Agricultural Science Research Journal Vol.

7(3): 96 – 102, March 2017

Available online at http://resjournals.com/journals/agricultural-science-research-journal.html
ISSN: 2026 –6073 ©2017 International Research Journals

Full Length Research Paper

Assessment of genetic diversity in Bambara groundnut

(Vigna subterranea (L.) Verdcourt) landraces in Burkina
Faso using microsatellite markers (SSR)
*Adjima Ouoba1, Serge Félicien Zida1, Mahama Ouédraogo1, Hervé Nandkangre1, Hamed
Mahamadi Ouédraogo3, Romaric Kiswendsida Nanéma3, Nerbéwendé Sawadogo3, Elisabeth P
Zida2, Konaté N’golo Moussa1, Congo Abdou Kader1, Romain W Soalla2 and Mahamadou
Sawadogo3
¹Institut de l'Environnement et de Recherches Agricoles (INERA), Département de Productions Végétales, Laboratoire de
Génétique et de Biotechnologies Végétales, 04 BP 8645 Ouagadougou, Burkina Faso
2
Institut de l'Environnement et de Recherches Agricoles (INERA), Département de Productions Végétales, Laboratoire de
Phytopathologie et de Biotechnologies Végétales, 04 BP 8645 Ouagadougou, Burkina Faso
3
Université OUAGA I Professeur Joseph KI ZERBO, UFR-SVT, Ecole Doctorale Sciences et Technologies, Laboratoire
Biosciences, Equipe de Recherche Génétique et Amélioration des Plantes, 03 BP 7021 Ouagadougou 03, Burkina Faso

*Corresponding Author: Email: adjimaouoba@yahoo.fr, Tel: +226 72 82 30 09 / +26 76 25 23 37

Abstract

Bambara groundnut is an important source of protein to the rural majority in sub-Saharan Africa. However, small
farmers grow locally adapted landraces which are generally low yielding. Adequate knowledge of variability
within Bambara groundnut germplasm collections is crucial for crop improvement and has important
implications for conservation, management and future usefulness of germplasm resources. The objective of this
study was to assess the genetic diversity of Bambara groundnut cultivated in Burkina Faso. A total of 92
Bambara groundnut accessions mainly collected in the three agro-climatic zones of Burkina Faso were estimated
using 10 SSR markers. For all the loci analyzed, results showed a low average value of gene diversity and
polymorphic information content of 0.307 and 0.298 respectively and a low average number of 4.9 alleles per
locus. A Neighbour-joining dendrogram based on the dissimilarity matrix clustered the 92 landraces into two
major mixed groups supported by accessions from the three agro-ecological zones. Thus, the study gave the
genetic diversity profile of the collection which can be used for selection of appropriate parental genotypes for
breeding program.

Key words: Vigna subterranea, accessions, gene diversity, dissimilarity matrix analysis.

