Jeffrey D. Ehlers

COWPEA (VIGNA UNGUICULATA L. WALP.)

Abstract Cowpea (Vigna unguiculata L. Walp.) is a widely adapted, stress tolerant grain legume, vegetable, and fodder crop grown on about 7 million ha in warm to hot regions of Africa, Asia, and the Americas. This review focuses on major breeding achievements, current objectives, and future opportunities for cowpea improvement. Early maturing cultivars have been developed with regionally acceptable grain quality and resistance to some important diseases and pests including bacterial blight (Xanthomonas campestris), cowpea aphid-borne mosaic virus (CABMV), cowpea aphid (Aphis craccivora), cowpea curculio (Chalcodermus aeneus), root-knot nematodes (Meloidogyne incognita and M. javanica), cowpea weevil (Callosobruchus maculatus) and the parasitic weeds Striga gesnerioides and Alectra vogelii. earliness is important in Africa and other regions because early cultivars can escape drought and some insect infestations, can provide the first food and marketable product available from the current growing season, and can be grown in a diverse array of cropping systems. New early maturing cultivars with indeterminate growth habits have been very effective in the extremely dry and hot environment of the Sahel. Heat tolerant breeding lines have been developed which have markedly higher pod set than most cultivars under high night temperature conditions. Development of cultivars with multiple resistances to biotic and abiotic stresses is an important current breeding objective. Earliness, delayed leaf senescence, and indeterminate growth habit are characteristics which are being combined to improve drought adaptation. In the future, high levels of resistance to very important insect pests such as flower thrips (Megalurothrips sjostedti), maruca pod borer (Maruca testulalis), lygus (Lygus hesperus), and pod bugs (Clavigralla tomentosicollis and others) need to identified. Genes from wild cowpeas or related Vigna species or genetic engineering may be necessary to develop cultivars with high levels of resistance to several of the major insect pests.

A consensus genetic map of cowpea [Vigna unguiculata (L) Walp.] and synteny based on EST-derived SNPs

Consensus genetic linkage maps provide a genomic framework for quantitative trait loci identification, map-based cloning, assessment of genetic diversity, association mapping, and applied breeding in marker-assisted selection schemes. Among “orphan crops” with limited genomic resources such as cowpea [Vigna unguiculata (L.) Walp.] (2n = 2x = 22), the use of transcript-derived SNPs in genetic maps provides opportunities for automated genotyping and estimation of genome structure based on synteny analysis. Here, we report the development and validation of a high-throughput EST-derived SNP assay for cowpea, its application in consensus map building, and determination of synteny to reference genomes. SNP mining from 183,118 ESTs sequenced from 17 cDNA libraries yielded ≈10,000 high-confidence SNPs from which an Illumina 1,536-SNP GoldenGate genotyping array was developed and applied to 741 recombinant inbred lines from six mapping populations. Approximately 90% of the SNPs were technically successful, providing 1,375 dependable markers. Of these, 928 were incorporated into a consensus genetic map spanning 680 cM with 11 linkage groups and an average marker distance of 0.73 cM. Comparison of this cowpea genetic map to reference legumes, soybean (Glycine max) and Medicago truncatula, revealed extensive macrosynteny encompassing 85 and 82%, respectively, of the cowpea map. Regions of soybean genome duplication were evident relative to the simpler diploid cowpea. Comparison with Arabidopsis revealed extensive genomic rearrangement with some conserved microsynteny. These results support evolutionary closeness between cowpea and soybean and identify regions for synteny-based functional genomics studies in legumes.

Identification and comparative analysis of drought-associated microRNAs in two cowpea genotypes

BackgroundCowpea (Vigna unguiculata) is an important crop in arid and semi-arid regions and is a good model for studying drought tolerance. MicroRNAs (miRNAs) are known to play critical roles in plant stress responses, but drought-associated miRNAs have not been identified in cowpea. In addition, it is not understood how miRNAs might contribute to different capacities of drought tolerance in different cowpea genotypes.ResultsWe generated deep sequencing small RNA reads from two cowpea genotypes (CB46, drought-sensitive, and IT93K503-1, drought-tolerant) that grew under well-watered and drought stress conditions. We mapped small RNA reads to cowpea genomic sequences and identified 157 miRNA genes that belong to 89 families. Among 44 drought-associated miRNAs, 30 were upregulated in drought condition and 14 were downregulated. Although miRNA expression was in general consistent in two genotypes, we found that nine miRNAs were predominantly or exclusively expressed in one of the two genotypes and that 11 miRNAs were drought-regulated in only one genotype, but not the other.ConclusionsThese results suggest that miRNAs may play important roles in drought tolerance in cowpea and may be a key factor in determining the level of drought tolerance in different cowpea genotypes.

