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Ministerial Conference on Harnessing Science and Technology to Increase Agricultural Productivity in Africa:  West African Perspectives
 Ouagadougou, Burkina Faso
June 21 – 23, 2004  

J.T. Ouédraogo.1 Institut de l’Environnement et de Recherches Agricoles (INERA), 01 BP 476 Ouagadougou 01, Burkina Faso;

Striga gesnerioides (Willd.) Vatke is the most important flowering parasitic plant causing substantial yield reduction in cowpea in the dry savannas of sub-saharan West and Central Africa. Collaborative trials with international and national research institutes have identified the presence of five races of Striga gesnerioides. Race 1 is found in Burkina Faso and Mali, race 2 in Mali, race 3 in Niger, Nigeria and probably in Burkina Faso, race 4 in Benin and race 5 in Burkina Faso, Nigeria and Cameroon.

To overcome the damage caused by this noxious plant, several methods have been developed and compose an integrated control strategy. The most important component of this strategy is the genetic resistance of the cowpea varieties. Several resistance genes were identified and should be well characterized and pyramidized in one superior variety resistant to all races. New biotechnology tools such as DNA markers, gene cloning and sequencing can help in the breeding programmes.

The present paper reports the utilization of the Amplified Fragment Length Polymorphism technique to identify DNA markers linked to the resistance gene for different races, the transformation of some of those markers in routinely used marker form and the construction of an improved genetic linkage map of cowpea.

Several DNA markers were found that are linked to the resistance genes to Striga races 1 and 3 the more prevalent ones. These markers will be appropriate substitutes to the natural infestations/inoculations required during the striga resistance breeding process. Everywhere, the breeder can select for Striga resistance based on the presence of the marker, without any physical contact with the Striga seeds. This type of selection called marker assisted selection is more efficient and faster than the conventional one. Based on these markers, the genes can be cloned and used to transform other crops such as sorghum and millet that are lacking Striga resistance genes.

Molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and resistance genes analogues were also placed on the new map, including markers for resistance to Striga gesnerioides races 1 and 3, virus resistance, Fusarium wilt, and root-knot nematodes.

The identification and mapping of DNA markers linked to Striga resistance is a stepping stone for the development of tools for use in marker-assisted selection programs and the eventual cloning and characterization of the genes encoding resistance to this noxious parasitic weed.

Cowpea (Vigna unguiculata (L.) Walp.) is a major food legume grown in the semi-arid regions of West and Central Africa, South America, and India. For many people in these regions it constitutes an important dietary protein source. Like other food crops, cowpea is attacked by a variety of pathogens, among which the parasitic flowering plant Striga gesnerioides (Willd.) Vatke is a major constraint to cowpea production. S. gesnerioides parasitism causes severe chlorosis, wilting, and stunting of susceptible hosts resulting in yield losses estimated in the millions of bushels annually (Aggarwal and Ouedraogo 1989; Muleba et al. 1997). Control of the parasite by chemical and cultural treatments is difficult and expensive and, therefore, significant effort has been put into the identification of natural sources of genetic resistance within cowpea cultivars and to the selection and breeding for improved lines. The first S. gesnerioides resistant cowpea varieties identified were Suvita-2 (Gorom local) from Burkina Faso and 58-57 from Senegal. Subsequently, additional sources of resistance were identified including B301, a landrace from Botswana (Singh and Emechebe 1990a), 872, a landrace from Niger, APL1, a landrace from Nigeria (Lane et al. 1997a,b) and two breeding lines developed at the International Institute of Tropical Agriculture (IITA), IT81D-994 and IT82D-849. Although resistant in Burkina Faso, when grown in Nigeria, Suvita-2 proved to be susceptible to S. gesnerioides parasitism subsequently showed that differences exist in the pathogenicity of S. gesnerioides populations from different locations in West Africa leading to our current understanding that at least five different races of S. gesnerioides are present in West and Central Africa (Lane et al. 1997b). Well adapted, high-yielding cultivars resistant to all five races of S. gesnerioides are under development, but not widely available. Among cultivars in use, Suvita-2 and IT81D-994 are resistant to races 1, 2, and 4, whereas B301 and IT82D-849 are resistant to races 1, 2, 3, and 5, (Lane et al. 1997b).

Currently, as revealed by studies on inheritance of resistance, evidence exists for three independent dominant resistance genes to S. gesnerioides (designated as Rsg1, Rsg2, and Rsg3) in varieties B301, IT82D-849, and Suvita-2, respectively (Atokple et al. 1995; Singh and Emechebe 1990a, 1996). Although good sources of resistance, cowpeas such as B301 and 58-57, have poor seed and agronomic qualities (Atokple et al. 1995; Lane et al. 1997a). Using traditional breeding and selection methods, attempts to create commercially acceptable cultivars, free of undesirable genetic traits and resistant to all five races of S. gesnerioides have not yet been successful. Using backcross breeding methods, several generations of backcrossing, taking up to seven or more field seasons, may be necessary to obtain lines carrying the desired character. The recent development of the DNA marker systems has contributed to a better understanding and utilization of many genes of resistance to plants enemies (Michelmore, 1995; Kumar, 1999). The DNA molecular markers have been wildly used to construct genetic linkage maps of many important crop plants including cowpea (Menéndez et al. 1997). DNA marker types such as amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995), and simple sequence repeat (SSRs) combined with the bulked segregant analysis (BSA) (Michelmore et al. 1991) made possible to rapidly identify molecular markers within plant genomes linked to agronomically important genes. Following their identification, the linked markers can be used in a marker-assisted selection program or as a stepping point to map-based cloning of the related genes (Kumar, 1999; Young, 1999).