Introduction grown in Asia and elsewhere, its cultivation is rare outside

the African continent (Hillocks et al., 2012).
Bambara groundnut (Vigna subterranea (L) Verdc.) is Its seed highly nutritious (63% of carbohydrate, 19% of
originated from the african continent. The exact area of protein and 6.5% of oil) making it a good supplement to
origin of the crop is in the region of northeastern Nigeria diets of a large part of the population, particularly to poorer
and northern Cameroon. This hypothesis is supported by people who cannot afford expensive animal protein
the distribution of wild Bambara groundnut which is (Ouédraogo et al., 2008; Bamshaiye et al., 2011). The
known to extend from Jos Plateau and Yola in Nigeria, to crop is widely adapted to all agro ecological areas of
Garoua in Cameroon (Goli, 1997). Although occasionally Burkina Faso including Sahelian, Sudan-sahelian and
96
Sudanian zones. It is the second most food legume after Environmental and Agricultural Researches and Training
cowpea (Vigna unguculata) and the main food for some Centre in Burkina Faso.
communities in western part of the country (Nadembèga,
2016). Burkina Faso is one of the main producers of the Methods
crop with Mali, Niger, Tchad, Ivory Coast, Ghana and
Nigeria (CIRAD-GRET, 2002, Ouédraogo et al., 2012). In DNA isolation
2014, the crop production was estimated around 50 000
tonnes for Burkina Faso One seed of each accession was sown in an hole (4 cm
(www.fao.org/faostat/en/#data/QC). diameter) of plastic planting plate of 72 holes containing
In most producing countries including Burkina Faso, industrial substrate NFU 44-551 with pH 6.5 (Jardinova
the great majority of the production is consumed locally. Company, France) as a planting medium. Leaves were
Despite the importance of the crop as a food legume in harvested from 10 to 21 days old plants and used for DNA
the country, Bambara groundnut production is limited on isolation. Genomic DNA was extracted following the
small scale (Ouoba et al., 2016). Flinders Technology Associates (FTA) plant cards method
The crop genetic diversity at global scale has been as described by Borman et al. (2008). Plant DNA was
studied by using different markers including nuclear DNA collected on the FTA card by placing the leaf over the
markers (Amadou et al., 2001; Ntundu et al., 2004; Basu card, overlaying it with plastic film and crushing the tissue
et al., 2007; Ouédraogo et al., 2008; Touré et al., 2013; with a hard object and the paper was air-dried for 24 h at
Odongo et al., 2015). However, relatively very few studies room temperature. Using a Harris Micro Punch instrument,
have been conducted using specially SSR markers to disks of 2-mm in diameter were punched out of FTA
assess Bambara groundnut genetic diversity. matrix impregnated with plant material. The sample disks
Nevertheless, these studies showed that SSR markers were placed in separate Eppendorf tubes and were
can be effectively employed to assess the genetic washed with FTA purification reagent. The washed disks
diversity of Bambara groundnut (Basu et al., 2007; were air-dried for 1 h at room temperature and stored at
Molosiwa, 2012; Odongo et al., 2015). Moreover, in –20°C.
Burkina Faso the work undertaken on Bambara groundnut
landraces variability were focused only on Molecular analysis
agromorphological characterization (Ouédraogo et al.,
2008; Déné, 2011; Ouoba, 2012). Also, no study on the Ten microsatellite primers selected for their high
genetic diversity of Bambara groundnut in Burkina Faso polymorphism (Molosiwa, 2012; Odongo et al., 2015)
using SSR markers was reported. (Table 1) were used to assess the genetic diversity of 92
The SSR markers tend to be largely used today in Bambara groundnut accessions. The PCR amplification
plants improvement strategies for their efficiency in was performed in 20 µl of PreMix (Bioneer corp, Republic
diversity assessment. The objective of this study is to of Korea), 2 μl of each primer (1 µM) (Integrated DNA
assess the genetic diversity in Bambara groundnut Technologies,) (foward-reverse) and one disk (2mm) of
landraces in Burkina Faso using microsatellite markers FTA card containing genomic DNA in BIO-RAD
(SSR). thermocycler (BioRad MyCycler PCR System, Texas,
United States). The PCR conditions included an initial
Material and methods denaturation at 94°C for 5 min followed by 35 cycles of
denaturation at 94°C for 30 s, annealing at 54-59.7°C
Plant material (depending on the primer) for 1 min, extension at 72°C for
1 min followed by one cycle of final extension at 72°C for
The plant material consisted of 92 Bambara groundnut 10 min, cooling at 10 °C. The PCR product were
accessions. The great part of them (86 accessions) were separated on agarose gel (2%) (Norgen Biotek Corp,
collected from farmers (March to April, 2014) in different Canada) stained with ethidium bromide (Norgen Biotek
sites situated in the three agro-ecological zones (sahelian, Corp, Canada) under a tension of 100 V during 1h 30 min
sudan-sahelian and sudanian zones) of Burkina Faso. in a 0.5x TBE (Tris-Borate-EDTA) buffer. The separated
Few of them (six accessions) were obtained from the gene amplicons were visualized on a gel visualization system.
bank of Environment and Agricultural Researches Institute The alleles were scored as absent (0) or present (1)
of Burkina Faso (INERA). The study was carried out at the based on the size of the amplified product using a 25 bp
ready to use DNA Ladder (Norgen Biotek Corp, Canada).

