RESEARCH
Diversity in 113 cowpea [V
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malnutrition. mate, pathogen populations and agricultural practices
a SpringerOpen Journal
Egbadzor et al. SpringerPlus 2014, 3:541
http://www.springerplus.com/content/3/1/541of Ghana. Most of these germplasm were collected in the
2CSIR – Plant Genetic Resources Research Institute, Bunso, Ghana
Full list of author information is available at the end of the articleCowpea is known to be relatively drought tolerant
(Boukar et al. 2011; Muchero et al. 2009) and this attri-
bute results in its cultivation mainly in the savanna and
forest – savanna transitional zones of West Africa. Re-
source inputs in cowpea production are relatively low
(Manifesto et al. 2001). The source of genetic resources
for crop improvement is the available germplasm in gene-
banks and this need to be assessed for availability of useful
traits for crop improvement (Tan et al. 2012).
Cowpea is one of the most researched crops at the
genebank of the Council for Scientific and Industrial
Research – Plant Genetic Resources Research Institute
(CSIR – PGRRI) (Bennett-Lartey 1992; Asare et al. 2010).
CSIR – PGRRI is situated at Bunso, in the Eastern Region
* Correspondence: kenfafa@yahoo.com
1West Africa Centre for Crop Improvement, University of Ghana, Legon,
Accra, Ghanapackage structure was used to separate accessions into three groups, and the programme correctly identified
varieties that were known hybrids. The hybrids were those accessions with numerous heterozygous loci. The
structure plot showed closely related accessions with similar genome patterns. The SNP markers were more efficient
in discriminating among the cowpea germplasm than morphological, seed protein polymorphism and simple
sequence repeat studies reported earlier on the same collection.
Keywords: Characterization; Cowpea; Diversity; Genetic; Genotype; Markers; SNPs
Introduction
Cowpea [Vigna unguiculata (L) Walp] is an important
staple food crop in Ghana and many other parts of the
world (Obembe 2008; Timko and Singh 2008). The crop
is also used as animal feed. As a legume, cowpea fixes
nitrogen and therefore, contributes to soil improvement.
Compared with other important staples such as maize,
rice, yams and plantains in Ghana, most cowpea varieties
have shorter maturity period (55 days for some varieties)
making it a crop of choice to address hunger and
compared to those used in the production of other major
staples, making its cultivation affordable by resource
poor farmers (Muchero et al. 2009).
Cowpea is primarily a self-pollinating crop and its
genetic base is considered to be narrow (Sharawy and
El-Fiky 2002; Fang et al. 2005; Asare et al. 2010). Pres-
ence of diversity in the germplasm of crops is essential
for successful crop improvement (Varshney et al. 2007).
Limited genetic diversity poses a threat to the survival of
a species as this limits ability to respond to changes in cli-Walp] accessions assesse
Kenneth F Egbadzor1,2*, Kwadwo Ofori5, Martin Yeboah1,
Eric Y Danquah1 and Samuel K Offei4
Abstract
Single Nucleotide Polymorphism (SNP) markers were used
of 108 from Ghana and 5 from abroad. Leaf tissues from
genotyped at KBioscience in the United Kingdom. Data w
polymorphism. The results were used to analyze genetic
software. The markers discriminated among all of the cow
from 0.006 to 0.63 were used for factorial plot. Unexpecte
of the accessions. Accessions known to be closely related
method. A maximum length sub-tree which comprised o© 2014 Egbadzor et al.; licensee Springer. This
Attribution License (http://creativecommons.or
in any medium, provided the original work is pOpen Access
igna unguiculata (L)
with 458 SNP markers
wrence M Aboagye2, Michael O Opoku-Agyeman3,
n characterization of 113 cowpea accessions comprising
nts cultivated at the University of Ghana were
generated for 477 SNPs, out of which 458 revealed
similarity among the accessions using Darwin 5
ea accessions and the dissimilarity values which ranged
high levels of heterozygosity were observed on some
lustered together in a dendrogram drawn with WPGMA
8 core accessions was constructed. The softwareis an Open Access article distributed under the terms of the Creative Commons
g/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
roperly credited.
1980s and 90s from different parts of Ghana. These have
been characterized based on morphological (Bennett-Lartey
1992) seed protein (Oppong-Konadu et al. 2005) and Sim-
ple Sequence Repeat (SSR) differences (Asare et al. 2010).
Single Nucleotide Polymorphism markers (SNPs) are
powerful tools in genetic diversity study in living organ-
isms (Deulvot et al. 2010). SNPs are more effective in di-
versity assessment compared with other markers such as
Bunso, Ghana, while one accession (WACCI01) was ob-
tained from West Africa Centre for Crop Improvement
(WACCI), University of Ghana. Four accessions were
breeding lines selected from accession GH4524 based on
seed coat colour differences (Figure 1). Six of the acces-
sions were improved varieties in cultivation in Ghana,
namely: ‘Asontem’, ‘Nhyira’, ‘Zaayura’, ‘Tona’, ‘Paddy Twua’
and ‘Bawuta’. In addition there were two lines each from
Egbadzor et al. SpringerPlus 2014, 3:541 Page 2 of 15
http://www.springerplus.com/content/3/1/541AFLPs and SSRs (Varshney et al. 2007). Using morpho-
logical markers, Cobbinah et al. (2011) observed multiple
duplicates within cowpea germplasm in Ghana. Reason
for the high number of duplicates was the limited num-
ber of morphological markers and the low genetic vari-
ability these markers revealed. Asare et al. (2010) using
SSRs could also not discriminate between some acces-
sions of the cowpea germplasm in reference. It is critical,
for the purposes of efficiency, that the best available tool
for genetic diversity assessment is deployed.
