Cowpea [Vigna unguiculata (L.) Walp] is an early maturing crop that tolerates poor soil fertility and water stress  . This makes it an important food crop in areas hard hit with climate change, famine, poor soil fertility, poverty and high population growth rate   . Cowpea has evolved from native wild types and its genetic diversity is greater than that of any other crop in the dry African savannah  . However, the cultivated cowpea has been shown to have a narrow genetic base suggesting the crop went through a “genetic bottleneck” during domestication  . Awareness of existing plant genetic diversity in the cowpea germplasm is therefore fundamental for effective management of cowpea genetic resources  . The establishment of the forensic identity of crop varieties has become vital for protecting plant breeders’ and farmers’ right following the enactment of the Convention of Biodiversity Conservation particularly in developing countries  . DNA fingerprinting which is also known as molecular characterization is a useful tool for genotype identification, diversity studies and associating molecular markers to phenotypic traits  . Fingerprinting with molecular markers allows precise, objective and rapid variety identification of plant varieties  . Unlike phenotypic based markers, molecular markers are stable and detectable in all tissues regardless of growth differentiation, development, pleitropic effect, epistatic effects and not confounded to environment where they grow   . SSRs, also known as Microsatellites, are co-dominant markers that are routinely used to study genetic diversity in different crop species  . These markers occur at high frequency and appear to be distributed throughout the genome of higher plants  .
Several successful molecular characterization and genetic diversity study works have been conducted on many cowpea accessions in the past using SSR markers in Ghana and the world at large      . However, new varieties have been released by the CSIR-Crops Research Institute of Ghana (CRI) and CSIR- Savannah Research Institute of Ghana (SARI) in recent times and there is therefore the need to record a forensic fingerprint of these newly released varieties together with old varieties using SSR markers for purposes of conservation, patent rights and genetic diversity studies. The aim of this study was therefore to fingerprint and evaluate genetic diversity among 22 cowpea varieties grown in Ghana, including four newly released varieties with the aid of 20 SSR markers.
2. Materials and Methods
2.1. Planting Materials
Seeds of 22 cowpea varieties were collected from the gene banks of CSIR-SARI and CSIR-CRI, sowed and nurtured in sterile soil in a greenhouse. Germplasm for this study was composed of 15 cultivars released by CSIR-CRI, 4 cultivars released by CSIR-SARI, 1 advanced line from CSIR-SARI, 1 landrace and 1 exotic variety grown in Ghana (Table 1).
2.2. Deoxyribonucleic Acid (DNA) Extraction
The laboratory studies were conducted in the Biotechnology laboratory of CSIR-CRI, Kumasi. Leaf explants were harvested from the 22 cowpea varieties two weeks after planting and kept in liquid nitrogen. Total DNA was extracted according to the prescribed protocol of the DNeasy™ Plant Mini extraction kit (Qiagen, Germany). DNA quality was checked using 0.8% (w/v) agarose gel electrophoresis. The concentration of the DNA was determined on a Nanodrop (spectrophotometer 2000C). The genomic DNA samples were diluted to a final concentration of 20 ng/μL with 1× TE buffer (10 mM Tris-HC1, pH 8.0; 1 mM EDTA) and stored at −20˚C for further use.
Table 1. Cowpea varieties used for the study.
2.3. Polymerase Chain Reaction Using SSR Markers
A total of 20 SSR primers (Table 2) were used to fingerprint the 22 cowpea varieties. Information about the primers was obtained from the SSR panel reported by Timko  . The primers were synthesized by Inqaba Biotechnical Industries Ltd., Pretoria, South Africa. Polymerase chain reaction (PCR) amplification was conducted in 20 μl volume tubes. Each PCR reaction contained 6 µl “One Taq Quick-Load 2x Master Mix” (composed of 20 mM Tris-HCl, 1.8 mM MgCl2, 22 mM NH4Cl, 22 mM KCl, 0.2 mM dNTPS and 25 units/ml One Taq DNA Polymerase), 2.0 µl Molecular Grade Distilled Water (MGDW), 0.5 µl of each primer pair and 2 ng/μL of genomic DNA sample to make a total volume of 10 µl. The PCR amplifications were performed in a thermal cycler C1000 (Seegene, Korea). The thermal cycler was set to initial denaturation at 94˚C for 1 minute followed by 35 cycles of denaturation for 30 seconds at 94˚C, annealing at 55˚C for 30 seconds, extension at 72˚C for 1 minute and ended with final extension at 72˚C for 10 minutes.
