African Journal of Biotechnology Vol. 10(15), pp. 2811-2819, 11 April, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Assessment of genetic diversity among accessions of
two traditional leafy vegetables (Acmella uliginosa (L.)
and Justicia tenella (Nees) T.) consumed in Benin using
amplified fragment length polymorphism (AFLP)
markers
Adéoti K.1, Rival A.2, Dansi A.3*, Ahohuendo B.C4, Santoni S.5, Beule T.2, Nato A.6, Henry Y.7,
Ahanchédé A.4, Vodouhè R.8, Hounhouigan D.J.4 and Sanni A.1
1
Laboratory of Biochemistry and Molecular Biology, Faculty of Sciences and Technology (FAST), University of AbomeyCalavi (UAC), BP 526 Cotonou, Republic of Benin.
2
Cirad-BioS, UMR DIAPC, Centre IRD. 34394 Montpellier Cedex 15, France.
3
Laboratory of Agricultural Biodiversity and Tropical Plant Breeding, Faculty of Sciences and Technology (FAST),
University of Abomey-Calavi (UAC), 071BP28, Cotonou, Republic of Benin.
4
Faculty of Agriculture (FSA), University of Abomey-Calavi (UAC), BP 526 Cotonou, Republic of Benin.
5
Centre INRA, UMR DIAPC, Unité de Génétique et Amélioration des plantes, Atelier de Marquage Moléculaire, 34060
Montpellier Cedex 01, France.
6
Institut de Génétique et Microbiologie, Bâtiment 360, Université Paris-Sud 11, 91405, Orsay, France.
7
Institut de Biotechnologie des Plantes, Bâtiment 630, Université Paris-Sud 11, 91405 Orsay, France.
8
Bioversity International, Office of West and Central Africa, 08 BP 0931, Cotonou, Republic of Benin.
Accepted 18 February, 2011
Amplified fragment length polymorphism (AFLP) markers were used to evaluate the genetic diversity
and explore the genetic relationship among accessions of Acmella uliginosa and Justicia tenella, two
leaf vegetables collected from different areas in the northwest and northeast parts of Benin (West
Africa). The total number of exploitable amplicons revealed with genomic DNA from A. uliginosa was
224 with an average of 50.5% polymorphic amplicons. Using DNA from J. tenella, we obtained 34% of
polymorphic amplicons from a total of 418. The coefficient of dissimilarity varied from 0.01 to 0.67 and
from 0.17 to 0.62 for Acmella and Justicia, respectively. Low genetic diversity was observed among
Acmella accessions although three distinct clusters could be differentiated. Contrarily, a great genetic
diversity was observed among J. tenella accessions. In addition to this, most of the clusters were
heterogeneous and showed the relationship between accessions collected from northeast and
northwest. Our results confirm the robustness of AFLP techniques for genetic diversity studies and
they provide the first set of molecular data for these two species.
Key words: Amplified fragment length polymorphism (AFLP), genetic diversity, leafy vegetable, Benin.
INTRODUCTION
Traditional leafy vegetables (TLVs) are plants whose
leaves are socially accepted, used and consumed
*Corresponding author. E-mail: adansi2001@gmail.com or
agrobreed.fast@gmail.com.
(Shippers, 2000). In the Republic of Benin, they occur as
cultivated and semi-cultivated crops or weedy and wild
plants, with ecological, social and cultural values, playing
a significant role in the daily food and nutritional
requirements of local people mainly in rural areas, but
more increasingly in urban zones. Recent surveys implemented throughout the country revealed 187 species of
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Afr. J. Biotechnol.
TLVs of interest, among which are Acmella uliginosa and
Justicia tenella (Dansi et al., 2008a, 2009). A. uliginosa
and J. tenella are distributed throughout Africa (Shippers,
2000) where they are known at both cultivated and wild
state. In Benin, these neglected and underutilized species (NUS) are locally cultivated mainly in the northern
part of the country, where they are intensively consumed
(Dansi et al., 2008b; Adéoti et al., 2009). A. uliginosa is a
key nutraceutical for local rural populations. It is used as
an antibiotic, which stimulates milk production and
facilitates the elimination of blood clots in women after
delivery (Dansi et al., 2009). J. tenella is also an
appreciated leafy vegetable. The leaves are cooked and
consumed as spinach. For medicinal purposes, the
leaves are used to treat cardiac disorder, diarrhoea, fever
and indigestion (Denton, 2004; Dansi et al., 2008c). The
plant possesses anti-inflammatory activity and is also
used as antidepressant (Sanmugapriya et al., 2005).