SNPs are numerous in the genome of plants and other
living organisms (Galeano et al. 2009; Deulvot et al. 2010)
and they serve as good tools for diversity studies (Acquaah
2007; Varshney et al. 2007). SNPs may be the best choice
for diversity studies at the moment. As of 2012, there had
been no report on cowpea diversity studies that used SNPs
markers (Tan et al. 2012). However, in 2013, Huynh et al.
(2013) and Lucas et al. (2013b) reported their diversity
work on worldwide cowpea collection. The objectives of
this study were to use SNP markers to:
1. Assess genetic diversity within cowpea germplasm
assembled from CSIR – PGRRI, Bunso, Ghana and
abroad.
2. Use diversity information to select a core cowpea
germplasm collection for breeding purposes.
3. Help guide future international research in cowpea
breeding.
Materials and methods
Plant materials
A total of 113 cowpea accessions were characterized.
These included 102 accessions collected from different
parts of Ghana. One hundred and one accessions of the
102 are being conserved at CSIR – PGRRI genebank at
B BBE
Figure 1 Different lines of Gh4524 based on seed coat colour differen
mottle eye respectively.University of California Riverside (UCR779 and CB27)
and International Institute of Tropical Agriculture (IITA)
in Nigeria (IT97K-556-6 and IT82E-18). The accession
labeled “market” is one of the popular cowpea imported
to Ghana from Togo and was, therefore, included in the
imported accessions. All the accessions are listed in Table 1.
Seeds were germinated in sterilized top soil contained
in nursery boxes at the Crop Science Department Gar-
den, University of Ghana. Leaf discs of one week old
plants were sampled from one plant per accession and
shipped to the laboratory of KBiosciences in the United
Kingdom where genomic DNA was extracted. The DNA
samples were genotyped using 500 SNPs from the cow-
pea panel (Muchero et al. 2009; Lucas et al. 2011).
Markers used
The SNP markers used were distributed across the cow-
pea genome. Figure 2 shows a map of the eleven linkage
groups of cowpea and indicate the positions of the
markers on the cowpea genome. The length of each
linkage group and their respective number of markers
are inserted. Twelve out of the 477 SNP markers were
unmapped, thus summing up to 465 instead of 477
markers (Figure 2).
Data analysis
The software Darwin (Perrier and Jacquemoud-Collet
2006) was used to analyze the data. Dissimilarity was
calculated using simple matching coefficient after Perrier
et al. (2003) as follows:
L
dij ¼ 1‐1=L
X
ml=π
I ¼ 1
Where:
M ME
ces: B, BBE, M and ME for black, big black eye, mottle and
Table 1 Passport data of the cowpea accessions used for the experiment
GH number Local name Collection date Region Lat Long Seed colour Structure ID % Hetero
GH1622 Ayi-dze 21-12-82 Volta 06°35'N 00°27'E Black 81 0.43
GH1630* Asedua 01-12-83 Volta 06°29'N 00°10'E Black 59 0.71
GH1665* Asedua 26-11-82 Eastern Cream 80 31.45
GH1667 AduaNsawa 30-11-82 Eastern 06°40'N 01°20'W Brown 29 0.21
GH2279 Sanji 14-10-87 North 09°15'N 00°37'W Red 96 0.67
GH2280* Sanji 14-10-87 North 09°15'N 02°10'W Brown 85 0.43
GH2281* Sanji 14-10-87 North Dark 95 0.24
GH2282* Sanji 14-10-87 North 09°55''N 00°07'W Black 84 0.00
GH2284* Sanji 14-10-87 North 09°18'N 02°25'W Red 107 6.99
GH2288* Isagi 14-10-87 North 09°26'N 02°00'W Black 41 0.85
GH2291* Sanji 15-10-87 North 09°13'N 01°02'W Red 68 3.81
GH2293 Sanji 15-10-87 North 09°29'N 01°13'W Red 74 0.22
GH2294* 15-10-87 North 09°29'N 01°13'W Mottled 102 0.51
GH2296 Sanji 15-10-87 North 10°21'N 00°35'W Red 53 0.21