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 Table 1: List of 92 Bambara groundnut accessions used in the study and their origin

Accessions N° Number Origin

In-1; In-2; In-3; In-4; In-5; In-6 INERA
06 Genebank(In)
Zs-7; Zs-8; Zs-9; Zs-10; Zs-11; Zs-12; Zs-13; Zs-14; Zs-15; Zs-16 ;Zs-17; Zs-18; Zs- 18 Sahelian
19; Zs-20; Zs-21; Zs-22; Zs-23; Zs-24 zone (Zs)
Zsd-25; Zsd-26; Zsd-27; Zsd-28; Zsd-29; Zsd-30; Zsd-31; Zsd-32; Zsd-33; Zsd-34; Sudan zone
Zsd-35; Zsd-36; Zsd-37; Zsd-38; Zsd-39; Zsd-40; Zsd-41; Zsd-42 18 (Zsd)

Zss-55; Zss-56; Zss-57; Zss-58; Zss-59; Zss-60; Zss-61; Zss-62; Sudan

;Zss-63;Zss-64; Zss-65; Zss-66; Zss-67;Zss-68; Zss-69; Zss-70; Zss-71; ,Zss-72; Sahelian
Zss-73; Zss-74; Zss-75; Zss-76; Zss-77; Zss-78; Zss-79; Zss-80; Zss-81; Zss-82; 50 zone
Zss-83; Zss-84;Zss-85; Zss-86; Zss-87; Zss-88; Zss-89; Zss-90; Zss-91; Zss-92 (Zss)

Table 2: Primer information of ten SSR markers used for amplification of DNA isolated from 92 accessions of Bambara
groundnut germplasm

Markers Forward (F) and reverse (R) primer sequences (5’- Annealing Alleles size (bp)
3’) temperature
(°C)
F : TTCACCTGAACCCCTTAACC
Primer 3 57.6 247
R : AGGCTTCACTCACGGGTATG