Table 2. SSR primers used for molecular characterization and their sequences
2.4. Gel Electrophoresis
PCR products were resolved on 6% (w/v) polyacrylamide gel electrophoresis (PAGE) in 1 X Tris/borate/EDTA buffer stained with 0.5% Ethidium bromide (Criterion cell model, vertical centrion tank) for 1 hour 30 minutes at a voltage of 80 V. The gel was photographed under Ultraviolet light with the aid of Alpha Innotech MultimageTM Light Cabinet for further analysis.
2.5. Scoring of Bands and Data Analysis
The bands observed on the gels after staining were scored on the basis of presence/absence (1/0) with the aid of the Alpha Imager version 3.41 software along with a 100-bp DNA ladder (Invitrogen®) to identify the molecular-weight of the DNA samples. Following the gel scoring, the molecular data was fed into Darwin software  to generate a dissimilarity matrix and a dendrogram.
The primers generated a total of 481 bands across the selected varieties out of which 428 (88.98%) were polymorphic. Only 1 (SSR 6336) out of the 20 primers did not show polymorphism among the varieties, and therefore was excluded from the analysis. A representative picture of the amplification product of primer SSR6243 showing pattern of allelic bands across the 22 cowpea varieties on 6% polyacrylamide gel is presented in Figure 1.
The size of amplified alleles ranged from 90 bp to 391 bp (Table 3). The primers SSR-6613 and SSR-6608 recorded the highest and lowest number of polymorphic bands of 79 and 5 respectively. The number of alleles varied from 1 to 6. The allele frequency ranged from 0.136 (SSR-6371 to 0.841 (SSR-6608) with mean of 0.445 among the varieties. The polymorphic information content (PIC) representing the allele diversity for a specific locus ranged from 0.107 (SSR-6608) to 0.656 (SSR-6613) with a mean of 0.293.
Figure 1. SSR 6243 marker scored on ethidium bromide stained PAGE gel (6%) for 22 cowpea varieties M = 100bp ladder; 1 = SARC-1-57-2; 2 = Apabgaala; 3 = Nhyira; 4 = Hewale; 5 = Asomdwee; 6 = Adom; 7 = Soronko; 8 = Bengpla; 9 = Hansadua; 10 = Agyenkwa; 11 = Padi-Tuya; 12 = Zaayura; 13 = Nketewade;14 = Zamzam;15 = Ayiyi; 16 = Songotra; 17 = Videza; 18 = Asontem; 19 = Tona; 20 = Asetenapa; 21 = Sanzi; 22 = Bra 01.
Table 3. Resolving power analysis of SSR primers.
Dissimilarity Matrix and Cluster Analysis Based on SSR Markers
The genetic distances within pairs of the 22 varieties were evaluated in a dissimilarity matrix generated from the molecular data using Darwin software. From the dissimilarity matrix (Table 4), there was no distance between Videza and Asomdwoe which implies that the two varieties are very similar. A short distance of 0.109 was found between Zamzam and Agyenkwa while the highest distance (most divergent) was found among three pairs of varieties: Agyenkwa and Adom; Hewale and Ayiyi; Zamzam and Helwale at genetic distance of 0.652. The dendrogram grouped the 22 varieties into four major clusters (A, B, C and D) at genetic distance of 0.20 (Figure 2). Cluster A was made up of only Hewale. Cluster B had two sub-clusters diverging at genetic distance of 0.125. Sub-cluster I consisted of only Zaayura while subcluster II consisted of Asomdwoe and Videza. The third major cluster was cluster C which consisted of 9 varieties grouped into two subclusters diverging at genetic distance of 0.15. Cluster D also consisted of two sub-clusters. The first sub-cluster comprised of 5 varieties (Adom, Asontem, Tona, Nhyira and Soronko) while the second sub-cluster consisted of 3 varieties (Hasnsadua, Bengpla and Songotra).
Figure 2. Dendrogram of 22 cowpea varieties using 19 SSR primers based on Unweighted Pair Group Method with Arithmetic Mean (UPGMA) generated by Darwin 6.0.010 software.