Thus, is of paramount importance that the agro biodiversity of these important NUS be precisely
characterised with the use of modern molecular tools.
Genetic resour-ces must be preserved based on this
accurate characterization for the benefit of local
populations. The assessment of intraspecific genetic
diversity and the understanding of its structure are a
prerequisite for any further action. To the best of our
knowledge, such information has never been reported
on these species.
There are now a number of molecular markers which
have proven very efficient in assessing plant genetic
diversity (Santoni et al., 2000). Among them, amplified
fragment length polymorphism (AFLP) (Vos et al.,
1995) has been found to be the most appropriate in
many cases. AFLPs were described as a powerful and
efficient approach in population genetics and diversity
analysis, molecular taxonomic classification, gene
mapping and marker-assisted breeding for various
crops (Ayele et al., 1999; Carr et al., 2003; Uptmoor et
al., 2003). AFLP analysis provides an effective means of
covering large area of the genome in a single assay
(Ayad et al., 1997; Milbourne et al., 1997; Zhang et al.,
1999; Muminovic et al., 2004). It is highly reproducible
and discriminative (Rafalski and Tingey, 1993; Savelkoul
et al., 1999; Soleimani et al., 2002), and generate a
virtually unlimited number of genetic markers (Blears et
al., 1998; Gaudeul et al., 2000). AFLP has been already
used to assess genetic diversity in many crops such as
hibiscus (Tiang et al., 2003), peach (Xu et al., 2006),
linseed (Adugna et al., 2006), soybean (Tara Satyavathi
et al., 2006), Rice (Mackill et al., 1996), wheat (Shoaib
and Arabi, 2006), sesame (Laurentin and Karlovsky,
2007) and fonio (Adoukonou-Sagbadja et al., 2007).
In this study, we used AFLP markers to assess the
genetic diversity and analyse the relationship among
accessions of A. uliginosa and J. tenella collected from
different agroecological zones of Benin, West Africa.
MATERIALS AND METHODS
Plant material
The plant materials under study consists of seventeen accessions
of A. uliginosa and fourteen accessions of J. tenella collected from
various villages (Table 1) located in the northern part of Benin
(Adeoti et al., 2009). Accessions were maintained as field collections at the Biological Control Station of the International Institute of
Tropical Agriculture (IITA) based in Cotonou, Benin.
DNA extraction
In order to take into account possible individual genetic variability
within each accession, total genomic DNA was extracted from
bulked young leaves (100 to 200 mg FW per accession) collected
from ten 2- to 3-week-old plants. DNA from freshly collected
material was extracted following the MATAB (mixed alkyltriméthylammonium bromide) procedure according to Doyle and Doyle
(1990). After RNAse treatment, DNA content was fluorometrically
quantified (GENIOS PLUS TECAN Scientific Instruments) using
Hoechst 33258 dye and diluted to 25 ng/µl working solution.
AFLP protocol
AFLP analysis was performed as originally described by Vos et al.
(1995) with minor modifications. Here, 250 ng of genomic DNA (10
µl of working solution) were digested using EcoRI and MseI
restriction enzymes and the generated fragments were ligated with
double-stranded site-specific adapters using T4 DNA ligase.
Following ligation, a pre-amplification was carried out with primers
containing one selective nucleotide cytosine and adenine for MseI
and EcoRI primers, respectively. PCR was performed for 30 cycles
which consisted of 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C
with final extension for 3 min at 72°C. Resulting PCR products were
10 times diluted with sterile double distilled water and used as
templates for the selective amplification step. This was carried out
with a couple of selective primers (EcoRI/MseI) containing three
selective nucleotides at the side. EcoRI was labelled with fluorescent dye. Selective amplification was performed on two steps in a
total volume of 20 µl containing 5 µl of diluted pre-selective PCR
product, 2 µl of each primer at 1 pmole/µl for EcoANN and 5
pmole/µl for MseCNN and 0.2 µl of 1 U of Taq polymerase. The first
step of selective amplification consisted of 12 cycles, 3 min at 94°C,
45 s at 94°C, 45 s at 65°C and 1 min at 72°C for final extension.