GH2306 Bonda 16-10-87 Upper West 10°40'N 02°01'W Black 101 0.21
GH2307 Bondawa 16-10-87 Upper West 10°53'N 02°07'W Black 92 0.21
GH2309* Bibitakone 17-10-87 Upper West 10°50'N 03°15'W Black 25 7.86
GH2312 Dapiala 17-10-87 Upper West 10°15'N 02°27'W Red 17 0.45
GH2314* Bengah 17-10-87 Upper West 10°0'N 02°24'W Mottled 76 2.50
GH2316* Bengah 17-10-87 Upper West 09°26'N 02°30'W Mottled 106 0.23
GH2317 Achibe 17-10-87 North 09°32'N 02°36'W Mottled 90 0.21
GH2323* Bengah 19-10-87 North 08°25'N 02°17'W White 88 1.49
GH2325 Asedua 30-11-87 Eastern 06°22'N 00°24'W Cream/Mixed 91 0.43
GH2326 Asedua 30-11-87 Eastern 06°22'N 00°24'W White 46 0.21
GH2328 Asedua 30-11-87 Eastern 06°22'N 00°24'W Red 86 0.22
GH2329 Asedua 30-11-87 Eastern 06°22'N 00°24'W White 55 0.21
GH2330 Asedua 30-11-87 Eastern 06°22'N 00°24'W Red 27 0.64
GH2331 Asedua 30-11-87 Eastern 06°22'N 00°24'W Mottled 54 0.42
GH2332 Asedua 30-11-87 Eastern 06°22'N 00°24'W White 47 0.21
GH2333 AduaNsawa 30-11-87 Eastern 06°22'N 00°35'W Mottled 112 0.85
GH2334* AduaNsawa 30-11-87 Eastern 06°22'N 00°35'W Cream 103 0.21
GH2335 AduaNsawa 12-1-87 Eastern 06°22'N 00°35'W Red 100 0.21
GH2340 AduaNsawa 12-1-87 Eastern 06°34'N 00°35'W Black 42 0.00
GH2341 AduaNsawa 12-1-87 Eastern 06°38'N 00°34'W White 24 0.21
GH2342* AduaNsawa 12-1-87 Eastern 06°38'N 00°34'W Red 83 0.63
GH2347* Yor 18-12-87 Eastern Black 97 0.00
GH2492* - 20-10-87 Ashanti White 62 1.35
GH3667* Ayi 23-10-93 Volta 06°18'E 0°11'N Dark 89 0.21
GH3668 Ayi 23-10-93 Volta 06°18'E 0°11'N Black 43 0.43
GH3669 Ayi 23-10-93 Volta 06°18'E 0°11'N Black 28 0.21
Gh3670* Ayi 23-10-93 Volta 06°35'N 0°06'E Brown 94 0.43
Gh3673 Ayi 24-10-93 Volta 06°41'N 0°17'E Black 69 0.64
Gh3674* Eveyi 24-10-93 Volta 06034'N 0°18' Black 77 0.63
Gh3675 Ase fita 25-10-93 Eastern 06°13'N 0°5'E Black 26 0.64
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Table 1 Passport data of the cowpea accessions used for the experiment (Continued)
Gh3677* Yor 25-10-93 Eastern 06°06'N 0°12'W Brown 99 0.43
Gh3685 Asedua 30-10-93 Eastern 07°16'N 02°19'W Black 108 0.43
Gh3689 Sanji 1-11-93 Eastern 08°54'N 0°39'N Brown 34 0.43
GH3701 Sanji 11-2-93 Eastern 06°10'w 0°03'N Brown 93 0.42
Gh3706* Benga 4-11-93 North 09°50'N 0°29'W Brown 87 0.00
GH3703 Tua 11-2-93 North Red 50 0.47
GH3708* Sega 11-4-93 North 09°20'W 02°20'N Mottled 105 0.45
GH3710* Tua 11-5-93 North Dark 109 0.21
Gh4028 Adua nsawa 19-5-96 North Red 57 0.23
GH4524 Yor 29-7-96 Accra Black 1,2,3,4 0.24
GH4529 Tolonye 30-7-96 Accra Mottled 65 0.43
GH4530 Yor 30-7-96 Accra Brown 18 0.43
GH4532 Ayiyibor 31-7-96 Volta Red 61 0.65
GH4533* Ayi 31-7-96 Volta Cream 82 1.32
GH4537* Ayi 08-1-96 Volta Brown 60 0.22
GH4541 Ayi 08-1-96 Volta Mttled 52 0.63
GH4546 Ayi 08-3-96 Volta Dark 70 0.21
GH4769* Bianga 22-10-96 Upper West Red 63 0.42
GH4771 Bianga 22-10-96 Upper West Black 38 0.21
GH4778 Gonja 27-10-96 North Red 23 0.21
GH5038 Vakli 11-4-96 Volta Cream 33 0.21
GH5039 Ekye 11-4-96 Eastern Red 66 0.44
GH5040* Yor 11-5-96 Eastern Red 40 0.21
GH5044 Yor 11-6-96 Eastern Red 39 0.21
GH5045 Yor 11-6-96 Eastern Red 44 0.66
GH5048 AduaNsadua 11-6-96 Eastern Red 37 0.21
GH5049 Asedua 11-6-96 Eastern Red 72 0.21
GH5050 Asedua 11-8-96 Ashanti Red 32 0.42
GH5344 Asedua 11-6-96 Ashanti Red 48 0.42
Gh5346 Asedua 9-11-96 Ashanti Brown 51 0.64
GH6045* Soronko Ashanti Reddish Brown 30 0.63
GH6060* Ayiyi 28-8-98 Eastern Cream 22 0.21
GH7167 Tue 18-2-03 Upper East White 104 0.42
GH7174 Tse 18-2-03 Upper East Cream 49 0.42
GH7178* Benga 19-2-03 Upper East Red 21 36.49
Gh7185 Sona 19-2-03 Upper East Black 71 0.21
GH7187 Goatana 20-2-03 Upper East Black 13 0.21
