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Assessment of genetic diversity in Vigna unguiculata L. (Walp) accessions using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) polymorphic markers

Background Assessment of genetic diversity of Vigna unguiculata (L.) Walp (cowpea) accessions using informative molecular markers is imperative for their genetic improvement and conservation. Use of efficacious molecular markers to obtain the required knowledge of the genetic diversity within the local and regional germplasm collections can enhance the overall effectiveness of cowpea improvement programs, hence, the comparative assessment of Inter-simple sequence repeat (ISSR) and Start codon targeted (SCoT) markers in genetic diversity of V. unguiculata accessions from different regions in Nigeria. Comparative analysis of the genetic diversity of eighteen accessions from different locations in Nigeria was investigated using ISSR and SCoT markers. DNA extraction was done using Zymogen Kit according to its manufacturer’s instructions followed by amplifications with ISSR and SCoT and agarose gel electrophoresis. The reproducible bands were scored for analyses of dendrograms, principal component analysis, genetic diversity, allele frequency, polymorphic information content, and population structure. Results Both ISSR and SCoT markers resolved the accessions into five major clusters based on dendrogram and principal component analyses. Alleles of 32 and 52 were obtained with ISSR and SCoT, respectively. Numbers of alleles, gene diversity and polymorphic information content detected with ISSR were 9.4000, 0.7358 and 0.7192, while SCoT yielded 11.1667, 0.8158 and 0.8009, respectively. Polymorphic loci were 70 and 80 in ISSR and SCoT, respectively. Both markers produced high polymorphism (94.44–100%). The ranges of effective number of alleles (Ne) were 1.2887 ± 0.1797–1.7831 ± 0.2944 and 1.7416 ± 0.0776–1.9181 ± 0.2426 in ISSR and SCoT, respectively. The Nei’s genetic diversity (H) ranged from 0.2112 ± 0.0600–0.4335 ± 0.1371 and 0.4111 ± 0.0226–0.4778 ± 0.1168 in ISSR and SCoT, respectively. Shannon’s information index (I) from ISSR and SCoT were 0.3583 ± 0.0639–0.6237 ± 0.1759 and 0.5911 ± 0.0233–0.6706 ± 0.1604. Total gene diversity (Ht), gene diversity within population (Hs), coefficient of gene differentiation (Gst) and level of gene flow (Nm) revealed by ISSR were 0.4498, 0.3203, 0.2878 and 1.2371 respectively, while SCoT had 0.4808, 0.4522, 0.0594 and 7.9245. Conclusions Both markers showed highest genetic diversity in accessions from Ebonyi. Our study demonstrated that SCoT markers were more efficient than ISSR for genetic diversity studies in V. unguiculata and can be integrated in the exploration of their genetic diversity for improvement and germplasm utilization. Electronic supplementary material The online version of this article (10.1186/s12863-017-0567-6) contains supplementary material, which is available to authorized users.


Background
Vigna unguiculata (L.) Walp (cowpea, 2n = 2× = 22) is a dicotyledonous annual crop in the family Fabaceae and it is commercially cultivated in both tropical and subtropical parts of the world [1][2][3]. It provides 53% carbohydrate, 2% fat and 22 to 33% protein, including a high content of lysine, an essential amino acid for human metabolism [4][5][6][7]. It is used as a ground cover crop for erosion control, weed suppressor due to its allopathic constituents, and as animal feed [8]. About 10% of the grainy-crop is grown as green leafy vegetable and fodder in Africa [9]. It fixes 80% nitrogen for its growth demand from the atmosphere [10][11][12]. This quality of nitrogen fixation contributes to sustainability of cropping systems and improvement of soil fertility in marginal lands (11)(12). The global production of cowpea was estimated to be 2.27 million tonnes, out of which Nigeria accounted for 850, 000 t [13,14]. Nigeria, Burkina Faso, Niger, Brazil, Haiti, India, Sri Lanka, Australia and the United States are the principal producers and account for over 70% in the production of the crop [15][16][17]. Nigeria has been reported to be the second greatest consumer of cowpea in the world [18]. It is widely cultivated due to its palatability and low cost of production [19,20]. The crop adapts to sandy soils and warm conditions but very sensitive to low temperatures [16,21]. It is an essential constituent of cropping systems of the drier tropics such as Asia, the Middle East, Southern Europe, Africa, Southern USA, and Central and South America [22]. The crop has relatively a feature of drought tolerance and this enhances its cultivation in the savanna and forest savanna zones of West Africa [6,23,24]. Cowpea management requires low resource inputs compared to other staple crops and is affordable to poor farmers [23]. Factors including drought stress, high salinity and changes in temperature can negatively affect production yield [25,26]. Effects of these factors on the crop can be minimized through effective breeding programme with unique accessions identifiable and selectable using informative molecular markers. Cowpea is known to be self-pollinating and the presence of diverse accessions is imperative to improve its low genetic base which is narrow [27][28][29]. However, identification of individual accession into different groups is mostly done based on the morphological features that do not necessarily demonstrate the real genetic relatedness and often affected by environmental factors hence, the need for use of molecular markers. Molecular markers such as Randomly amplified polymorphic DNA (RAPD), Simple sequence repeat (SSR), Amplified fragment length polymorphism (AFLP) and Inter-simple sequence repeat (ISSR) have been applied in the estimation of genetic diversity, genetic relationship and germplasm management and conservation in V. unguiculata [29][30][31]. Knowledge of the genetic diversity available within the local and regional germplasm collections can enhance the overall effectiveness of cowpea improvement programs [32]. Use of SCoT markers that were developed to target the conserved regions of genome across various plant species due to their longer primer lengths and high annealing temperatures would be more efficient and reliable compared to other arbitrary markers [33]. Inter-simple sequence repeat markers are arbitrary markers that target multiple genomic loci and amplify DNA segments present between two identical microsatellite regions that are opposite with each other in orientation [34]. Apparently, to the best of our knowledge, comparative assessment of ISSR and SCoT markers has not been applied in V. unguiculata for its genetic diversity assessment. The objective of this study was to compare the efficacies of ISSR and SCoT polymorphic markers to assess the genetic diversity in accessions of V. unguiculata from different regions in Nigeria.