The second step was performed for 25 cycles which consisted of 45
s at 94°C, 45 s at 56°C, 1 min at 72°C and 3 min at 72°C. The PCR
products of selective amplification were diluted 10 times and an
aliquot (2 µl) of diluted solution was mixed with 18 µl of a ROXlabeled internal size standard (AMM 524). Then the mixture was
denaturated for 5 min at 95°C, loaded and separated on an ABI
PRISM 3130X Genetic Analyzer sequencer (Applied Biosystems).
Scoring and analysis of AFLP data
Electrophoregram generated by the sequencer were analysed
using the GeneMapper version 3.7 software package (Applied
Biosystem, 2004). Clear and unambiguous peaks were considered
as AFLP markers and scored as present (1) or absent (0) in order
to generate a binary data matrix. The total number of scored
markers and the number and percentage of polymorphic markers
were determined for each primer pair used. Polymorphic markers
were used for further data analysis. With the binary matrix (0, 1)
compiled, pairwise relatedness between all accessions was esti-
Adéoti et al.
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Table 1. Studied accessions of A. uliginosa and J. tenella analysed and
corresponding collecting sites.
Species
Acmella
uliginosa
Justicia tenella
Accession
number
AA1
AA2
AA3
AA4
AA5
AA6
AA7
AA8
AA9
AA10
AA11
AA12
AA13
DA1
DA2
DA3
DA5
Collecting
sites
Boukoumbé
Koutagou
Cobly
Souomou 1
Tiélé
Tchakalakou
Péporiyakou 1
Pam-Pam
Dangoussar
Bajoudè
Koupagou
Péporiyakou 2
Souomou 2
Djougou
Belléfoungou
Borondy
Kawado
AJ1
AJ4
BJ1
BJ2
BJ3
BJ4
BJ5
DJ1
DJ2
L1S
LJ2
LJ3
LJ4
Nouagou
Péporiyakou
Sonoumon
Bori
Wèrèkè
Boroyerou
Ina
Nalohoun I
Dangoussar
Gogounou
Toumè
Sérou
Guéssou-sud
mated using Dice index of similarity (Dice, 1945). Using DARwin5
software package Version 5.0.158 (Perrier and Jacquemoud-Collet,
2006) and the Neighbor-joining method, a dendrogram was
generated with the aim of analysing the relationship between
accessions. The binary matrix was also used to undertake a
factorial coordinate analysis (FCA) with the same software in order
to obtain a graphical representation of the genetic diversity patterns
existing among accessions.
RESULTS
Diversity among A. uliginosa accessions
A total of four pairs of primer combinations were
screened. Among these, only three generated useful
amplification products with a high polymorphism; these
County
Region
Atacora
Atacora
Atacora
Atacora
Atacora
Atacora
Atacora
Atacora
Atacora
Donga
Atacora
Atacora
Atacora
Donga
Donga
Donga
Donga
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Northwest
Atacora
Atacora
Borgou
Borgou
Borgou
Borgou
Borgou
Atacora
Atacora
Borgou
Borgou
Borgou
Borgou
Northwest
Northwest
Northeast
Northeast
Northeast
Northeast
Northeast
Northwest
Northwest
Northeast
Northeast
Northeast
Northeast
were selected for DNA profiling. Sequences of selected
primers, total number of generated markers and associated polymorphism are shown in Table 2. The number
of amplicons obtained per primer combination ranged
from 51 to 88 and the percentage of polymorphic amplicons generated by each primer combination ranged from
30 to 71% (Table 2). A total of 224 amplicons (50 to 500
pb) was generated, from which 50.50% (114 amplicons)
were found to be polymorphic. The genetic dissimilarity
index calculated between accessions ranged from 0.01 to
0.67 (Table 3). The lowest value was obtained between
AA12 collected at Perporiyakou 2 and AA11 collected at
Koupagou, while the highest value was calculated
between AA1 and AA7 sampled at Boukoumbé and
Péporiyakou1, respectively. The generated dendrogram
based on the dissimilarity matrix (Table 3) using the
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Table 2. Number of AFLP amplicons and corresponding rate of polymorphism for the two species under study.
Percentage of
polymorphism (%)
88
85
Number of polymorphic
amplicons
27
61
EcoACG TAM / MseCAG
51
26
50
EcoACT FAM / MseCAT
EcoACG TAM / MseCAG
EcoACG / MseCTA
Eco ACA / MseCAA
EcoAGG / MseCTC
92
92
93
81
60
15
45
31
27
26
16
49
33
33
43
Species
Primer combination
A. uliginosa
EcoACT FAM / MseCAT
EcoAAG HEX / MseCTC
J. tenella
Number of
amplicons
30
71
Table 3. Dissimilarity matrix between accessions of A. uliginosa as revealed by AFLP markers.