Gh7218 Sona 04-1-03 Upper West Dark 58 0.00
GH7222 Bondabene 04-1-03 Upper West Brown 56 0.66
GH7224 Sompla 04-1-03 Upper West Dark 31 0.21
GH7226* Bene 04-1-03 Upper West White 111 0.43
GH7228 Bene 04-2-03 Upper West Dark 45 3.24
GH7229 Bene 04-2-03 Upper West Dark 19 0.21
GH7230* Bene 04-2-03 Upper West Brown 79 0.24
GH7231 Bene 04-2-03 Upper West Dark 67 2.44
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Table 1 Passport data of the cowpea accessions used for the experiment (Continued)
GH7233 Bene 04-2-03 Upper West Brown 35 0.42
GH7234* Bene 04-2-03 Upper West Dark 110 20.09
GH7235 Bene 04-2-03 Upper West Dark 75 0.21
GH7243 04-2-03 Upper West Dark 78 0.42
Gh7245* Sonorni 1-4-03 Upper West Brown 98 0.43
Gh7273 1-4-03 Upper West White 73 0.63
GH7875 Asedua 15-3-06 Eastern Red 20 0.21
GH7888* Asetenapa 16-8-06 Ashanti Cream 36 0.63
IT97K556 IITA, Nigeria Brown 5 0.43
IT82E-18* IITA, Nigeria Brown 6 0.88
UCR 779* UCR, USA Brown 7 0.21
CB 27* UCR, USA White 8 0.64
Bawuta* CSIR – SARI White 14 0.21
Tona* CSIR – CRI Brown 9 0.42
Nhyira* CSIR – CRI Cream 10 0.42
PaddyT* CSRI – SARI White 15 0.21
Asontem* CSIR – CRI Red 16 2.77
Market* Volta (Ho) White 64 2.70
WACCI01* Legon, Accra Dark 11 0.66
Zayuraa* CSIR – SARI White 12 1.07
Note: Lat Latitude, Long Longitude, Hetero Heterozygosity. * - Accessions selected as core for further studies.
1 3 5 7 9 11
0
20
40
60
2
80
6
100
1084
Linkage 
group
No. of 
markers
Length 
(cM)
1 39 58.97
2 44 71.05
3 43 104.46
4 45 44.17
5 41 62.35
6 41 57.64
7 44 50.05
8 41 57.21
9 45 41.85
10 44 63.17
11 38 47.94
Linkage group
Po
si
tio
n 
(cM
)
Figure 2 The SNP markers used for the experiment – Length of linkage group, number of markers and their positions.
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dij: dissimilarity between unitsi and j
L: number of loci
π: ploidy
ml: number of matching alleles for locus l (Perrier et al.
2003).
The calculated dissimilarity coefficient was used to
construct a tree using the hierarchical clustering of
Weighted Paired Group Method with Arithmetic Mean
(WPGMA). It was used for a factorial plot and a Max-
imum Length sub-tree was constructed to select a repre-
sentative core accessions.
Detection of the underlying genetic population among
the studied cowpea accessions was carried out with the
Structure software (Pritchard et al. 2000). Three popula-
tions (K = 3) were assumed and indicated with blue,
green and red colours. Different numbers were tried for
K and finally 3 accepted with admixture ancestry model.
Length of burnin was 5000 and the number of MCMC
was set at 10000.
Results
Allelic diversity
Out of the 477 SNPs, 458 were polymorphic. SNP data
revealed that some of the markers although poly-
morphic, only few, sometimes just one genotype had
accessions that shared common allele per locus, thus,
varied greatly: from 0% versus 100% to 50% versus 50%
(refer to Additional file 1).
Heterozygosity
Some of the cowpea accessions were heterozygous at
some of the marker loci. Heterozygosity at a locus may
indicate accessions undergoing segregation. Many of the
accessions in the collection had at least one heterozy-
gous site (Table 1, column 9).
Factorial plot of the cowpea accessions
General diversity of the germplasm is displayed in a fac-
torial plot in Figure 3 and Additional file 2. Lines and a
circle were drawn in Figure 3 to aid in explanation.
Three major clusters were identified in the Figure de-
marcated by the “y” shaped two green lines. Members of
each cluster were characterized mostly by similar seed
coat colour.
