Genetic diversity in cowpea [ Vigna unguiculata ( L . ) Walp . ] varieties determined by ARA and RAPD techniques

Cowpea, Vigna unguiculata (L.) Walp. presents phenotypical variabilities and in order to study the genetic diversity of cultivated Senegalese varieties, two experimental approaches were used. First, a physiological characterization based on nitrogen fixation was used to assess cowpea breeding lines. Inoculation with two Bradyrhizobium strains (NGR234 and ISRA312), showed a difference in nitrogen fixation potential between the cowpea varieties. Diongoma is the highest nitrogen fixing variety, whereas Mouride is the lowest. The second approach employed genetic characterization based on DNA polymorphism to screen. Results suggest that random amplified polymorphic DNA (RAPD) technology can be used to reorganize the national germplasm in order to eliminate the putative duplicates, and to identify elite varieties.


INTRODUCTION
Cowpea, Vigna unguiculata (L.) Walp. is a tropical grain legume which plays an important nutritional role in developing countries of the tropics and subtropics, especially in sub-saharan Africa, Asia, Central and South America (Singh et al., 1997). Because of its high protein content (20-25%), cowpea has been referred to as "poor man's meat." Cowpea young leaves, pods and peas contain vitamins and minerals which have fuelled its usage for human consumption and animal feeding (Nielsen et al., 1997;Rachie, 1985). Despite its importance, the production of cowpea which is about 1000 kg/ha in Sub-saharian regions, does not meet the need of consumers (Ehlers and Hall, 1997). The low yield is the result of poor soil, particularly in nitrogen and the high cost of chemical fertilizers. However, cowpea establishes symbiotic association with Bradyrhizobium bacteria enabling it to fix atmospheric nitrogen. Nevertheless, breeders and agronomists have not given high priority to cowpea varieties with high nitrogen fixing potential in breeding program in order to improve production and maintain soil fertility. Attempts have, however, been made to screen for a cowpea variety with *Corresponding author; E-mail: ddiouf@ucad.sn Abbreviations: ARA, Acetylene Reduction Activity; RAPD, Random Amplified Polymorphic DNA; AFLP, Amplified Fragment Length Polymorphism. higher nitrogen fixing potential (Gueye and Ndiaye, unpublished).
The objective of our work is to identify cowpea varieties with high nitrogen fixing potential, to verify the pattern of variation within cowpea breeding lines in Senegal, and to evaluate the applicability of ARA and RAPD techniques in the screening of cowpea varieties. The highest nitrogen fixing varieties will be selected for subsequent breeding programs.

In vitro culture, plant nodulation and nitrogen fixation assessment
Cowpea seeds were sterilized with 70% alcohol for 15 min and washed 3 times with sterile distilled water. Following sterilization, the seeds were imbibed for 3 h with sterilized distilled water and germinated in Petri dish containing 8 g/l of Agar-Agar (Sigma) for 48 h in the dark at 30ºC. Cowpea were grown in a greenhouse with a photoperiod of 12 h, 20 w/m 2 at 28ºC in Leonard apparatus containing sterilized sand from Cambérène. The plants were watered daily with Jensen medium (Vincent, 1970). In order to assess their nitrogen fixation, the cowpea plants were inoculated with fresh cultures of Bradyrhizobium strains, NGR234 and ISRA312. NGR234 is a promiscuous strain because it nodulates many legumes, whereas ISRA312 is only effective on V. unguiculata (Léwin et al., 1987). After 5 weeks of growing, nodules were harvested, the aboveground parts of plant weighed, and nitrogen fixation assessed by ARA method according the protocol described by Hardy et al. (1973).

Molecular characterization of cowpea with RAPD
DNA was extracted from leaves of seven cowpea varieties (Table 1) from the Senegalese national germplasm according to Fulton et al., (1995). PCR amplifications were performed in a 0.2 ml tube containing 2 mM MgCl2, 1 mM of each primer, 200 mM of each dNTP, 1.5 U of lyophilized Taq (Amersham Pharmacia Biotech) and 25 ng of genomic DNA in a final volume of 25 ml. Forty four primers (Genosphere Biotechnologies) were used in this study. The reaction were placed in a PTC-100 thermocycler (MJ Research Inc.) programmed for predenaturating step of 3 min at 94°C followed by 45 cycles of 1 min 94°C, 1 min at 36°C, 2 min at 72°C and a final extension of 5 min at 72°C. Amplification products were electrophoresed on 1.8% agarose gel, stained for 30 min with 1 mg/l of ethidium bromide and photographed under UV light.