Sample collection and DNA extraction
Eighteen cowpea accessions were collected from different local government areas in Abia, Enugu and Ebonyi States including three samples from International Institute of Tropical Agriculture, (IITA), Ibadan, Nigeria (Table 1). Approximately 100 mg of fresh young leaves of V. unguiculata were collected from the screenhouse for DNA extraction using ZYMOGEN KIT (Zymo Research Corporation, USA), according to its manufacturer's instructions.  Tables 2. The PCR cycling profile used for the reaction consisted of an initial step at 94°C for 5 min., followed by 35 cycles of 94°C for 30 s, 72°C for 1 min, and a 10 min final extension at 72°C. Eight (8) μl of the PCR reaction products were electrophoresed in a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide and photographed on Transilluminator UV light (Fotodyne Incorporated, Analyst Express, USA).

Data analyses
Data matrices of Inter-simple sequence repeat and start codon targeted polymorphic marker profiles were generated by scoring (1) for presence and (0) for absence of individual allele. The obtained data matrices were used for phylogenetic reconstruction using Unweighted Pair Group Mean with Arithmetic (UPGMA) and dissimilarity index in Jaccard's option [35] were used for the analysis. The analysis was conducted using NTSYSpc software version 2.02. Furthermore, the genetic diversity, allele frequency and polymorphic information content (PIC) were computed using PowerMarker (Version 3.25). Genetic diversity and population structure analyses of the accessions were analysed using POPGENE software version 1.32.

Genetic diversity revealed by inter-simple sequence repeat (ISSR) markers
To access the level of genetic diversity in 18 accessions of V. unguiculata, a total of 10 ISSR primers were tested. Four (4) out of the 10 primers produced scorable bands that were used in the diversity analysis (Fig. 1). A dendrogram of the 18 accessions generated from UPGMA procedure, grouped the accessions into five major clusters at similarity index of 0.751 (Fig. 2). Cluster I consisted of AbCp-1, EbCp-1, and AbCp-5 consisting of accessions from Abia (AbCp), Ebonyi (EbCp) and Enugu (EnCp) States of Nigeria. Cluster II was subdivided into two subclusters, SCI and SCII. SCI had three accessions EnCp-2, EnCp-4 with EnCp-3 all from Enugu State. SCII contained accessions from different locations but showed that accessions EbCp-2 and EnCp-5 from Enugu were more closely related as well as accessions EbCp-4 and EbCp-5 from Ebonyi while accessions TVu-264 and Ife Brown from IITA were closer together. The accessions    Principal component analysis of the generated amplicons also resulted in five groups (Fig. 3) corresponding to the numbers of clusters obtained from the dendrogram. Each group was a representative of unique accession. The four (4) ISSR primers amplified a total of 32 alleles. The amplified alleles from each primer ranged from 4 to 14, with a mean of 9.4000 (Table 3). Polymorphic information content (PIC) values ranged from 0.2834-0.9073 with a mean value of 0.7192. The ISSR markers ISSR825, UBC816 and UBC826 were found to be polymorphic while UBC814 was monomorphic with the least PIC value. The genetic diversity values ranged from 0.2963-0.9136 with a mean of 0.7358 while major allele frequency spanned from 0.1667-0.8333 with a mean value of 0.3778. Allelic scores, counts and frequencies detected from the accessions of V. unguiculata using ISSR markers were recorded (Additional file 1: Table S1). The genetic diversity in Ebonyi (EbCp) population was identified to be the highest, with effective number of alleles (Ne), Nei's gene diversity (H) and Shannon's Information index (I) values of 1.9831, 0.4957 and 0.6888, respectively (Table 4). Contrarily, the genetic diversity in the IITA accessions using ISSR markers was found to be the lowest, with Ne, H, and I values of 1.7166, 0.3817 and 0.5551, respectively. The genetic diversity values of these populations were ranked as EbCp > EnCp > AbCp > IITA from high to low based on polymorphisms at the selected ISSR loci.
With the four ISSR markers, the number of polymorphic loci (NPL) and percentage polymorphic loci (PPL) obtained from the 10 cowpea accessions ranged from 17 to 18 and 94.44-100, respectively ( Table 5)

