Theor Appl Genet (2002) 104:358–366 © Springer-Verlag 2002 S. Coulibaly · R.S. Pasquet · R. Papa · P. Gepts AFLP analysis of the phenetic organization and genetic diversity of Vigna unguiculata L. Walp. reveals extensive gene flow between wild and domesticated types Received: 11 September 2000 / Accepted: 14 June 2001 Abstract Amplified fragment length polymorphisms mesticated forms that has led to a large crop-weed com- (AFLPs) were used to evaluate genetic relationships plex distributed over the entire African continent. In ad- within cowpea [Vigna unguiculata (L.) Walp.] and to dition, wild materials from northeastern Africa are still assess the organization of its genetic diversity. Nei’s lacking. Overall, the superiority of the AFLP technique genetic distances were estimated for a total of 117 acces- over isozymes resided in its ability to uncover variation sions including 47 domesticated cowpea (ssp. ungui- both within domesticated and wild cowpea, and should culata var. unguiculata), 52 wild and weedy annuals be a powerful tool once additional wild material be- (ssp. unguiculata var. spontanea), as well as 18 perennial comes available. accessions of the wild subspecies pubescens, tenuis and alba. AFLP variation was also used to study genetic Keywords Vigna unguiculata · Cowpea · Domestication · variation among and within domesticated and wild ac- AFLP · Genetic diversity cessions based on their geographical origin (western, eastern and southern Africa). Wild annual cowpea (var. spontanea) (HT=0.175) was more diverse than domesti- Introduction cated cowpea (HT=0.108). Wild cowpea was more di- verse in eastern (HS=0.168) than in western Africa Cowpea, Vigna unguiculata (L.) Walpers (2n=2x=22), is (HS=0.129), suggesting an eastern African origin for the an essential crop in less-developed countries of the tro- wild taxon. The AFLP data were consistent with earlier pics and subtropics, especially in sub-Saharan Africa, findings of a unique domestication event in cowpea in Asia, and Central and South America (Singh et al. 1997). the northern part of the continent and suggested that do- As a legume, cowpea is valued for the high protein con- mestication in eastern or southern Africa was unlikely. It tent of its grains, but also for the vitamins and minerals did not allow a more precise localization of domestica- present in the young leaves, pods and peas (Nielsen et al. tion due to extensive gene flow between wild and do- 1997). V. unguiculata includes annual cowpeas (ssp. unguiculata) and ten wild perennial subspecies. Sub- species unguiculata includes all the domesticated (var. Communicated by. J. Dvorak unguiculata), as well as wild and weedy, forms [var. S. Coulibaly · P. Gepts (✉) spontanea (Schweinf.) Pasquet] (Pasquet 1993). Among Department of Agronomy and Range Science, the perennial forms, subspecies pubescens (R. Wilczek) University of California, Davis, CA 95616-8515, USA Pasquet, tenuis ( E. Mey.) Maréchal, Mascherpa and e-mail: [email protected] Stainier, and alba (G. Don) Pasquet are genetically the Fax: +1-530-752-4361 closest to ssp. unguiculata (Pasquet 1999). A widely R.S. Pasquet used classification subdivided all domesticated forms in- Institut de Recherche pour le Développement, to four cultivar-groups based essentially on seed and pod International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya characters (Westphal 1974; Ng and Maréchal 1985). The cultivar-groups include Unguiculata grown as a pulse, R. Papa Biflora (catjang) used mainly as a forage, Sesquipedalis Dipartimento di Biotecnologie Agrarie ed Ambientali, Università degli Studi di Ancona, Via Brecce Bianche, (yardlong or asparagus bean) grown as a vegetable, and 60131 Ancona, Italy Textilis cultivated for the fibers of its long floral pedun- Present address: cles. Recently, Pasquet (1998) proposed the addition S. Coulibaly, Department of Vegetable Crops, of another cultivar-group, namely Melanophthalmus University of California, Davis, CA 95616-8746, USA (blackeyed pea). 359 The African origin of cowpea has never been a point The objectives of the present study were to assay of contention since wild forms are endemic to Africa. AFLP variation in a representative sample of the primary Moreover, of the wild forms, var. spontanea (Schweinf.) gene pool of cowpea to: (1) confirm the occurrence of Pasquet (also referred to as var. dekindtiana sensu a single domestication event and investigate a more- Verdcourt by some authors) is accepted as the most like- precise origin of domestication for cowpea; (2) assess ly progenitor of domesticated cowpea (Padulosi and Ng the importance of gene flow in the current organization 1997; Pasquet 1999). Its morphology and growth habit of domesticated cowpea; and (3) compare the AFLP