Introduction

Cowpea [Vigna unguiculata (L.) Walpers] is an important food legume grown in semi-arid regions, viz. Sub-Saharan Africa, Brazil and India. It is usually intercropped with cereals (sorghum or millet) but also cultivated in rotation as a sole crop. Cowpea is valued for its high protein content and is consumed as dry seeds, fresh peas, green pods or leaves (Menedez et al. 1997). In addition, the crop fixes 80% of its nitrogen requirement for growth from the atmosphere (Asiwe et al. 2009), thereby reducing nitrogen fertilizer demand and costs of crop production.

The study of genetic diversity is important in a crop breeding program for the selection of suitably diverse parents to obtain heterotic hybrids as well as for germplasm characterization and conservation. Various morphological, biochemical and molecular markers are used for the characterization of germplasms. Molecular marker-based characterization is a useful complement to morphological and physiological characterization of cultivars, because they are plentiful, independent of tissue or environmental effects and allow cultivar identification in the early stages of plant development (Miller and Tanksley 1990).

The diversity of different cultivars of cowpea has been accessed by morphological and physiological traits (Musvosvi 2009; Ghalmi et al. 2009), allozymes (Garba and Pasquet 1998; Pasquet 1999, 2000) and seed storage proteins (Panella et al. 1993; Fosto et al. 1994). DNA-based molecular markers like chloroplast DNA polymorphism (Vaillancourt and Weeden 1992), RFLP (Fatokun et al. 1993), AFLP (Fang et al. 2007) and simple sequence repeats (SSR) (Uma et al. 2009; Asare et al. 2010) have been used for genetic diversity analysis of cowpea.

Among various PCR-based molecular markers, RAPD markers had become more popular because their application does not need any prior information about the target sequences on the genome (Welsh and McClelland 1990). RAPD, being a multi-locus marker (Karp et al. 1997) with the simplest and fastest detection technology, has been successfully employed for determination of intraspecies genetic diversity. This has been used widely for the estimation of genetic variability as well as the cultivar identification/differentiation of cowpea (Akundabweni 1995; Menedez et al. 1997; Mignouna et al. 1998; Tosti and Negri 2002; Pasquet and Gepts 2004; Pandey et al. 2004; Diouf and Hilu 2005).

In this study, analysis of genetic diversity among ten Indian cultivars of cowpea using 18 sets of RAPD markers is reported.

Materials and methods

Ten Indian cowpea cultivars viz. IC 9883, IC 97704, VRC 32, VRC 7, PIC 246, IC 9739, IC 2875, VRC 11, N 106 and PG 1 seeds were collected and used in this study. The genomic DNA was isolated from leaves using the CTAB method (Rogers and Bendich 1988), analyzed and quantified by standard methods (Maniatis et al. 1989). Eighteen RAPD primers were used for setting up PCR reactions (Malviya and Yadav 2010), and resulting amplicons were analyzed on 1.8% agarose gels.

The amplicons generated from different primers were scored separately on the basis of the presence or absence of bands corresponding to each cultivar, i.e., using binary code 1 and 0 for the presence or absence of band, respectively, regardless of its intensity. The unweighted pair group method using arithmetic average (UPGMA) clustering was carried out by applying the Past software (Qyvind and Harper 2005). Support for clusters was evaluated by bootstrap analysis with Win Boot software (Yap and Nelson 1995) by generating 1,000 samples after resampling with replacement of characters within the combined 1/0 data matrix. This matrix also was reduced to two dimensions by projecting the cultivars in two-dimensional space (Cruz and Viana 1994) using the software Past (Qyvind and Harper 2005). Genetic diversity (H), arthimatic mean heterozygosity (H av) and average heterozygosity for polymorphic markers [(H av)p] were also calculated (Nei 1987; Powell et al. 1996).

Results and discussion

Among 18 RAPD markers, only 12 RAPD markers showed significant polymorphism and were used for analysis of genetic diversity in cowpea cultivars. The genetic diversity of 17 cultivars of another grain legume, pigeon pea [Cajanus cajan (L.) Millsp.], using the same 18 RAPD markers has been reported (Malviya and Yadav 2010). These 12 RAPD markers generated a total of 181 bands ranging in size from 82–7,634 bp with 148 (81.7%) bands showing polymorphism (Table 1). A 64.5% polymorphism has been reported for six cowpea genotypes using RAPD markers (Sharawy and Fiky 2003). The degree of polymorphism among the ten cultivars ranged from 69.2 to 92.3%. A maximum of 23 bands were obtained by primer 12ES10G23, while 17ES10C28 primer resulted in the generation of 18 bands. A minimum of 11 bands were obtained with 16ES10C27 primer. The RAPD profiling revealed an average of 15 bands per primer, while 6 out of 12 primers generated more than 15 bands. Variation in genetic diversity among cowpea cultivars using different primers ranged from 0.1742 to 0.4054. Genetic diversity ranging from 0.03 to 0.34 has been reported for 20 landraces of cowpea scattered throughout Algeria using RAPD markers (Ghalmi et al. 2009). The heterozygosity calculated for all the 181 bands across the cultivars revealed the H av and H avp values to be 0.0177 and 0.0216, respectively. Genetic variation with heterozygosity of 0.123 and 0.223 for domesticated cowpea and its wild progenitors respectively has been reported (Ba et al. 2004).

Table 1 The number of loci and genetic diversity detected in cowpea cultivars using RAPD markers
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The similarity index among the cowpea cultivars varied from 0.181 to 0.923 because of the diversification in morphology and parentages. Cultivars IC-2875 and VRC-7 as well as cultivars N-106 and IC-2875 revealed maximum similarity of 0.923, while cultivars VRC-7 and IC-9883 showed the least genetic similarity.

The dendrogram constructed with high bootstrap values revealed the two distinct clusters I and II comprised of 2 (IC-97704 and PIC-246) and 7 (VRC-32, VRC-11, VRC-7, IC-2875, N-106, PG-1 and IC-9739) cultivars, respectively, while one cultivar IC-9883 occupies a unique position (Fig. 1a). A similar pattern was reflected in the projection of the genetic distances among ten cultivars of cowpea in two-dimensional space (Fig. 1b). The RAPD profiling of these cultivars also resulted in identification of cultivar-specific unique bands. A total of 27 unique bands specific for different cultivars ranging in size from 149 to 6,642 bp have been observed (Table 1). Genetic variability among cowpea varieties using RAPD resulting in fragments of sizes varying from 300 to 2,000 bp has been reported (Zannou et al. 2008). In case of cultivars N-106, IC-9883, VRC-7 and VRC-32, unique bands were obtained with primers 14ES10A25, 19ES10T30, 20ES10A31 and 22ES10G33, respectively. These unique bands can be utilized as ready reference for cultivar identification and also can be converted to other molecular markers like SCAR after its subsequent cloning and sequencing.

Fig. 1
figure 1

Projection of the genetic distance among ten cowpea cultivars. a Dendrogram constructed using UPGMA method; b two-dimensional space representation

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The narrower genetic base obtained in this study is similar to earlier reports based on the fact that a single domestication event is involved in the origin of this crop (Doebley 1989; Pasquet 2000; Coulibaly et al. 2002; Ba et al. 2004). Seed conservation generally limits the exchange of germplasm throughout the country and also inhibits the integration of genotypes from other sources outside of India into local breeding programs. These factors might contribute to limiting genetic diversity.

In conclusion, the genetic diversity among ten Indian cowpea cultivars has been revealed using RAPD markers that could be further studied with more sets of RAPD markers and also by employing other DNA-based molecular markers.