern of genetic variability (Vos et al., 1995).AFLP technique can be used to assess genetic variations within a species or among closely related species.
Therefore, the major objective of this research is to assess the genetic diversity in forty (40) accessions of AYB mainly collected from different regions of Nigeria using AFLP molecular markers.

Plant material
27 accessions of AYB were obtained from the Genetic Resources Center of the International Institute of Tropical Agriculture (IITA) Ibadan which consists of 2 accessions from Ghana, 1 from Bangladesh and 24 from Nigeria, with 8 of them collected from Enugu State in South East Nigeria, while 13 accessions were from the Institute of Agricultural Research and Training (IAR&T) Obafemi Awolowo University, Moor plantation Ibadan, all of Nigerian origin (Table 1).Most of the accessions from IAR&T were collected from South West Nigeria except AYB 34, which is from Kaduna State in North West Nigeria (Figure 1).Seeds of each accession were grown in the greenhouse and young leaves from two weeks old plants were collected for DNA extraction.

DNA extraction
DNA samples were extracted from fresh leaf samples using a ZYMO ZR plant/seed DNA Miniprep extraction kit No. D6020 *Corresponding author.E-mail: omenabernojus@gmail.com.Tel : 2348075927894 or 2348185183876.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abbreviation: AFLP, Amplified fragment length polymorphism; AYB, African yam bean; FCO, factorial coordinate analysis; NUS, neglected underutilized species; PIC, polymorphic information content; TSs, tropical sphenostylis stenocarpa; UPGMA, unweighted paired group method using arithmetic average.(Inqaba Biotech, South Africa) following the manufacturer's instructions.The qualities of the extracted DNA samples were checked using agarose gel electrophoresis (1.5%) stained with GR Green (Excellgen, Inqaba Biotechnical, South Africa).The DNA purity and integrity was assessed using Nanodrop spectrophotometer (ND-1000).

Amplified Fragment Length Polymorphism (AFLP) analysis
AFLP analysis was conducted according to Vos et al. (1995).Five AFLP primer combinations were screened and four were selected for PCR amplification (Table 2).Genomic DNA (100 ng/µl) were digested with 1 μL of EcoR1/MseI unit enzymes (Invitrogen AFLP Table 2. Adapters and primers used for pre-amplification and selective amplification of AFLP procedure.

MseI adapter GACGATGAGTCCTGAG TACTCAGGACTCAT
EcoRI primer Core reagent kit) incubated at 37ºC for 5 h after which the enzymes were inactivated at 70°C for 15 min.Pre selective ampli-amplification was carried out by ligating digested fragments with adapters following manufacturer's instruction.10 µL of ligation reaction master mix was added to each of the tubes containing the digested genomic DNAs.PCR amplification was carried out using diluted ligations (1:10) mix.The PCR program in the thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, USA) was denatured for 2 min at 94°C, annealing at 56°C for 60 s and extension at 72°C for 60 s for a total of 25 cycles.Pre-selective PCR products were stored at 4°C.For selective amplification, preselective products were diluted (1:50) and then selective amplification was run using a touchdown programme {94°C for 30 min, 94°C for 30 s, 65°C for 30 s (-0.7 per cycle), 72°C for 60 s for a total of 12 cycles; 94°C for 30 s, 56°C for 30 s and 72°C for 60 s and 72°C for 2 min for a total of 23 cycles}.

Data collection and Analysis
Amplified DNA fragments were separated on a 6% denaturing polyacrylamide gel.The presence/absence of unequivocally scorable bands was transformed into a binary character matrix (1 for presence and 0 for absence of a band at a particular position).
For cluster analysis, pair wise distance matrices were compiled by the NTYSYSpc 2.1 software packages (Rohlf, 2000), using the Jaccard's similarity coefficient (Jaccard, 1908).A dendrogram was constructed by unweighted paired group method using arithmetic average (UPGMA).For Factorial Coordinate Analysis (FCO) DARwin software package Version 5.0.158(Perrier and Jacquemoud-Collet, 2006) was used.Polymorphic information content (PIC) was also calculated using the method described by Anderson et al. (1993) Thus: Where; p i is the frequency of the i th allele.

Polymorphism as detected by amplified Fragment Length Polymorphisms (AFLPs)
The four informative primer combinations generated a total of 1730 amplification fragments across all AYB accessions of which 1647 were polymorphic (95.20%).
The number of amplified polymorphic AFLP bands per primer pair varied from 360 to 520 with an average of 411.75.An average percentage polymorphism of 95.6% was also generated.E-AGC/M-CAG produced the highest number of polymorphic bands 520, followed by E-ACG/M-CAT ( 384), E-ACT/M-CAG (383 bands), and E-AAC/M-CAG (360).Primer combination E-ACG/M-CAT produced the highest number of monomorphic bands (82) (Table 3).Primer combinations E-AAC/M-CAG and E-ACT/M-CAG displayed the highest percentage polymorphism (100%).Polymorphic information content (PIC) values ranged from 0.9447 to 0.9626.

Cluster analysis of AYB accessions
The dendrogram grouped the accessions into two major clusters with an overall similarity of 67.5% (Figure 2).The level of similarity between the accessions ranged from 0.66 to 0.91.TSs 138 and TSs 139 were the most closely related accessions with a similarity index of 0.91.Cluster 1 was further grouped into two sub clusters 1.1 and 1.2.Twenty seven accessions were found to cluster together in sub cluster 1.1, while six accessions were grouped in sub cluster 1.2.Out of these six accessions, a pair of accessions; TSs 68 (from Ghana) and TSs 66 (from Bangladesh), and TSs 107 and TSs 88 (both from Nigeria) were found to be genetically close to each other compared to TSs 78 and TSs 123 which displayed some degree of genetic distinctness.The pattern of clustering of accessions appears to be based on their geographical location.Thus, for example, some of the accessions that cluster together in sub cluster 1.