Genetic diversity revealed by start codon targeted (SCoT) polymorphic markers
Similarly, a total of 10 SCoT polymorphic markers were used to access the level of genetic diversity in the same 18 accessions of V. unguiculata analyzed for genetic diversity using ISSR markers to compare the powers of the two marker systems to discriminate the accessions. Out of the 10 primers used, only five (5) produced scorable bands that were used in the analysis (Fig.4). A dendrogram of the 18 accessions generated by UPGMA procedure similarly grouped the accessions into five major clusters at a similarity index of 0.72 (Fig. 5). Cluster I consisted of accessions AbCp-1 and AbCp-5 from Abia State (AbCp). Cluster II contained only accession AbCp-3 also from Abia State. Cluster III grouped together accessions AbCp-2 and AbCp-4 (Abia), EnCp-1, EnCp-2 and EnCp-4, (Enugu), and IT84S-2246 from IITA, and. Cluster IV contained only accession EnCp-5 from Enugu State while the fifth cluster was subdivided into two subclusters SCI and SCII. SCI contained accession EnCp-3 (Enugu), and accessions EbCp-2, EbCp-3 and EbCp-4 from Ebonyi with accessions EbCp-2 and EbCp-3 from Ebonyi being more closely related. Accessions EbCp-1 and EbCp-5 from Ebonyi, and TVu-264 and Ife Brown from IITA were grouped together in subcluster II (SCII). Cluster V comprised of a mixture of accessions from Enugu, Ebonyi and IITA. Principal component analysis of the generated amplicons from SCoT markers resulted in five clusters (Fig. 6). Each cluster is a representative of unique accession or accessions.
The five (5) SCoT markers amplified a total of 52 alleles. The number of alleles amplified by each marker ranged from 8 to 15, with a mean of 11.1667 (Table 7)

