Being a legume, cowpea is nitrogen-fixing (Sanginga, 2003) and fits perfectly in the traditional intercropping systems that are common in Africa, especially given its ability to tolerate shade. The total area under cowpea cultivation is more than 12.5 million hectares worldwide, with an annual production of around 4.5 million metric tons (Singh et al., 2002).

Cowpea remains one of the most important summer adapted food grain legumes grown under rained conditions in Sudan. Despite its importance in Sudan, the yields remain extremely low at an average of 0.26 tons/ha FAO STAT (2010). This production is mainly limited by a wide range of biotic and abiotic constraints.

Cowpea is believed to have been introduced into the western regions of Sudan from West Africa from where it spread to other regions. A national effort to conserve Sudanese cowpea collections resulted in the conservation of more than 250 accessions from different agro-ecological zones at the central gene bank in Wad Medani. These cowpea accessions have been morphologically characterized using the International descriptor list for cowpea published by International Board for Plant Genetic Resources (IBPGR). Traditional selection methods in cowpea depended mainly on the observed morphological variations even though morphological characteristics are easily influenced by the environment (Meglic et al., 1996). The genetic diversity information is extremely important, accurate assessment of genetic variability is important for the preservation and utilization of germplasm resources (Huaqiang et al., 2012).

There is an urgent need to undertake more detailed genetic characterization of cowpea germplasm in order to optimally exploit the resources for improved cowpea production in Sudan. Such analysis would also reveal the true origin of Sudanese cowpea germplasm and establish the extent at which the global cowpea collection at IITA would benefit the cowpea breeding programs in Sudan.

Simple sequence repeat (SSR) markers are one of the most frequently used markers in the genetic diversity analysis of cowpea (Li et al., 2001; Ogunkanmi et al., 2008; Lee et al., 2009; Asare et al., 2010; Badiane et al., 2012). The earliest cowpea SSR research is conducted by Li et al. (2001) and 27 SSR primers have been developed. Comparative studies in plants have shown that SSR markers, which are single locus markers with multiple alleles, provide an effective means for discriminating between genotypes (Powell et al., 1996; Li et al., 2001). This study assessed the genetic diversity of 245 Sudanese cowpea accessions alongside 22 global accessions obtained from IITA, Nigeria using SSR markers. The main objectives were to understand the extent of genetic variation and likely origin of Sudanese germplasm as well as create a mini-core collection based on.

Plant materials

Seeds of 231 Cowpea (V. unguiculata L. Walp.) accessions obtained from Plant Genetic Resources Unit of the Agricultural Research Corporation of Sudan representing six different agro-ecological zones of Sudan that is, Northern, River Nile, South Kordofan, North Kordofan, Blue Nile and Bahr Eljabel State (Figure 1) in addition to 36 global cowpea accessions obtained from International Institute of Tropical Agriculture (IITA)-Ibadan-Nigeria (Table 1) were used in the present study. These materials (267 accessions) were planted in the greenhouse of Bioscience Eastern Central Africa BecA-ILRI Hub-Kenya for seedling establishment.15 accessions failed to germinate in the green house; a rest of 252 accessions was successfully grown and used.

DNA extraction

Young leaves sampling were taken eight days after sowing in 1.5 mL Eppendorf tube, frozen immediately in liquid nitrogen and stored in -80°C, then leaves samples were manually grinded using micropestle. Genomic DNA isolated from young seedlings leaves following ZR plant/seed DNA protocol. DNA quality and quantity check done using Nano-drop spectrophotometer and 1% Agarose gel electrophoresis stained with Gel red was used to run the gel. The DNA was normalized by adjusting its concentration to 25 ng μL^-1 in an optical 96-well Reaction plates using sterile de-ionized water.

Microsatlite amplification

A total of 18 polymorphic SSR markers were used to screen 252 cowpea DNA samples (Table 2). The forward and reverse primers for each of the 18 SSR markers were labeled at the 5´ end of the oligonucleotide using fluorescent dyes to enable detection. PCR reaction were performed in 10 µL final volume in a mixture containing (Tag DNA polymerase1U, dNTPs 1 mM and Reaction buffer 1x) in bulk Polymerase chain reaction (PCR) premix, 5 mM reverse and forward primers, 2 µL DNA, 0.2 µL of 25 mM MgCl₂ and 7.2 µL of double distilled water. The optimal annealing temperature varied according to the T_m of the primer pairs and was determined using gradient PCR. For each amplification process, an initial denaturation of DNA at 95°C for 3min was followed by 30 cycles consisting of 30 sec at 94°C, 30 s at 50 to 60°C for annealing temperature (Table 2) 2 min at 72°C extensions a final extension of 15 min at 72°C was performed and the amplification products analyzed on 2% agarose gels in Tris Borate buffer stained with Gel red for visualization to establish polymorphism (Figure 2).