From the study it was observed that, 19 informative SSR primer pairs produced 1 to 6 alleles per primer pair with an average of 2.53. This collaborates with the 1 to 6 alleles per primer reported by Asare et al.  when they assessed the genetic diversity in cowpea germplasm from Ghana using SSR primers including some of the primers used in this study. There is however, reports of number of allele per locus ranging from 1 to 9  , 5 to 12  , 1 - 16  , 2 to 5  , 5 to 12  and 2 to 17  in previous cowpea variability studies. According to Ali et al.  , such variations in numbers of alleles can be attributed to the types of primers used in each study and/or the rate of polymorphism of each primer pairs.
According to the Bostein et al.  scale of informativeness, PIC value ≥ 0.5 is highly informative, 0.25 - 0.5 reasonably informative and ≤0.25 is slightly informative, and marker loci with many alleles and a PIC value near 1 are most desirable. In this study, the polymorphic information content (PIC) ranged from 0.107 to 0.631 with an average of 0.293. Asare et al.  also reported PIC range from 0.07 to 0.66 in a variability study of cowpea germplasm from Ghana. However, other researchers have reported PIC values in the range of 0.02 to 0.73  , 0.08 to 0.33  , 0.61 to 0.92  and 0.33 to 0.83  . Based on the Bostein et al.  scale of informativeness, the most desirable markers used in this study were SSR-6265 and SSR-6613.
Three of the primers used in this study (i.e. SSR-6258, SSR-6243 and SSR- 6323) were also used by Badiane et al.  to study the genetic relationship among cowpea varieties from Senegal. The three primers showed polymorphism just as was reported by Badiane et al.  . Badiane et al.  further observed that the primer that gave the highest allele frequency also recorded the lowest genetic diversity as well as lowest polymorphic information content (PIC). A similar situation was observed in this current study where SSR primer 6608 scored the highest allele frequency but the lowest PIC value. Previous studies conducted by Badiane et al.  and Doumbia et al.  reported low levels of polymorphism among SSR primers. This present work reports high level of polymorphism among cowpea varieties as reported also by other authors    .
Dissimilarity Matrix and Cluster Analysis Based on SSR-Markers
The delineation of cowpea germplasm into groups of genetic relatedness is a valuable resource for guiding introgression efforts in breeding programmes and for improving the efficiency of germplasm management  . The results from the dissimilarity matrix showed that there was no distance between Videza and Asomdwoe (Table 4) implying that the two varieties are very similar. The two varieties were released by CSIR-CRI, Ghana in the year 2012. There is however enough evidence in the Catalogue of Crop varieties released and registered in Ghana  to prove morphological variation among the two varieties. Future fingerprinting involving these varieties should include more informative primers to detect differences within the varieties.
The dendrogram generated from the molecular data grouped the varieties into four clusters. Nketewade, Agyenkwa and Zamzam (three out of four newly released varieties by CSIR-CRI) were in the same subcluster CII (Figure 2). Adom and Asontem were found in the same subcluster DI in this study. In terms of morphology, these two varieties share several morphological traits  . The trend re-enforces the closeness of the two varieties. They share several characteristics in common and this may be due to the fact that they were developed from a common ancestor. Three pairs of varieties (Agyenkwa and Adom; Hewale and Ayiyi; Zamzam and Hewale) recorded high genetic distance of 0.652 each. The varieties in each pair have different ancestry, different agro-ecological zones of production and different release times  . These varieties will serve as good materials from which suitable parental lines could be selected for hybridization programme.
This present work has provided additional molecular information about 22 cowpea varieties grown in Ghana including four newly released varieties (Agyen- kwa, Zamzam, Hansadua and Nketewade). The information has been made available to CSIR-CRI and CSIR-SARI in Ghana for the purpose of varietal protection and patent right. The 19 polymorphic SSR cowpea primers were able to detect genetic diversity among cowpea germplasm grown in Ghana except for Videza and Asomdwoe. Future diversity studies involving these two varieties should include additional polymorphic primers in other to detect the differences between the two varieties.
This work was supported by research grants provided by the West Africa Agricultural Productivity Programme (WAAPP) to the Council for Scientific and Industrial Research-Crops Research Institute of Ghana. The authors wish to thank Dr Francis Kusi of CSIR-Savannah Agricultural Research Institute (SARI) for providing germplasm materials from Northern Ghana.
Compliance with Ethical Standards
The datasets generated and analysed in this current study are available from the corresponding author on reasonable request.
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