Discussion
Sustainability in food security within sub-Saharan Africa and Nigeria in particular, is dependent on development,   [36]. It has been established that cowpea possesses the characteristic high variability, drought tolerance and adaptability to support food security [37][38][39]. Therefore, genetic diversity study using informative markers is critical in the management, genetic improvement, identification of unique genotypes/accessions and utilization of germplasm [40][41][42]. In this study, ISSR and SCoT markers were used to assess the degree of genetic diversity and relationship among the accessions of V. unguiculata within the Southern and Western Nigeria. Comparing different markers for genetic diversity studies can provide a more informative classification than a single method alone [43]. Our results show both markers have strong discriminating power on the cowpea accessions as indicated by the high values of the genetic diversity indices including number of alleles per primer, PIC values, percent polymorphism, among others. The markers differentiated the accessions into five major groups or clusters which is in agreement with the previous work in which SCoT and ISSR markers were used to study the genetic diversity of 38 grain legume crop, annual Cicer species, though SCoT identified four groups contrary to five groups detected in this study [44]. Also, five clusters have been reported in 35 Vigna accessions using ISSR markers [45]. Some of the accessions got clustered with IITA materials (TVu-264, Ife Brown and IT84S-2246) popularly used as breeding and research materials due to possession of unique traits. The mean activities on seed beetle, Callosobruchus maculatus (F.) using Ife Brown and IT84S-2246 varieties were reported show susceptibility and resistance, respectively [46,47]. This suggests that V. unguiculata accessions identified in the same cluster were more genetically similar while those found on different clusters might be more genetically diverse. The farther away accessions are from each other, the more the possibility of possessing wider genetic diversity which also reflects their locations on clusters as previously reported [48]. It has been noted that populations  having high genetic diversity of neutral markers and alleles could be utilized as suitable candidates for high adaptive variation, fitness and conservation [49][50][51]. From previous studies, allelic richness is an indicator of genetic diversity and it is mostly used in assessment of molecular markers to identify populations for conservation and breeding purpose [51,52]. In this study, both marker types demonstrated high values for total number of alleles mean numbers of alleles, PIC, genetic diversity and total number of polymorphic loci with higher values in SCoT markers in comparison with ISSR. This showed that SCoT markers may be more efficient in studying genetic diversity and relatedness of V. unguiculata. The total number of alleles (ISSR: 4-14; SCoT: 7-15) and PIC (ISSR: 0.2834-0.9073; SCoT: 0.6304-0.9210) values obtained were in agreement with the values reported by Amirmoradi et al. [44] in 38 accessions of grain legume crop (annual Cicer species). A research on a legume crop, chickpea, using 9 SCoT markers revealed as high as 145 alleles and PIC range of 0.43-0.47 in 48 genotypes [53] and this contracts the number of alleles obtained in this study possibly due to the larger number of genotypes and species used. According to Singh et al. [45], 83 alleles were identified in 35 Vigna species using 17 ISSR markers but in this study, as high as 70 alleles were detected with just 4 ISSR markers. The variations in the number of alleles and PIC range observed in this study (SCoT: 80 alleles; 0.6304-0.9210 PIC) with respect to earlier reports in the literature could be attributed to different crop species and number of genotypes involved. However, it is interesting that both the ISSR and SCoT markers used in this work identified as high as 94-100 percentage polymorphic loci (% polymorphism) in the accessions studied. These values are very close to the earlier reports by Amirmoradi et al. [44] wsho reported 85.7-100% and 86.6-100% in ISSR and SCoT, respectively. Zhang et al. [54] reported similar values (ISSR: 82.35-96%; SCoT: 81.25-100%) in a different plant species called Switch grass. Many other reports with other marker systems showed lower percent polymorphisms relative to our findings in this study. For instance, RAPD and microsatellite markers revealed 71.2% [52], 90.0% [55], 55.5% [56], 46.5% [57,58], 64.5% [59] and 58.44% polymorphism [30]. The percentage polymorphism obtained from this present study is quite high indicating the higher informative nature of the markers used (ISSR and SCoT). It has been revealed that high polymorphism identifiable by molecular markers was hinged on the presence of repeated sequences of AC, CA, AG and GA [30]. It has been observed that genetic parameters including Nei's genetic diversity, numbers of effective alleles as well as Shannonʼs information index are very crucial in the study of genetic diversity in plant species [60,61]. They are essential measures of degree of genetic diversity. Among the populations of V. unguiculata accessions evaluated in this study, we found that the Nei's genetic diversity (Ne), effective number of alleles (H) and Shannonʼs information index (I) were highest in Ebonyi population followed by Enugu and Abia (all in the Southeastern Nigeria), while

Conclusion
This finding suggests that accessions from Ebonyi State are probably more genetically distinct and may not have been subjected to hybridization with other accessions and might have originated from various ancestors of cowpea in the past as opined by other researchers [52,62]. The study also revealed that Start codon targeted (SCoT) markers are more efficient in resolving genetic diversity and relatedness than ISSR in cowpea. This is indicated by the higher values of Ne, H, I, Ht, Hs and Nm obtained with SCoT markers compared to those obtained with ISSR markers. The SCoT markers generally exhibited higher values of all the diversity parameters evaluated than ISSR markers, except in major allele frequency, allele count, allele frequency and gene flow. This finding demonstrates that these exceptional parameters may not be good indicators of genetic diversity in V. unguiculata species. The comparative genetic diversity in the accessions of V. unguiculata from Southern Nigeria using ISSR and SCoT polymorphic markers demonstrated the high efficiencies of both markers in discriminating cowpea accessions with SCoT markers being more efficient than ISSR markers. Cowpea accessions from Ebonyi were found to be most genetically diverse compared to those obtained from other States and IITA. In this study, ISSR and SCoT markers have proved to be efficient for genetic fingerprinting and other  potential applications including determination of seed purity, efficient utilization and management of genetic resources in cowpea. They played major roles in the identification of diverse accessions collected by linking some to already known IITA breeding and research accessions (Ife Brown, TVu-264 and IT84S-2246) in which some have been known to be susceptible or resistant to some disease attacks. These can be utilized when making a choice for hybridization to provide genetic barriers against different biotic stressors to improve cowpea breeding. Thus, the results obtained in this study demonstrated a wide genetic diversity of the cowpea accessions. Based on the outcome of this study, we suggest that selection could be made from the accessions in each of the unique clusters detected by the polymorphic SCoT markers as stock for novel gene exchange in crop breeding.

Additional files
Additional file 1: Table S1. Allelic scores, count and frequencies obtained from Vigna unguiculata accessions using Inter-simple sequence repeat (ISSR) markers. (DOC 58 kb) Additional file 2: Table S2. Allelic scores, count and frequencies obtained from Vigna unguiculata accessions using Start codon targeted (SCoT) markers.