PCR analysis

PCR products of 4 primers with different dyes coloaded together in 96-well working plate vortexes and spined then a sub sample of 1.4 ml from the mixture added to 8 ml Hi-Di (Formamide) and 500 LIZ mixture in 96 PCR plate then the reaction vortexes spines and loaded on PCR machine at 95°C as Denaturation temperature for 3 min the product fast cooled in ice for 10 min before analysis. Fragment analysis was performed on the ABI 3730 sequencer machine, and peaks were sized and the alleles classified using the Gene mapper software (Applied biosystems). The informativeness of each primer pairs was realized using the polymorphic information content (PIC) using the Power Marker software program. The genetic structure of the accessions was investigated by Analysis of Molecular Variance (AMOVA) (Input as Allelic Distance Matrix for F-Statistics using GenAlEx software program; whereas the principal coordinate analysis (PCoA) was performed to identify genetic variation patterns among the cowpea genotypes.

Genetic distance

Generally, genetic distances among cowpea genotypes are low, re?ecting the initial bottleneck during domestica-tion, and maintained by the inherent self-pollination mechanism in the crop (Asare et al., 2010). On the whole, the genetic difference observed among the different ecological zones was varied, the least GD 0.031 observed between North and South Kordofan while the greater distance 0.303 was observed by two pairs; IITA; Bahr Eljabel and Northern; Bahr Eljabel state. The lowest GD between Sudanese regions and the out-group was observed by North Kordofan 0.131 then South Kordofan with 0.165, this closely related Genetic Distance to the out group germplasm can confirm the earlier suggestion that cowpea crop introduced to Sudan from West African Countries to the Western part of Sudan (Darfur and Kordofan) and from there it spread to the rest of the county, never the less this result showed a light bar of possibility of other sources of Sudanese cowpea a part from West African countries this can be clear from phylogenetic tree where Group A is mostly dominated by South Kordofan while IITA cluster together with Sudanese germplasm in other two groups (Figure 4). Blue Nile germplasm was observed to be more closely related to South Kodofan with GD of 0.034 then to North Kordofan with 0.071 GD (Table 6).

Three pairs of accessions (HSD 5700 Blue Nile, HSD 4854 South Kordofan; HSD 5701 Blue Nile, HSD 4854 South Kordofan and HSD 4568 South Kordofan, HSD 6560 South Kordofan) possessed genetic distances 0.000, suggesting that the members of these pairs may in fact be either separately collected with different names in the same Region or gene flow between Regions.

Variability in SSR markers

The delineation of cowpea germplasm into groups of genetic relatedness will be valuable for guiding introgression efforts in breeding programs and for improving the efficiency of germplasm management (Bao-Lam Huynh et al., 2013).

In the present study, the 16 informative SSR primer pairs used to analyze the 252 cowpea germplasm from Sudan and IITA-Nigeria resulted in 2 to 17 alleles per primer pairs with an average of 8.1. This result is in agreement with Badiane et al. (2012), who found number of alleles in Senegal cowpea varied from 1 to 16. Fatokun et al. (2008) detected alleles ranging from 4 to 13 alleles among 48 wild cowpea lines collected from different Agro- ecological zones in Africa with an average of 7.5 alleles per primer. However, previous works in Ghana, Burkina Faso, Senegal and Nigeria have revealed detections of allels ranging from 1 to 6, 5 to 12, 1 to 9 and 2 to 5 respectively (Asare et al (2010), Sawadogo et al. (2010) Diouf and Hilu (2005) and Adetiloye et al. (2013)). These 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.

In this study the polymorphic information content (PIC) ranged from 0.33 to 0.83 with a mean of 0.56. Fatokun et al. (2008) observed PIC ranging from 0.29 to 0.87 with a mean of 0.68 among the 48 wild cowpea lines. However other cowpea Researchers (Li et al. (2001), Badiane et al. (2012) and Asare et al. (2010) reported PIC ranging from (0.02 to 0.73, 0.08 to 0.33 and 0.07 to 0.66) respectively. The informativeness of PIC value measured by Botstein et al. (1980) scale revealed that the mean PIC value ≥ 0.5 is highly informative, 0.25~0.50 reasonably informative and < 0.25 is slightly informative, and Loci (Marker) with many alleles and a PIC value near 1 are most desirable (Botstein et al., 1980).  