F : ACGCTTCTTCCCTCATCAGA
Primer 4 57.6 197
R : TATGAATCCAGTGCGTGTGA

F : TCAGTGCTTCAACCATCAGC
Primer 5 55.3 260
R : GACCAAACCATTGCCAAACT

F : CCGGAACAGAAAACAACAAC
Primer 6 57.6 189
R : CGTCGATGACAAAGAGCTTG

F : TGTGGGCGAAAATACACAAA
Primer 7 59.7 198
R : TCGTCGAATACCTGACTCATTG

F : CAAACTCCACTCCACAAGCA
Primer 8 57.6 250
R : CCAACGACTTGTAAGCCTCA

F : TGACGGAGGCTTAATAGATTTTTC
G358B2-D15 59.0 193
R :GACTAGACACTTCAACAGCCAATG

F : GAGTCCAATAACTGCTCCCGTTTG 59.0
mBam2co80F 220
R : ACGGCAAGCCCTAACTCTTCATTT

F : GAGGAAATAACCAAACAAACC
G180B2-D11 59.0 198
R : CTTACGCTCATTTTAACCAGACCT

F : TGACGGAGGCTTAATAGATTTTTC
G358B3-D15 59.0 196
R : GACTAGACACTTCAACAGCCAATG

98
Data analysis based on Fst was calculated between the clusters using
Genetix 4.04 (Weir et Cockerham, 1984).
Molecular data was recorded in binary fashion for SSR
marker loci analyzed and scoring was based on presence Results
and absence of band for each primer set with one (1) and
zero (0) being the respective scores. Only reproducible Polymorphism
bands were scored. The summary statistics on total
allele’s number (Na), number of effective alleles (Ne), The analysis of ten SSR loci revealed a total of 49 alleles
expected heterozygosity or gene diversity (He) and across the 92 Bambara groundnut accessions and all of
polymorphic information content (PIC) were performed them were polymorphic (Table 3). The number of alleles
using GenALEX version 6.41. (Na) per primer pair varied from 2 (primer 7FR) to 7
Genetic dissimilarity between all the accessions was (primers 3FR and 5FR) alleles with an average value of
calculated with DARwin 6.0.4 (Perrier and Jacquemoud- 4.9. Expected heterozygosity (He) ranged from 0.176
Collet, 2006) using simple matching coefficient. The (primer B2D15FR) to 0.440 (primer 4FR) with an average
dissimilarity coefficients were then used to generate an of 0.307 per primer. Polymorphic information content (PIC)
unweighted neighbor-joining tree with Jaccard’s Similarity ranged from 0.174 (primer B2D15FR) to 0.435 (primer
Coefficient with a bootstrapping value of 1,000 using the 4FR) with an average of 0.298 per primer.
same software (DARwin 6.0.4). Genetic differentiation

Table 3: Estimate of genetic diversity of Bambara groundnut germplasm using ten SSR markers

Markers Na Ne He PIC
Primer 3FR 7 5.335 0.331 0.328
Primer 4FR 4 3.622 0.440 0.435
Primer 5FR 7 4.986 0.276 0.273
Primer 6FR 6 4.175 0.231 0.229
Primer 7FR 2 1.698 0.411 0.406
Primer 8FR 4 2.982 0.337 0.278
G180B2-D11FR 4 2.977 0.265 0.262
B2D15FR 5 3.129 0.176 0.174
OAB3D15FR 4 3.006 0.303 0.299
mBam2co80FR 6 4.357 0.299 0.296

TOTAL 49 36.267 3.069 2.98

MEAN 4.9 3.627 0.307 0.298
Na = number of alleles, Ne= number of effective alleles, He = Expected heterozygosity or Nei’s genetic diversity index, PIC =
polymorphic information content.

Genetic diversity mixture of accessions from different origins. The four

groups define the highest level of genetic divergence of
The dendrogram based on Neighbour-joinning methods the set of accessions. Genetic differentiation indexes (Fst)
partitioned the 92 landraces into four clusters (Figure 1). between the groups showed significant difference
The genetic diversity parameters of each cluster are between the four genetic groups (Table 5). All the clusters
shown in Table 4. Each cluster was composed of a had sub-clusters.

99
 Figure 1: Neighbour-joinning (DARwin version 6.0.4) tree based on allelic data from ten SSR loci among 92 Bambara groundnut
accessions from Burkina Faso.
Cluster given in red was cluster I, Blue cluster II , green cluster III and black cluster IV

Table 4: Diversity parameters of the four genetic groups estimated on GenALEX version 6.41

Clusters N Na Ne P (0,95) He
92 49 - - -
Cluster1 9 18 1.157 26.53% 0.114
Cluster2 44 35 1.337 71.43% 0.219
Cluster3 14 36 1.397 73.47% 0.247
Cluster4 25 42 1.425 85.71% 0.261

Table 5: Genetic differentiation (Fst) of four groups, estimated using Weir and Cockerham (1984) on Genetix version 4.04

Clusters Differentiation index (Fst)

Cluster1 Cluster2 Cluster 3
Cluster2 0.25* 0 -
Cluster3 0.35** 0.16* -
Cluster4 0.37** 0.20* 0.27**
*(high differentiation); ** (Very high differentiation)