Dendrogram of the cowpea accessions
Figure 4 is a dendrogram of the cowpea accessions
drawn with the calculated values of dissimilarity using
hierarchical clustering with WPGMA method. Acces-
sions written in black represent genebank materials ob-
AM
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http://www.springerplus.com/content/3/1/541them in the collection. The percentage of the cowpea
CRE
DARKFigure 3 Factorial display of 113 cowpea accessions.tained from Bunso and WACCI01 while those in blue
 TO WHITE
RED TO BROWN
Figure 4 Dendrogram of 113 cowpea accessions constructed
from dissimilarity using 458 polymorphic SNP markers.
Egbadzor et al. SpringerPlus 2014, 3:541 Page 7 of 15
http://www.springerplus.com/content/3/1/541and green are improved varieties from Ghana and
Gh4524 lines respectively. Accessions in red are from
IITA, UCR and the one named “market”. For legibility
purposes, different portions of Figure 4 were shown in
Figures 5, 6 and 7.
Result of structure analysis
The result of analysis made with Structure is presented
in Figure 8. Each accession is represented by a vertical
line, which is partitioned into coloured segments indicat-
ing the estimated membership fractions for it. Estimated
Ln Prob. of data = -32014.2: Mean value of ln likelihood =
701.5: Mean value of alpha = 0.071: Mean value of Fst 1 =
0.7: Mean value of Fst 2 = 0.56: Mean value of Fst 3 = 0.49.
Accessions in the same population in Figure 8 were col-
lected from across the different agro-ecological zones of
Ghana with no known history of genetic relationship be-
tween most of them.
Core 48 accessions
Maximum length sub tree method (Perrier et al. 2003)
was used to identify forty-eight core accessions for
breeding purposes (Figure 9). Accessions in red repre-
sent foreign materials; black for genebank materials, blue
for improved varieties and green for Gh4524 line. These
48 accessions are very diverse morphologically. The core
48 accessions include UCR779, CB27, IT97K-556-6 and
IT82E-18 which are internationally known cowpea lines.
These materials have unique alleles that are not likely
to be available in the genebank of PGRRI. Five of the
improved varieties from Ghana such as ‘Asontem’ and
‘Nhyira’ are also in the core 48.
Discussion
For purposes of discussion, we denote those alleles
present in not more than 10% of the studied collection
as ‘rare alleles’. The cowpea accessions, GH7888 (a gene-
bank material), ‘Zaayura’ and IT97K-556-6 shared a rare
allele. IT97K-556-6 is IITA line while ‘Zaayura’ is a com-
mercial variety released by CSIR - Savanna Agricultural
Research Institute. Another rare allele was shared by
UCR779 and ‘Zaayura’. UCR 779, a Botswana landrace,
resistant to aphid (Muchero et al. 2009) was one of the
most unique accessions in the collection. It was the only
line with “A” against “T” for one marker. “Asontem”
(IT82E-16) which is one of the improved varieties in
Ghana developed by IITA in collaboration with CSIR-
Crops Research Institute, also had a rare allele at a locus.
The last example of a rare allele observed in the collec-
tion was “T” for GH7167, GH2288 and CB 27 where all
other accessions had “C”. The allelic diversity thus varied
greatly for the studied cowpea accessions.
The 458 SNP markers were able to discriminate
between all the cowpea accessions studied. Previous
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http://www.springerplus.com/content/3/1/541studies (Bennett-Lartey 1992; Oppong-Konadu et al.
2005; Asare et al. 2010) could not discriminate all ac-
cessions, but the increase number of markers used here
and their high levels of polymorphism allowed the dis-
crimination of even closely related accessions such as
BBE, M, B and ME (segregated lines of Gh4524). This
confirms the robustness of SNP markers in diversity
studies as reported by Varshney et al. (2007). The separ-
ation of the four Gh4524 lines on the other hand could
most probably be due to segregation as Lucas et al.
(2013a) were able to identify duplicates from the USDA’
core cowpea collection with SNP markers.
Figure 5 First 38 cowpea accessions in Figure 4.Definite patterns were identified in the cowpea collec-
tion. Both Ghanaian and foreign elite accessions clus-
tered together (Figures 3 and 4). Patterns could also be
seen in Figure 4 based on the seed coat colour similarities
of the cowpea accessions. However, accessions collected
from different regions of Ghana did not cluster together in
most cases. Asare et al. (2010) also did not observe strong
geographic relationship in the PGRRI cowpea collection
when they used SSR in diversity studies. Tanhuanpaa and
Manninen (2012) in their studies on Phleum pretense
with SSRs also did not observe significant correlation be-
tween the various accessions and their geographic
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http://www.springerplus.com/content/3/1/541origins. Geography does not always reflect underlying
genetic structure (Rosenberg et al. 2002).
Only 150 markers which are about 30% did not have
any cowpea showing heterozygosity. Some markers gen-
erally revealed higher levels of heterozygosity. There
were 23 accessions heterozygous for a particular marker.