AA2
AA3
AA4
AA5
AA6
AA7
AA8
AA9
AA10
AA11
AA12
AA13
DA1
DA2
DA3
DA5
AA1
AA2
AA3
AA4
AA5
AA6
AA7
AA8
AA9
AA10
AA11
AA12
AA13
DA1
DA2
DA3
0.41
0.41
0.51
0.48
0.52
0.67
0.48
0.40
0.43
0.43
0.44
0.46
0.49
0.46
0.49
0.39
0.04
0.08
0.05
0.22
0.49
0.09
0.03
0.03
0.02
0.03
0.07
0.07
0.05
0.07
0.05
0.10
0.09
0.19
0.45
0.10
0.03
0.07
0.04
0.05
0.10
0.10
0.07
0.09
0.04
0.06
0.23
0.50
0.11
0.07
0.08
0.08
0.09
0.07
0.05
0.05
0.03
0.09
0.23
0.49
0.08
0.06
0.05
0.06
0.08
0.06
0.05
0.03
0.04
0.08
0.37
0.23
0.19
0.23
0.22
0.23
0.24
0.24
0.22
0.23
0.19
0.50
0.45
0.50
0.50
0.50
0.48
0.51
0.50
0.50
0.45
0.07
0.08
0.09
0.09
0.09
0.09
0.08
0.10
0.09
0.05
0.03
0.04
0.07
0.07
0.04
0.06
0.02
0.03
0.05
0.08
0.08
0.05
0.07
0.06
0.01
0.07
0.07
0.04
0.06
0.04
0.07
0.07
0.05
0.08
0.06
0.03
0.04
0.05
0.09
0.04
0.04
0.09
0.02
0.06
0.08
neighbour-joining approach of the UPGMA method
showed three distinct clusters (Figure 1):
1. Cluster
I contains accessions collected at
Djougou,Borondy, Bellefoungou (County of Donga) and
at Souo-mou, Tiele and Pam Pam (East of the County of
Atakora).
2. Cluster 2 groups together accessions from Dangoussar, Cobly and Kawado located along the Benin/Togo
border.
3. Cluster 3 joins individuals collected at Perporiyakou1,
Boukoumbé and Tchakalakou situated at the centre of
the County of Atakora.
Axis 1 of the factorial coordinate analysis (FCA) separates the accessions analysed into two groups (Figure 2)
of which one is exactly cluster 2 and the second one, the
other accessions (Cluster 1 and Cluster 3) which are not
structured into differentiated groups.
Diversity among J. tenella accessions
Five primers combinations were used to assess the
genetic diversity among J. tenella accessions (Table 2). A
total of 418 fragments were produced among which, only
144 were found to be polymorphic. The average polymorphic rate was 34%. The number of amplicons per
primer combination ranged from 60 to 93 and the
percentage of polymorphic amplicons varied from 16 to
49% (Table 2). The dissimilarity indexes among accessions varied from 0.17 to 0.62 (Table 4). The lowest
Adéoti et al.
I
II
III
Figure 1. Neighbor-joining analysis of A. uliginosa accessions. The dendrogram was generated from UPGMA cluster analysis
of dissimilarity data.
Figure 2. Factorial coordinate analysis for A. uliginosa accessions as generated by DARwin software using
dissimilarity coefficient matrix calculated from AFLP data.
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Table 4. Dissimilarity matrix of J. tenella accessions based on AFLP data.
AJ1
AJ4
BJ1
BJ2
BJ3
BJ4
BJ5
DJ1
DJ2
L1S
LJ2
LJ3
AJ4
0.52
BJ1
0.52
BJ2
0.52
0.40
0.37
BJ3
0.51
0.35
0.30
0.37
BJ4
BJ5
DJ1
0.51
0.51
0.51
0.37
0.46
0.44
0.32
0.39
0.45
0.42
0.54
0.48
0.34
0.45
0.47
0.41
0.44
0.45
DJ2
0.59
0.54
0.53
0.53
0.52
0.62
0.60
0.55
L1S
0.48
0.51
0.49
0.50
0.48
0.51
0.53
0.51
LJ2
0.56
0.23
0.24
0.44
0.37
0.38
0.45
0.48
0.55
0.50
LJ3
0.53
0.19
0.21
0.39
0.31
0.36
0.44
0.43
0.55
0.51
0.27
LJ4
0.55
0.26
0.29
0.46
0.36
0.39
0.47
0.50
0.55
0.52
0.32
0.27
LJ5
0.48
0.46
0.39
0.51
0.46
0.44
0.31
0.46
0.57
0.52
0.46
0.44
LJ4
0.17
similarity index (the highest dissimilarity) was found
between the accession collected at Boroyerou (County of
Borgou) and the one sampled at Dangoussar (County of
Atakora). The highest similarity index was calculated
between the individual taken at Péporiyakou (Atakora)
and the one collected at Sonoumon (Atakora). The dendrogram revealed three genetic groups, designated as I,
II and III (Figure 4). Apart from the group III which joins
two accessions from the County of Borgou, the other two
groups are composed of samples from both Borgou and
Atakora.