The cluster I was the smallest in terms of number of Sudanian zone and 22 from Sudan-Sahelian zone. It was
accessions with a certain genetic proximity. It consisted of subdivided into sub-groups reflecting a high level of
nine accessions including one from the sahelian zone, six variability within this group. This would explain the high
from the Sudanian zone, and two from the Sudan- values of the genetic parameters observed for this group.
Sahelian zone. The parameters of genetic diversity within The cluster III consisted of 14 accessions including two
the accessions of this group yielded 18 alleles in total and from Sahelian zone, three from Sudanian zone and nine
26.53% for polymorphism rate. Expected heterozygosity from Sudan-Sahelian zone. It was organized into
and effective alleles per locus were respectively 0.114 and subgroups and had higher genetic parameters than group
1.57. The parameters for this group were the lowest II, even though the number of its accessions was three
indicating a low variability of the accessions that less than that of cluster II. Despite its lower number of
composed it. accessions, this group had a high potential genetic
The cluster II had the highest number of accessions diversity.
(44) with high diversity parameters. In fact, this group The cluster IV was the most genetically diversified.
displayed 71.43% of polymorphism, a total allele’s number Indeed, it gave the highest genetic parameters with a
of 35 and an effective allele number of 1.337 per locus for number of alleles of 42, a polymorphism rate of 85.71%, a
an expected heterozygosity of 0.219. It included five number of effective alleles of 1.57 per locus and an
accessions from INERA, 12 from Sahelian zone, five from expected heterozygosity of 0.114.
100
Discussion Expected heterozygosity which gave for all accessions
an average of 0.307, was relatively low and indicates a
Microsatellite markers were developed and used to moderate variability within accessions. This value was
evaluate genetic diversity of several leguminous plant close to that of Odongo et al., 2015 who found for this
species such as cowpea (Vigna unguiculata), common parameter an average of 0.345. However this value was
bean (Phaseolus vulgaris), groundnut (Arachis hypogaea lower than that found in common bean (Phaseolus
L.) and in particular Bambara groundnut (Vigna vulgaris) by Blair et al. (2009) who observed an expected
subterranea) (Diouf and Hilu, 2005; Masi et al., 2003; heterozygosity of 0.64 among 604 genotypes analyzed
Basu et al., 2007; Molosiwa, 2012; Odongo et al., 2015, using a set of 36 SSR markers. The size of the population,
Gaikpa et al., 2015). In Burkina Faso, to our knowledge, it the number of markers and the type of species could
is the first time that SSR markers are used to evaluate the explain differences observed. The value relatively low of
genetic variability of Bambara groundnut cultivated in this expected heterozygosity calculated in our study, indicates
country. a moderate variability within accessions.
The 10 microsatellite markers used here showed The structuration of 92 accessions diversity into four
polymorphism among the set of 92 Bambara groundnut genetically distinct groups, obtained from the dendrogram
accessions. According to Shete et al. (2000), the with Neighbor-Joining method gave the links existing
heterozygosity and the polymorphism information content between the accessions. The accessions have been
(PIC) are two distinct values which can be employed to grouped independently from their origins and formed
determine the level of polymorphism in markers. composite groups. The mixture of accessions in all the
According to Botstein et al. (1980), markers with clusters made them more heterogenic groups. The
polymorphic information content above 0.5 are considered grouping of the genotypes of these accessions into sub-
highly informative. The average PIC of 0,298 found among clusters in each cluster indicated substantial level of intra-
the 10 markers in the present study proves that the landrace polymorphism.
majority of markers were slightly informative in this Gathering of the accessions independently of their
germplasm. origins was also observed with several accessions (105)
The average value of PIC (0.298) for this study was from different localities in Kenya (Odongo et al., 2015).
close to that found by Odongo et al. (2015) which was According to these authors, this is explained by the
0.280. On the contrary, this value is very lower than that of existence of a very high flow of accessions following the
Molosiwa (2012) who found an average value of PIC of exchange of seeds between farmers from different