Most of these accessions clustered together (Figures 3
and 4). Gh7234 for instance had as many as 90 hetero-
zygous sites. This suggests that some of the genebank
cowpea accessions are not pure. Phenotypic analysis
strengthened the assertion that seeds of Gh7234 were
different in terms of seed coat colours with the domin-
ant as dark mottling. Similar observation was made for
Gh7231 which had 13 heterozygous sites. However,
Figure 6 Middle 38 cowpea accessions in Figure 4.some of the improved varieties including the foreign
ones (IT97K-556 and CB27) also had one or more het-
erozygous sites. High heterozygosity known in such
crops as plantains (Tenkouano et al. 1999), Scot pines
(Gupta et al. 2001) and cassava (Dyer et al. 2011) was
unanticipated in this study. The high heterozygosity ob-
served in some of the cowpea accessions might be due
to outcrossing (Lucas et al. 2011; Kouam et al. 2012)
during regeneration at the genebank and to the fact that
some of them have hybrid origin. There were only five
accessions (Gh2282, Gh2340, Gh2347, Gh3706 and
Gh7218) that were homozygote for all the loci.
Three major clusters are identified on the factorial dis-
play of the accessions indicated by two green lines which
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http://www.springerplus.com/content/3/1/541formed a “y” shape (Figure 3). Accessions of same clus-
ter generally have similar seed coat colours with only
few exceptions. Some of these exceptions are Gh2281
and Gh7185 with dark seed coat colours clustering in
the red to brown seed coat colour group while Gh2284
and Gh5048 with red seeds clustered with dark colours.
Seed coat colour is a frequently used as a morphological
trait in classifying crop varieties (Adesoye and Ojobo
2012) and may also be linked with other important traits
(Atis et al. 2011). The clusters according to the seed coat
colours are; Dark, Cream to White and Brown to Red
(Figure 3). The boundaries between the dark seed coat
colour cluster and the other two were very conspicuous.
However, the boundary between the white and red seed
coat colour clusters was not very clear. Six accessions in
Figure 7 Final 37 accessions of Figure 4.Gh5049
Gh7875
Gh4028
Gh4769
Gh5346
Gh4778
Gh4530
Gh5045
Gh5039
Asontem
Gh3674
Gh3673
Gh3669
Gh2347the purple outlined circle formed a sub-cluster between
the white and red seed coat colour clusters. Even
though, the six accessions formed a sub-cluster, each in-
dividual was closely linked to its respective major cluster,
with the exception of IT82E-18 (Figure 3). In contrast,
Asare et al. (2010) did not observe clustering pattern
based on seed coat colour when they characterized cow-
pea collection with SSRs. However, in this study clear
pattern based on seed coat colour was observed. Simi-
larly in maize, SNP markers were used to identify kernel
colour gene (Sharma et al. 2011).
All the foreign accessions fell on a straight line (red).
Meaningfully, they also fell in their appropriate colour
seed coat clusters. These are elite germplasm (improved
varieties) and have been selected for similar traits over a
Gh4532
Gh3670
Gh4537
Gh3703
Gh3679
Gh4546
Gh4529
Gh4541
Gh3667
WACCI01
Gh2281
UCR779
Gh6045
Tona
Gh7888
Nhyira
Gh2280
Gh7185
Gh3677
IT97K5566
Gh6060
IT8E18
Gh4533
Figure 8 Estimated population structure for the cowpea accessions studied.
Egbadzor et al. SpringerPlus 2014, 3:541 Page 11 of 15
http://www.springerplus.com/content/3/1/541
Egbadzor et al. SpringerPlus 2014, 3:541 Page 12 of 15
http://www.springerplus.com/content/3/1/541long period of time. The improved varieties from both
Ghana and abroad are found on or above the red line
(Figure 3). Local accessions that clustered with these
elite accessions could be very useful materials for cow-
pea breeding programmes, especially in Ghana, for being
genetically close to the elite varieties and being adapted to
the local climate. For instance, the dissimilarity between
GH7888 (a genebank material), and ‘Zaayura’ was as
small as 0.026. GH7167, GH2288 and CB 27 clustered
together. CB27 was released in California in 1999 and is
resistant to Fusarium wilt race 3 and moderately suscep-
tible to aphid (Muchero et al. 2009). Phenotypically
CB27 did not share much similarity with Gh2288. Seed
mass of CB27 was twice that of Gh2288. The kidney
Figure 9 Dendrogram of 48 core cowpea accessions identified throug
breeding purposes.Gh3710
Gh2288
Gh2282
Gh2342
Gh2334
Gh2291
Gh4524
Gh2323
Gh2492
Gh2309
Gh7226
Gh7234
Gh3708
Gh3706
Gh7245
Gh2316shaped seed of CB27 had white seed coat with black eye.
This type of cowpea has a high preference in Ghanaian
markets (Langyintuo, 2003). Gh2288 on the other hand
had dark mottling seed coat colour. Accession CB27 was
erect while Gh2288 was prostrate. Few traits shared by
CB27 and Gh2288 are, pigmented immature pod tip
which dry up to straw, pendant pods and sub-hastate ter-
minal leaflet that were slightly curved.