Physiological genotyping of cowpea
The effect of cowpea varieties on dry weight of aboveground parts and nodules, as well as the number of nodules are presented in Table 1. Ndiambour and Diongoma varieties have the highest dry weight of the aboveground parts, while Mouride had the lowest. The number of nodule per plant and their dry weight also differed between the cowpea varieties. Table 1 also showed that the varieties with low number of nodules have higher nodule dry weight. We also observed that nodulation by ISRA312 was more profuse than that of NGR234 (data not shown). There were also differences in nitrogen fixation between the cowpea varieties when inoculated with the two Bradyrhizobium strains (Table 2). Diongoma variety has the highest nitrogen fixation (with ISRA312), while Mouride has the lowest.

Molecular genotyping of cowpea varieties
Forty-four primers were screened with DNA samples from seven cowpea varieties. Of these, ten primers did not Fall et al. 49 amplify the DNA; nine amplified but did not show any discrimination, while 25 gave polymorphic RAPD patterns. Eleven (A1, A13, A14, B6, B10, B11, B12, B15, F4, F13, F16) of the 25 polymorphic primers were selected because they gave very clear bands. In total, 331 bands (amplification products) were produced by the PCR reactions. Sixty-one of the bands were polymorphic (Figure 1).

DISCUSSION
Assessment of nitrogen fixation in cowpea has been conducted in some Nigerian and Senegalese varieties (Gueye, 1982). However, varieties which are mainly cultivated in Diourbel, Thiès, Louga and Saint-Louis (North West regions of Senegal) where the lands are poor, were not represented in this study. The main objective of our study is to identify a better cowpea variety-Bradyrhizobium strain symbiotic combination for improving the yield of the farmer and soil fertility. Our results confirmed that biological nitrogen fixation is dependent on both the plant and the nodulating bacteria, as have been reported in other studies (Gueye and Bordeleau, 1988;Kishinevsky and Zur, 1997). Successful inoculation of the cowpea varieties with ISRA312 and NGR234 indicates that cowpea could be nodulated by more than one Bradyrhizobium strain. In general, ISRA312 induced more nodules and fixed more nitrogen than NGR234. Two main varieties, Diongoma having a higher nitrogen fixation, and Mouride which is a much lower nitrogen fixing variety, were identified in this study.
Afr. J. Biotechnol. Variation in nitrogen fixation has also been reported in other legumes such as Faidherbia albida (Gueye et al., 1997;Sanginga et al., 1990). Other studies using 15 N in both semi-controlled and field conditions also indicate that Diongoma belongs to the group of higher nitrogen fixing cowpea varieties (Gueye M., unpublished). This difference in nitrogen fixation may be due to the genetic framework of the different varieties.
Physiological assessment is not sufficient for adequate characterization of plant varieties because nitrogen fixation can be influenced by many environment factors (Antolin et al., 1995). For these reasons, molecular markers offering more valid results were used in our studies in order to improve the physiological approach. Previous studies using alloenzymes, RAPD and microsatellites, indicate a low level of variation in cowpea (Li et al., 2001;Pasquet, 2000;Tosti and Negri, 2002). However, high level of polymorphism has been observed in lentil, soybean and cowpea using AFLP technique (Coulibaly et al., 2002;Maughan et al., 1996;Sharma et al., 1996). In addition, a high level of polymorphism was observed in Phaseolus using RAPD (Bai et al., 1998). In addition to physiological assessment, our data showed that some DNA fragments could be specific to the higher or lower nitrogen fixing varieties suggesting that some genes could govern the higher nitrogen fixation character in cowpea. Our findings also provide an alternative avenue for understanding the biological nitrogen fixation process and the genetic identification of parent plants in a breeding program.