The contraints to cowpea breeding for Striga resistance are the existence of several races (five identified to date), the long time and the difficulty to pyramidize the different resistance genes into one single cowpea cultivar and the need to select for resistance in a field infested with Striga seeds. All these constraints can be overcome by using molecular tools such DNA markers linked to the resistance genes, their usage in a marker assisted program and the cloning and use of the resistance genes to transform susceptible cowpea varieties or even crops like sorghum, millet or maize.

The most important Striga races are race 1 and race 3. For this reason, the first studies have been undertaken for the resistance genes to these two strains (Ouedraogo et al. 2001; 2002a). Using the four cultivars, Suvita-2, B301, IT81D-994 and IT82D-849 resistant to race 1, four F₂ segregating populations were generated by self-fertilizing the F₁ individuals in order to identify the amplified fragment length polymorphism (AFLP) markers.The parents and 150 F₃ lines obtained from F₂ plants of a cross between Tvu 14676 (resistant) and IT84S-2246 (susceptible) were grown under S. gesnerioides race 3 and used to identify the AFLP markers linked to the gene conferring the resistance to that striga race.

Three AFLP markers tightly linked to the resistance gene to race in the cultivar IT82D-849 have been identified. Seven markers were found linked the resistance gene from the cultivar Gorom, following analysis of an F₂ population from the cross between Gorom and the susceptible cultivar Tvx 3236. Analysis of a population derived from the cross between Tvx3236 and the resistant cultivar IT81D-994 identified five markers linked to the resistance gene of this cultivar. Five markers linked to the resistance gene in the cultivar B301 were identified. The distances between the markers and the genes varied from 0.9 to 10 cM.

Tvu 14676, a S. gesnerioides race 3 resistant line showed that resistance to S. gesnerioides race 3 was controlled also by a single dominant gene. Six AFLP markers linked that gene were identified. All these markers are highly suitable for use in a marker assisted selection program (MAS) to introgress resistance genes to Striga race 1 or race 3 into promising breeding lines. To this end we are currently attempting to make these markers more informative tools by transforming them into sequence characterized amplified regions (SCAR).

Conversion of the AFLP markers into PCR-based markers

The AFLP markers being dominant markers, they cannot allow the breeder differentiate heterozygous from dominant homozygous individuals. The sequence characterized amplified region (SCAR) marker developed from the dominant markers is codominant. It therefore distinguish the heterozygotes from the either of the two homozygotes. This type of PCR-based marker is well suited for a MAS programme. Figure 1 shows a PCR profile of a SCAR marker derived from an AFLP marker linked to the IT82D-849 cowpea cultivar resistance gene (Figure 1). Used in MAS program, this type of marker has three major advantages : first it is an excellent substitute for screening under infestation or inocculation. This screening without any inocculum can be done at any developmental stage of the plants; second, the big constraint in introgressing genes using the conventional methods is the transfer of too large blocks of genes not necessarily of interest. The MAS allow the transfer and selection of the desired traits and the quick recovery of the reccurent parent genotype. Third, gene marking through resistance genes identification in many cultivars, allows their deployment and pyramiding in superior cultivars. Today, very cheap technologies exist and markers like SCARs are promising tools for breeding programmes.

For striga, there is no need to infest the plot with striga seeds to select for the resistance. The presence of the marker implies the presence of the resistance gene in the individual. The breeding is made more efficient and faster compared to the conventional methods.

A direct application of genetic linkage maps has been in tagging genes of economic importance with molecular markers.

High-resolution genetic maps provide breeders with powerful tools for analyzing the inheritance of genes of interest, for monitoring the transmission of specific genes or genomic regions from parents to progeny, and for map-based cloning of those genes (Kumar 1999). Moreover, the DNA markers present in these maps can often be used in marker-assisted selection strategies capable of increasing breeding efficiency and overcome some limitations of conventional breeding methods (Young 1999).

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata (L.) Walp) based on the segregation of various DNA markers, biochemical and biological resistance traits. The new genetic map of cowpea consists of 11 linkage groups spanning a total of 2670 cM, with an average distance of 6.43 cM between markers (Ouédraogo et al., 2002b). Molecular markers associated with various biological resistance/tolerance traits, resistance genes, and RGAs were also placed on the map including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes.

Six of the markers linked to striga resistance genes were located on to the linkage group 1. These markers are linked to the S. gesnerioides race 1 and race 3 resistance genes found in the resistant cowpea lines B301, IT82D-849 and Tvu 14676 (Ouédraogo et al. 2001). Three other markers linked to Striga resistance genes mapped to linkage group 6. These markers are linked to the S. gesnerioides race 1 resistance genes found in cultivars Gorom and IT81D-994 (Ouédraogo et al., 2002a).

Locating some markers on the cowpea map opens ways to the resistance genes map-based cloning. Map-based cloning of genes of interest has been facilitated by the recent development of the DNA markers systems. Genetic mapping is the first step toward map-based cloning of genes and the actual cowpea map makes the markers reported in the present work the most more useful stepping point for that purpose. The laboratory of Pr M.P. TIMKO of the University of Virginia is closely working with INERA on this matter.

Biotechnology tools such as DNA markers linked to resistance genes have greatly contributed to the cowpea improvement. MAS programme can be undertaken by the developping countries. Marker assisted selection: A fast track to increase genetic gain in plant. Based on the identified markers and the improved genetic linkage map constructed, more advanced laboratories can help clone the resistance genes and open ways to genetic transformation of cowpea and other crops susceptible to striga. International research institutes like the International Institute of Tropical Agriculture is on the way to develop a transformation and regenration protocols for cowpea. This will strengthen the national programmes capacities to undertake more biological works on striga and other traits resistance breeding.

Africa missed the green revolution and no excuse to miss the biotechnology revolution.

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