No elite genotype fell in the dark coat coloured cluster
(Figure 3). Commercial varieties of cowpea are mainly
white or brown to red coat coloured in Ghana as they
are the types preferred by consumers (Quaye et al. 2011;
Langyintuo et al. 2003). Separation of many Ghanaian
accessions away from elite and commercial varieties may
Gh2284
Gh2294
Gh2314
Gh7230
Gh1630
Gh1665
Gh7178
PaddyTwua
Bawuta
Zaayura
Market
CB27
Gh2347
Gh3674
Gh3670
Gh4537
Gh3667
WACCI01
Gh2281
Gh5040
Gh4769
Asontem
UCR779
Gh6045
Gh7888
Nhyira
Gh2280
Gh3677
IT97K566
Gh6060
IT82E18
Gh4533
h Maximum Length Sub Tree method for conservation and
Egbadzor et al. SpringerPlus 2014, 3:541 Page 13 of 15
http://www.springerplus.com/content/3/1/541mean availability of diversity that could be exploited for
cowpea improvement. Despite claims of limited genetic
variation in cowpea (Asare et al. 2010; Kumar et al.
2011 Tan et al. 2012), there is substantial morphological
and genetic evidence that cowpea is a very diverse taxon
(Huynh et al. 2013). This experiment has shown that the
studied germplasm has some amount of diversity that can
be used for cowpea improvement. Furthermore the cow-
pea community should consider the many subspecies of
cowpea and the tens of thousands of accessions collected
from more than 50 countries that are available through
different germplasm collections. Special interest would
be to use the landraces in broadening the genetic base of
the improved cowpea varieties similar to what was sug-
gested for asparagus bean in China (Tan et al. 2012).
Clustering of materials such as CB27, Paddy Twua
(Padi Tuya), ‘Bawuta’ and ‘Zaayura’ is very significant.
This is because Padi ‘Tuya’ and a number of varieties re-
leased by CSIR – SARI are known to have parentage
from California Black eye (Padi et al. 2004). Close rela-
tionship between “Market” and CB27 (Figures 3 and 4)
was also not surprising. “Market” was an imported cow-
pea picked from a market and was suspected to originate
from California Black-eye because of its seed features.
Clustering of CB27 and Market had confirmed their re-
latedness. The dendrograms in Figures 6 and 7 support
pedigree knowledge as seen in the clustering of Gh4524
lines and UCR779 with IT82E-18 which are both from
South/East Africa, Botswana and Mozambique, respect-
ively. The SNP markers were for that matter very reli-
able in this diversity study. An exception was that
IT82E-18 did not cluster with Asontem (IT82E-18 in
Ghana). It could probably be that the Asontem collected
was not the IT82E-18 as it has been in the hands of
farmers for a long time. Farmers might be calling a mor-
phological similar variety Asontem. Another possibility
resulting in the non-clustering of IT82E-18 and the sup-
posed Asontem is that the plant genotyped as Asontem
could be a rogue as described by Luca et al. (2013b).
Three populations were assumed and represented by
different colours; blue, green and red with 8, 22 and 38
accessions discretely coming from them respectively
(Figure 8). Thus the total number of accessions without
admixed genome was 68. Members in the blue popula-
tion, some of which are Gh2323, Gh7167 and Gh7174
clustered at the top left corner in Figure 3. In the excep-
tion of Gh7178 (13 in Figure 5), all the accessions with
entirely blue genome have white or cream seed coat
colour. Gh2323 and Gh7273 which are both members of
the blue population in Figure 8 were the closest relatives
in Figure 4. The cowpeas in the green population in
Figure 8 are mostly red seed coated and also showed
close relationship in the dendrogram in Figure 4. Ac-
cessions such as Gh5039, Gh5040 and Gh5049 in thegreen population clustered together in Figure 3. Similar
patterns were also observed for the red population in
Figure 8. However, accessions in this group are more di-
verse in terms of seed coat colour. The clustering pat-
tern in the dendrogram and factorial plot with “Darwin”
thus had some similarities with that of “Structure”. Some
authors believe that the software Structure does not al-
ways create clusters that are consistent with evolutionary
history of individuals in populations; however, it is one
of the most frequently used software for cluster analysis
(Kalinowski 2011). In this study the result of the struc-
ture analysis made biological sense especially when it is
compared to the phenotype of the cowpea accessions
and the analysis made with Darwin.
Different combinations of admixture genome for
different cowpea accessions were observed. Some of the
accessions had genome from two different populations
while others were from all the three. All of the improved
varieties had genome from different populations (Figure 5).
“Zaayura”, “CB27” and “Market” had similar patterns in
the exception of having slightly different proportions for
the various segments. These three varieties are believed
to have been bred from materials with common parent-
age (Padi et al. 2004). The four accessions obtained from
Gh4524 (numbers 1, 2, 3 and 4 in Figure 8) showed very
similar patterns and had portions of their genome from
different sources. Some other accessions from the gene-
bank as well showed inheritance of genome from differ-
ent populations. Cowpea is predominantly inbreeding
and it is shown by the mean apha value of 0.07 indicating
that most of the accessions are essentially from one
population. However, mean value of 3.4% outcrossing has
been reported (Kouam et al. 2012) which might be the
reason for some of the genebank materials to be
admixed. The observation in this study thus confirms this
phenomenon.