0.670 using 20 markers SSR within 105 Bambara geographically close zones. The autogamous mode of
groundnut accessions. The very high value of the PIC reproduction of the plant could be one of the reasons that
obtained by this author could be explained by the large favored the seeds exchange between farmers.
number of SSR primers selected for their high All the accessions (excepted one) from INERA were
polymorphism after a screening of 75 SSRs gathered in cluster II where the majority of accessions
The number of alleles revealed by the 10 SSRs within from Sudan-sahelian zone are located. The Sudan-
the 92 accessions which was of 49 with an allelic richness sahelian zone would be the origin of accessions from
of 4.9 was lower than that of Ntundu et al. (2004) and INERA. The works of Amadou et al. (2001) and of Ntundu
Amadou et al. (2001). Indeed Ntundu et al. (2004) had et al. (2004), have shown that accessions collected in
detected 346 alleles with an allelic richness of 31.5 within isolated localities or very distant from each other tend to
100 Bambara groundnut accessions using 11 AFLP group together according to their origins. This may be
markers. Amadou et al. (2001) obtained a total of 63 attributed to the fact that movement of plant germplasm
alleles with an allelic richness of 3.7 with 25 accessions between isolated localities may have been limited, which
using 17 RAPD markers. may have created isolation from the rest of Bambara
Several reasons could explain the discrepancies groundnut accessions (Ntundu et al., 2004).
observed on the allele’s number and allelic diversity as
compared to the present study. First, the size of the Conclusion
population used could be one of the reasons. Indeed the
number of accessions (92) used for the present study was The extent of genetic diversity within and among 92
higher than that of Amadou et al. (2001) (25 accessions) Bambara groundnut accessions mainly collected in the
but lower than that of Ntundu et al. (2004) (100 three agro-ecological zones of Burkina Faso has been
accessions). According to Kalinowski (2004) large investigated using 10 SSR markers. All markers showed
samples usually contain more alleles than small samples. relatively low polymorphism within the set of 92 Bambara
In addition the number and the type of markers used could groundnut accessions. The genetic diversity of Bambara
explain the difference observed in the allelic diversity. groundnut evaluated in this study is relatively low among
These authors used respectively FLP markers (17) and the accessions and moderate within them. These
RAPD markers (11 markers). Microsatellite markers landraces constitute an important source of genetic
compared with others markers has many advantages: variability to exploit. The application of cluster analysis
they are specific, highly polymorphic, co-dominant revealed that Bambara groundnut accessions mainly
inherited, multi-allelic, reproducible and reliable grouped into four mixed and composites clusters. The
(Missihoun et al., 2015; Li et al., 2015). genetic diversity profile of the collection given here can be
101
used for selection of appropriate parental genotypes for Kalinowski TS (2004), Counting alleles with rarefaction: private
breeding program. The selection for genetic improvement alleles and hierarchical sampling designs, Conservation
in Bambara groundnut depends on the availability of Genetics 5: 539 -543.
genotypes having a favorable allele for good agronomic Li P, Wang Y, Sun X , Han J (2015). Using microsatellite (SSR)
and morphological markers to assess the genetic diversity of
traits, which depends on the available genetic diversity. 12 falcata (Medicago sativa spp. Falcata) populations from
Thus, it is crucial to investigate about a possible genetic Eurasia. African Journal of Biotechnology 8(10): 2102-2108
relationship between morphological and molecular traits. Missihoun AA, Adoukonou-Sagbadja H, Sedah P, Dagba RA,
Ahanhanzo C, Agbangla C (2015). Genetic diversity of
Acknowledgements sorghum bicolor (L) Moench landraces from Northwestern
Benin as revealed by microsatellite markers. African Journal
The authors are grateful to Mcknight Foundation for of Biotechnology. 14(16): 1342-1353.
funding this research work through the Mcknight 14-152 Molosiwa OO ( 2012). Genetic diversity and population structure
project. The farmers also are highly acknowledged for analysis of bambara groundnuts
(Vigna subterranea (L.) Verdc.) landraces using morpho-
providing major part of accessions used in this study. agronomic characters and SSR markers. PhD thesis,
University of Nottingham. UK. 259p.
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