Gene diversity in this study was 0.60 on average ranging from 0.34 to 0.85. In Senegal cowpea gene diversity varied from 0.08 to 0.42 with mean of 0.28 (Badiane et al., 2012), whereas In Ghana cowpea germplasm gene diversity ranged from 0.12 to 0.68 with an average of 0.44 (Asare et al., 2010). The results of gene diversity reflect the proportion of polymorphic loci across the genome. Therfore according to the result of this study the markers used were highly polymorphic compared to those used by the Badiane et al. (2012), and Asare et al. (2010).

Heterozygosity in this study was observed ranging from 0.01 to 0.13 with a mean of 0.05, Asare et al. (2010) revealed Variation in heterozygosity among Ghanaian cowpea SSRs increasing from 0.01 to 0.84 with an average occurrence of 0.19. The low value of Heterozygosity agrees with previous series reported by several cowpea researchers who documented that cowpea in general has a narrow genetic base due to the result of a bottleneck induced by a single domestication event which involved in the origin of this crop, where the proportion of heterozygozity is likely to be low, and likely its inherent nature of self-pollination mechanism (Pasquet, 2000; Coulibaly et al., 2002; Ba et al., 2004).

Genetic relationship among population

Generally, genetic distances among cowpea genotypes are low, re?ecting the initial bottleneck during domestica-tion, which was maintained by the inherent self-pollination mechanism in the crop (Asare et al., 2010). In this study the shorter genetic distance of 0.031, which was found between North and South Kordofan states, might be due to gene flow resulting from the seed exchange practiced by farmers particularly within and between these two neighbouring states. The same phenomenon was observed in Northern and River Nile states, which are also neighbouring states in the northern region of the country, showing a shorter genetic distance of 0.087 mostly due to the same reasons.

The greatest genetic distance of 0.303, which was found among the accessions obtained from the IITA compared to the accessions collected from Bahr Eljabel state, as well as among accessions collected from Northern state compared to those collected from Bahr Eljabel state, can be attributed to the far distances between those non-neighbouring geographical regions, where the possibility of gene flow due to seed exchange is almost lacking. Accessions obtained from the IITA were representing materials that were originally collected from other countries in west Africa. Northern state is located in the far north of Sudan whereas Bahr Eljabel is within republic of South Sudan. On the other hand, the shorter genetic distance found between the IITA and North Kordofan accessions as well as IITA and South Kordofan accessions can interpreted by the earlier suggestion that cowpea crop was introduced to Sudan from West African Countries to the western part of Sudan (Kordofan and Darfur), from where it spread to the rest of the country. The closely genetic distance between Blue Nile state and North and South Kordofan states can be attributed also to seed and or grain exchange between farmers and inhabitants of these states for cultivation and food uses. Moreover, an ethnic relationship with similar cultural background exists between some groups of populations in these three states, which reflecting in similar nutritional and cultivation habits and practices within them.

Three pairs of accessions in this study were found to have genetic distances of 0.00, which indicate the common genetic make-up within each pair of accessions, though they were observed to have different farmers’ variety names. The same variety with the same morphological characters can have different names following the locality or ethnic groups. Consequently, a better understanding of the genetic variation and strong sound footing system of classification of the cowpea collection in Sudan with molecular markers is urgent need.

The current study suggested low level of genetic diversity among Sudanese Cowpea population with great diversity with individuals. Therefore broadening the genetic base of Sudanese cowpea population may be achieved through introgression of new alleles either from wild cowpea germplsam or by out breeding with more closely related species to Cowpea V. triphylla and V. reticulate.

A total of 11 markers (SSR 6569, VM 70, SSR 6577, VM 94, VM 37, VM 30, VM 35 BMD 17, VM 53, VM 51 and SSR 6573) were highly informative (0.5 <PIC<0.83) and can be used in future diversity studies as marker core set in cowpea; IITA germplasm grouped together with Sudanese germplasm revealing the similarity of Sudanese cowpea to West African countries. Sudanese Cowpea Core collection can be created from South Kordofan, River Nile and Bahr Eljabel germplasm.

The authors have not declared any conflict of interest.