and are very similar to that of cowpea landraces, but it also isozyme-derived organization of genetic diversity in possesses wild-like attributes such as shattering pods cowpea. with small seeds. Despite the wide distribution of var. spontanea throughout sub-Saharan Africa, molecular studies point to a unique domestication event (Panella Materials and methods and Gepts 1992; Pasquet 1999). A disagreement exists, however, as to where cowpea was domesticated. Two do- Plant materials mestication areas have been proposed, in western and northeastern Africa, respectively (Baudoin and Maréchal A total of 117 accessions, reflecting the geographical distribution and current taxonomy of V. unguiculata ssp. unguiculata and 1985; Ng and Maréchal 1985; Vaillancourt and Weeden closely related taxa, were evaluated (Table 1). The accessions in- 1992; Ng 1995; Pasquet 2000). A western African center cluded 47 domesticated annuals, 52 wild and weedy annuals, and of domestication has been proposed based on: (1) the 18 perennial forms. Domesticated materials (D) were divided into highest level of morphological diversity for cultivated three groups based on their geographical origin: western-central Africa (DW), eastern Africa (DE), and southern Africa (DS). cowpea, (2) the existence of weedy intermediates be- Likewise, wild accessions (W) were divided into western, eastern, tween wild and cultivated cowpeas, (3) the oldest arche- and southern African groups (WW, WE, and WS). The perennial ological evidence for cowpea in Ghana, and (4) the iden- accessions belonged to ssp. pubescens, ssp. tenuis, and ssp. alba. tification of a wild and a cultivated accession with an For comparative purposes, all accessions used in the AFLP work identical cpDNA in Nigeria (Ng and Maréchal 1985; had previously been surveyed with 36 isozyme loci (Pasquet 1999, 2000). Vaillancourt and Weeden 1992; Ng 1995). An alternative center of domestication of cowpea has been proposed in northeastern Africa based on: (1) the absence of true AFLP analysis ecologically wild cowpea in West Africa, (2) the high level of morphological diversity of wild cowpea in the Approximately 5 g of leaf tissue were harvested from plants grown in the greenhouse and used for extraction of total genomic region from Ethiopia to South Africa (Baudoin and Mar- DNA, based on the CTAB procedure described by Doyle and échal 1985), and (3) results from ethnobotanic, linguis- Doyle (1987). The amplified fragment length polymorphism tic, and isozyme studies (Pasquet 1996). Isozyme studies (AFLP) protocol followed was that described by Vos et al. (1995). have revealed the absence of a center of diversity in All adaptors and primers are listed in Table 2. AFLP banding pat- terns were revealed by exposure of X-ray films (Kodak X-Omat, West Africa (Vaillancourt et al. 1993) and a higher level Sigma Chemical Co., St. Louis, Mo.) for 1–2 days. Only the bands of genetic diversity in cultivars of Ethiopian origin that could be read unambiguously on each autoradiograph were (Pasquet 2000). considered for analysis. Some important shortcomings of previous studies in- clude the limited size and representativity of the samples Genetic distance estimates and cluster analysis used. In particular, of the 26 wild accessions surveyed by Vaillancourt and Weeden (1992), only 12 correspond to Each band was treated as a separate putative locus, and scored as the most-probable progenitor of cowpea, var. dekind- present (1) or absent (0) in each accession. Estimates of similari- tiana sensu Verdcourt. They include accessions from just ties were based on three different measures: (1) Nei and Li’s simi- larity index (1979) developed for endonuclease restriction-gener- five countries (Nigeria, Burundi, Tanzania, Malawi and ated fragments, also known as the Dice coefficient; (2) Jaccard’s Zimbabwe). Among the domesticated accessions, the (1908) coefficient; and (3) Nei’s (1972) similarity. cultivar-group Textilis was missing. Another caveat Cluster analysis was based on the unweighted paired-group shared by studies of proponents of both scenarios is the method using arithmetic averages (UPGMA) of the NTSYS-pc software (Rohlf 1992). The dendrogram was created with the limited level of polymorphism uncovered by isozymes TREE option of NTSYS and the goodness of fit of the clustering and cpDNA markers. For example, only two cpDNA to the data was calculated using the COPH and MXCOMP proce- patterns were identified among 32 domesticated acces- dures (Rohlf 1992). Similarly, the correspondence between iso- sions (Vaillancourt and Weeden 1992). In the study by zyme and AFLP-based clustering of the accessions surveyed was Panella and Gepts (1992), 14 of the 24 putative isozyme tested. In order to identify major cluster