Factorial coordinates analysis of 40 African yam bean accessions
Factorial coordinate analysis generated by DARwin software version 5.0 using dissimilarity coefficient matrix placed the 40 AYB accessions into four groups (Figure 3).Group 1 includes four accessions (TSs 134, TSs137, TSs 138 and TSs 139).These accessions also appeared as a subgroup in sub cluster 1.  and AYB 34 which was collected from Kaduna (North West Nigeria) displayed some degree of genetic isolation (Figure 2).The other fifteen accessions were found to be included in the 4 th group.The results of both cluster and factorial coordinate analysis showed some resemblance in the pattern of grouping the 40 AYB accessions studied.

DISCUSSION
The present study revealed the extent and organization of genetic diversity within 37 selected accessions of AYB from Nigeria, 1 from Bangladesh and 2 from Ghana assessed with AFLP markers.High levels of genetic variation were observed.Both cluster and FCO tend to group the accessions based on their geographic origin and this might indicate considerable genetic divergence between the accessions reflecting different growing environments.TSs 68 and TSs 123 both from Ghana were clustered in sub cluster 1.2 along with three Nigerian accessions TSs 88 and TSs 107 which showed close resemblance and TSs 78 with some level of genetic distinctness just as TSs 123.TSs 66 from Bangladesh was closely linked with TSs 68; it may have originated from Ghana.From the dendrogram, AYB 1 was found to be closely related to AYB 4. This could suggest that this accession could also be collected from Ado-Ekiti (Ekiti State), South West Nigeria.The same is true for AYB The accessions from Enugu were closely linked in group II of the scatter plot (Figure 3) while the two accessions from Umueze TSs156 and TSs 157 and TSs 140 from Agbani were clearly distinct.The FCO placed the accessions with known passport data into two geographically distinct regions South East (Group II) and (Group III) South West Nigeria.Accessions in Groups IV may have been from South South Nigeria where AYB is also widely cultivated and utilized.
Estimates of genetic distances based on differences in AFLP patterns are informative about genetic diversity (Greef et al., 1997;Powell et al., 1996), phylogeny (Hill et al., 1996) and the geographic origins of genotypes and gene pools of plants (Beismann et al., 1997, Paul et al., 1997).Moyib et al. (2008)  distinct cluster groups at 80% similarity index.The similarity indices ranged from 0.42 to 0.96 which were considered to be useful in facilitating the development of large number of new varieties through hybridization and transfer of useful genes, thus maximizing the use of such available germplasm as genetic resource materials for breeders.
In the work of Adewale (2011), 80 AYB accessions were characterized using five AFLP primer combinations (E-AAC/M-CAG, E-ACT/M-CAG, E-AGC/M-CAG, E-ACG/M-CAT and E-ACG/M-CTG) which generated a low percentage polymorphism of 26% due to the many monomorphic fragments obtained (168).This revealed higher similarities across some of the genomic loci of the tested population of AYB, an indication of low genetic diversity among the collected accessions by Adewale (2011).In the present study using four of the primer combinations, E-AAC/M-CAG, E-AGC/M-CAG, E-ACT/M-CAG and E-ACG/M-CTG, a higher average polymorphic content of 0.96 and percentage polymorphism of 95.6% were obtained which indicates a higher genetic diversity across the genomic loci of the 40 AYB accessions studied.The study also revealed that the 37 AYB accessions collected from different places in Nigeria represent a genetically diverse population.
Genetic fingerprinting using AFLP markers has been reported in various crops; Dolichos Bean (Venkatesha et al. 2010); Velvet bean (Capo-chichi et al. 2003); and in assessment of genetic diversity in sesame (Sesamum indicum L.) (Ali et al. 2007).It has also been used successfully for analyzing genetic diversity in some other plant species such as peanut (Herselman, 2003) and soy-bean (Ude et al. 2003).In these studies different primer combinations were used to assess genetic diversity.The AFLP primer combinations used for AYB in the present study are efficient for genetic diversity studies in the species.AFLP markers are the most ideal genetic markers for analysis of genetic diversity both at intraspecific and interspecific level particularly for closely related species and underutilized crops.
For selection of good parental material for heterosis breeding program, the diversity results obtained through AFLP can be used to correlate with the pedigree relationship and morphological traits for genetic improvement in AYB.Having a good understanding of the degree of genetic relationships among AYB accessions will be of paramount importance for genetic improvement of the crop.

Conclusion
The results obtained from the present study showed the robustness and the suitability of AFLP as a molecular tool for diversity analysis and for the assessment of genetic diversity among individuals of AYB species.The high percentage polymorphism obtained (95.6%), indicates the heterogeneity of the AYB accessions used.Primer combinations E-ACT/M-CAG and E-AAC/M-CAG which produced 100% level of polymorphism are the most useful primer combinations for diversity analysis of AYB accessions.The genetic relatedness among the accessions could provide useful information regarding selecting both potential cross parents and desirable phenotypic traits for breeding programs.

Figure 1 .
Figure 1.African yam bean accession collection sites in Nigeria.

Figure 2 .
Figure 2. Dendrogram of 40 African yam bean accessions revealed by UPGMA cluster analysis using Jaccard's similarity coefficient.

Figure 3 .
Figure 3. Factorial coordinate analysis for African yam bean accessions as generated by DARwin software version 5.0.0.158 using similarity coefficient matrix.

Table 1 .
List of passport data of accessions used in this study.

Table 3 .
Polymorphism obtained from four primer combinations on 40 AYB accessions.