Important sources of genetic resistance to Macrophomina were identified in cowpea genotypes assessed under mild drought-stress conditions. Based on field screening, genotypes IT98K-499-39, Suvita 2, IT93K-503-1, and Mouride exhibited the highest levels of resistance and are potential sources of host resistance in management strategies in response to Macrophomina infection.
Screening a RIL population derived from a cross between the resistant IT93K-503-1 and susceptible CB46 genotypes facilitated the genetic analysis of the resistance trait in one of these highly resistant germplasm sources. Our QTL mapping approach took advantage of the recently developed high density EST-derived SNP marker consensus map of cowpea  in which the IT93K-503-1 × CB46 RIL was a constituent map. The QTL mapping indicated a quantitative basis of this trait and also transgressive segregation for both resistance and susceptibility. The transgressive lines with higher resistance levels indicate the potential for selecting novel resistance forms in breeding populations in which the susceptible parent contributes to the resistance phenotype.
Furthermore, genic SNP markers with disease resistance gene annotation and disease resistance-associated genes were found in the genomic intervals defined by the QTL and these were located within syntenic regions in soybean and Medicago. Most interestingly, pectin metabolism related genes were identified in three major QTL intervals. The role of plant cell wall polysaccharides, including pectins, in defense against pathogenic microbes has been described , and Radha  demonstrated that French bean plants infected with Macrophomina had 50% less pectin compared to healthy ones, an observation attributed to the pectinolytic activity of enzymes released by the pathogen. Examples of the role of pectins in pathogen defense in other systems include the over expression of pectin methylesterase inhibitors in Arabidopsis that resulted in increased resistance to the necrotrophic fungus Botrytis cinerea . Similarly, disrupting a pectin methylesterase gene resulted in reduced virulence of B. cinerea on apple (Malus domestica), grapevine (Vitis vinifera) and Arabidopsis . Since Macrophomina and B. cinerea share similar pathogenicity mechanisms involving pectin degradation as described above, these results suggest an important role for pectin-related genes in defense against Macrophomina. However, this cannot be addressed in the present study, and additional functional genetic studies will be required to validate this potential role. QTL Mac-2 coinciding with a pectin esterase inhibitor had the highest level of expression and statistical significance of all eleven resistance loci mapped in this study, making it a suitable target for molecular characterization. The significant effect of Mac-2 on Macrophomina resistance will likely result in noticeable and quantifiable change in phenotype sufficient to support its role as a Mendelian factor. Several other QTL mapped in intervals where disease resistance genes were found based on the cowpea genic-SNP markers and synteny with soybean and Medicago. These provide excellent additional candidate gene targets for functional characterization.
This study demonstrated the co-location of Macrophomina resistance and maturity related senescence QTL in the RIL population used for mapping. This finding in cowpea supports the suggested association between early maturity and susceptibility to Macrophomina in other crops [22, 19, 23]. The same pattern of association between earliness and susceptibility to fungal pathogens has been described in other systems as well. Notably, early maturing potato cultivars have been shown to be more susceptible to the late blight disease caused by the fungal pathogen Phytophthora infestans [30–32]. Similar to our findings, QTLs independent of maturity effects have been reported providing the opportunity to breed for early-maturing, late blight-resistant potato cultivars [31, 32]. Gebhardt and Valkonen  suggested that this association was pleiotropic in nature with the same genes affecting both traits in potato. Interestingly, based on cowpea synteny with soybean and Medicago, both flowering and chlorophyll-metabolism related candidate genes were identified within the two maturity-associated QTL intervals and no apparent disease resistance genes were identified. Although this does not exclude the role of other genes within these QTL intervals, these findings may provide additional molecular evidence for the association between early maturity and Macrophomina susceptibility that has been described previously.
The association between early maturity and Macrophomina susceptibility was demonstrated further by the locus carrying Mac-7 and Mat-1. This locus had the largest effect on maturity-induced senescence in a previous study , and in the current study, also had the strongest effect on mean percent mortality in both single locus and pair-wise loci analyses. However, the co-location of the maturity and Macrophomina response QTL on this locus may limit its potential application in marker-assisted breeding programs due to tight linkage and possible pleiotropic effects. Breeding for Macrophomina resistant early-maturing varieties that are an important drought escape strategy in parts of West Africa will be complicated by the opposite effects that this locus confers to earliness and Macrophomina resistance. In our simulation study we selected Mac-2 and Mac-5 QTLs as possible target QTLs for pyramiding to improve Macrophomina resistance. The presence of these two QTLs resulted in a doubling in reduction of Macrophomina -induced mean percent mortality compared to the presence of either QTL individually. This gain in effect was, however, much less significant (gain of 1% - 3%) when we added a third QTL to this combination, suggesting that for breeding purposes, the value of pyramiding three loci compared to two may be markedly diminished.
Genetic overlap was observed in cowpea only between three QTLs mapped in response to drought-induced premature senescence and response to Macrophomina infection. This represented a relatively small proportion of overlap considering the ten QTLs mapped for the drought response  and nine QTLs mapped here for response to Macrophomina infection. Whether this co-location is due to linkage or pleiotropy will require further experimentation especially for Mac-5 QTL where both osmotic stress and disease resistance genes were found in the corresponding syntenic soybean genomic region. Similar findings were reported in sorghum where the non-senescence drought tolerance trait was shown to be largely independent from the Macrophomina resistance trait . These findings have important implications for cowpea breeding programs aimed at improving productivity under arid and semi-arid conditions. Macrophomina can cause significant crop losses even under mild drought stress conditions, and introgression of Macrophomina resistance loci should constitute an important component of the breeding schemes targeting genetic improvement with drought tolerance. Furthermore, because early maturing varieties are used as a drought escape strategy in rain-fed production systems, the introgression of non-maturity related resistance loci will be required to mitigate their susceptibility.