DISCUSSION
The amplified fragment length polymorphism (AFLP)
analysis revealed a high level of similarity between
accessions of A. uliginosa. This is an indication of low
genetic diversity among the collected accessions. Cluster
II of the UPGMA dendrogram (Figure 1) based on the
dissimilarity matrix assembles individuals from Dangoussar, Kawado and Cobly, three geographically distinct
bordering villages of Togo (Figure 3). Somehow, these
results are in agreement with the farmers’ assumptions
which depict this species as originating from Togo (Adéoti
et al., 2009). Three accessions from this cluster which
were collected at Péporiyakou, Boukoumbé and Tchakalakou seem to be genetically different. They probably
originate from another ancient introduction of the species
into Benin from Burkina Faso. A second hypothesis
suggests the existence of pure specimen or hybrids from
Acmella oleracea, a wild relative of A. uliginosa which is
well known by farmers. Nevertheless, a precise
morphological examination of these three samples by
taxonomists from the National Herbarium of Benin confirmed their identity as A. uliginosa, which makes more
0.50
0.46
plausible our first hypothesis. In fact, the Wama, Ditamari
and Natimba ethnic groups from these villages are all
originating from both Burkina Faso (mainly) and Togo
(Adam and Boco, 1993). The use of this species by the
Gourmantché ethnic group (from Burkina Faso), the
recent findings on the multiple origins of fonio in Benin
(Adoukonou-Sagbadja et al., 2006; Adoukonou-Sagbadja
et al., 2007; Dansi et al., 2010) as well as the grouping of
Benin ethnic groups based on their origin with regard to
the leafy vegetables species they consume (Dansi et al.,
2008a), concomitantly support this hypothesis. Then
diversification observed among A. uliginosa’ accessions
could be related to people migration through this part of
country.
Contrary to A. uliginosa, no clear genetic structuring
could be obtained within the accessions of J. tenella
(Figure 4) despite the high level of amplicons generated
per primer. Other AFLP markers could be tested to
confirm once again this result. The two major clusters
assemble individuals collected from Atakora as well as
those from Borgou. Therefore, the accessions collected
from the Northwest were not genetically very different
from the ones collected from the Northeast. This result
was rather expected when our previous report is considered (Adéoti et al., 2009). Indeed, we described this
species as mainly located in the Northeast with a spread
towards the Western region resulting from the migration
of people.
Conclusion
Results from the present study confirm the robustness
and the suitability of the AFLP approach for plant
diversity analysis and for the assessment of genetic
relationship among individuals of a given species
Adéoti et al.
Figure 3. Sampling sites for A. uliginosa (L.) and J. tenella (Nees) T in Northern Benin.
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I
II
III
Figure 4. Neighbor-joining analysis of J. tenella accessions. The dendrogram was generated from UPGMA cluster analysis
of dissimilarity data.
collected from different locations. We have applied this
technique to two different neglected and underutilized
species from Benin (A. uliginosa L. and J. tenella (Nees)
T.) for the first time. More investigations are needed to
clarify the origin (single or multiple) of A. uliginosa in
Benin taking into account accessions from Togo and
Burkina Faso. Likewise, other molecular markers such as
ISSR or microsatellites could be useful to deepen the
genetic relationship among accessions of these two species. The present study will be extended to two morphologically close species of TLVs (Sesamum radiatum and
Ceratotheca sesamoides) in order to examine their
genetic relationship and to explore the relationship
between ecology (soil, climate) and genetic diversity.
Abbreviations
AFLP, Amplified fragment length polymorphism; TLVs,
traditional leafy vegetables; NUS, underutilized species.
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