The establishment of a core germplasm collection helps
in easy management and identification of variations for
breeding purposes (van Hintum et al. 2000). Where the
germplasm collection is very large, management goes be-
yond core to mini core collection (Upadhyaya et al. 2010).
Forty-eight core accessions were consequently, identified
from the fingerprinting for conservation and crop im-
provement. The core 48 accessions include UCR779,
CB27, IT97K-556-6 and IT82E-18 which are internation-
ally known cowpea lines. These materials had unique al-
leles that are not likely to be available in the genebank in
Ghana. Five of the improved varieties from Ghana were
included in the 48 core accessions. These 48 accessions
include all of the 11 improved varieties in the study.
Bringing these improved accessions which were hitherto
not in the collection into the activities of the genebank
might mean expansion of the gene pool of the cowpea
which is considered to be narrow (Tan et al. 2012).
Egbadzor et al. SpringerPlus 2014, 3:541 Page 14 of 15
http://www.springerplus.com/content/3/1/541Expansion of gene pool is important for crop improve-
ment (Varshney et al. 2007).
The sphericity index as explained by Perrier et al.
(2003) was considered in choosing the 48 core cowpea
accessions. The sphericity index for all the 113 acces-
sions was 0.69. This figure meant that there was much
redundancy in the collection, compared to the final
three accessions which had the highest sphericity index
of 1. The core 48 accessions selected had sphericity
index of 0.79 which was quite low indicating much re-
dundancy which could permit further reduction in the
number of accessions included in the core. However, as
much as 10 improved varieties were included in the core
when the number of accessions was reduced to 20 with
sphericity index of 0.88. This meant that with 20 core
accessions, only 50% would be from the genebank.
To avoid further narrowing of the genetic base of the
cowpea germplasm for breeding purposes (Sharawy and
El-Fiky 2002; Fang et al. 2005; Asare et al. 2010; Tan
et al. 2012), the 48 core accessions were, therefore,
accepted to increase the genetic base of the core.
The core accessions varied in morphological traits
such as growth habit where there were a wide range
spanning from erect to spreading types. Plant pigmenta-
tion, leaf shape and flower colour also varied among the
core accessions. Seeds with different coat colours, sizes
and shapes were found within the core accessions. Some
of the accessions in the core collection had been reported
to have resistance to biotic stresses. Examples include
CB27 and UCR779 which are resistant to Fusarium wilt
and aphid respectively (Muchero et al. 2009). These ac-
cessions could be used as parents to develop varieties re-
sistant to biotic stresses such as aphid borne mosaic virus
which is a serious constraint to cowpea cultivation in
many parts of Africa (Orawu et al. 2012). Further evalu-
ation of the core 48 accessions may reveal other traits
that might be of interest to cowpea breeders.
Conclusion
This study was one of the earliest diversity studies in
cowpea using SNP markers. The markers were efficient
in discriminating between all the accessions used in the
study including closely related materials such as Gh4524
lines. Accessions known to be related by ancestry such
as CB27 and Paddy Twua, clustered together, demon-
strating the reliability of the markers. The information
provided in this diversity study could be useful in cowpea
improvement in Ghana and elsewhere. A total of 48 acces-
sions were identified as a core collection for breeding pur-
poses. These core accessions were morphologically very
diverse and included UCR779, CB27, IT97K-556-6 and
IT82E-18. These are elite materials obtained from differ-
ent countries. Improved varieties from Ghana such as
‘Asontem’ and ‘Nhyira’ are also in the 48 core accessions.The genetic diversity of the selected core could be of im-
portance for future plant breeding for the development
of superior varieties of cowpea.
Additional files
Additional file 1: Raw SNP Genotype Data used for Analyses.
Additional file 2: Factorial plot of the cowpea accessions.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KF Egbadzor is the Principal Investigator; K Ofori, EY Danquah, SK Offei and
M Yeboah were the principal investigator’s PhD. Supervisors. They, therefore,
gave guidance at each stage of the experimentation and writing of the
manuscript. MO Opoku-Agyeman assisted in the data analyses. LM Aboagye
corrected the first draft of the manuscript. All authors read and approved the
final manuscript.
Acknowledgement
We are indebted to the Generation Challenge Programme and the Alliance
for a Green Revolution in Africa for financing the research. We thank
management and staff of CSIR – PGRRI, Bunso, Ghana and Prof. J. Ehlers of
California Riverside University for providing the cowpea samples for the
experiment. We also thank Mr. Edward Addo for assisting in sampling and
packaging of the experimental materials for genotyping.
Author details
1West Africa Centre for Crop Improvement, University of Ghana, Legon,
Accra, Ghana. 2CSIR – Plant Genetic Resources Research Institute, Bunso,
Ghana. 3Cocoa Research Institute of Ghana, Tafo, Ghana. 4The Biotech
Centre – University of Ghana, Legon, Accra, Ghana. 5Department of Crop
Science, University of Ghana, Legon, Accra, Ghana.
Received: 24 July 2014 Accepted: 9 September 2014
Published: 20 September 2014
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