Development of cowpea cultivars and germplasm by the Bean/Cowpea CRSP

This paper reviews accomplishments in cowpea cultivar and germplasm development by the Bean/Cowpea Collaborative Research Support Program (CRSP) which was funded by the United States Agency for International Development for a period of about 20 years. Drought-adapted, pest and disease resistant cultivars ‘Mouride’, ‘Melakh’ and ‘Ein El Gazal’ were developed for rainfed production in the tropical Sahelian zone of Africa. Cultivars ‘CRSP Niebe’ and ‘Lori Niebe’ which have seed and pod resistance to cowpea weevil and some disease resistance were developed for rainfed production in the tropical Savanna zone of West Africa. Cultivar ‘California Blackeye No. 27’ was developed for irrigated production in subtropical California, USA and is a semidwarf with heat tolerance and broad-based resistance to root-knot nematodes and Fusarium wilt. Various cultivars with persistent-green seed color including ‘Bettergreen’ and ‘Charleston Greenpack’ were bred for use in the food freezing industry in the USA. Germplasms were developed with unique traits including: snap-type pods, green manure/cover crop capabilities, heat tolerance during reproductive development, chilling tolerance during emergence, delayed leaf senescence as a mechanism of adaptation to mid-season drought and high grain yields, differences in stable carbon isotope discrimination, harvest index, rooting and plant water- and nutrient-relations traits, broad-based resistance to root-knot nematodes and Fusarium wilt, and resistance to flower thrips, cowpea aphid, lygus bug and cowpea weevil, and various quality traits including all-white and sweet grain. These germplasms provide a valuable resource for breeding additional cowpea cultivars for Africa and the USA.

Genetic diversity of cowpea [ Vigna unguiculata (L.) Walp.] in four West African and USA breeding programs as determined by AFLP analysis

Cowpea is an important grain legume and hay crop of many tropical and subtropical regions, especially in the dry savanna region of West Africa. The cowpea gene pool may be narrow because of a genetic bottleneck during domestication. Genetic variation within specific breeding programs may be further restricted due to breeding methods, ‘founder effects’ and limited exchange of germplasm between breeding programs. Genetic relationships among 60 advanced breeding lines from six breeding programs in West Africa and USA, and 27 landrace accessions from Africa, Asia, and South America were examined using amplified fragment length polymorphism (AFLP) markers with six near infrared fluorescence labeled EcoRI + 3/1bases/MseI + 3/1bases primer sets. A total of 382 bands were scored among the accessions with 207 polymorphic bands (54.2%). Despite a diverse origin, the 87 cowpea accessions shared a minimum 86% genetic similarity. Principal coordinates analysis showed clustering of breeding lines by program origin, indicating lack of genetic diversity compared to potential diversity. Accessions from Asia and the Americas overlapped and were distinct from West African breeding lines, indicating that germplasm from Asia and the Americas have common origins outside West Africa. US and Asian breeding programs could increase genetic variability in their programs substantially by incorporating germplasm from West Africa, while national programs in West Africa should consider introgression of Asian germplasm and germplasm from other parts of Africa into their programs to ensure long-term gains from selection.

Genic SNP markers and legume synteny reveal candidate genes underlying QTL for Macrophomina phaseolina resistance and maturity in cowpea [Vigna unguiculata (L) Walp.].

BackgroundMacrophomina phaseolina is an emerging and devastating fungal pathogen that causes significant losses in crop production under high temperatures and drought stress. An increasing number of disease incidence reports highlight the wide prevalence of the pathogen around the world and its contribution toward crop yield suppression. In cowpea [Vigna unguiculata (L) Walp.], limited sources of low-level host resistance have been identified, the genetic basis of which is unknown. In this study we report on the identification of strong sources of host resistance to M. phaseolina and the genetic mapping of putative resistance loci on a cowpea genetic map comprised of gene-derived single nucleotide polymorphisms (SNPs) and amplified fragment length polymorphisms (AFLPs).ResultsNine quantitative trait loci (QTLs), accounting for between 6.1 and 40.0% of the phenotypic variance (R2), were identified using plant mortality data taken over three years in field experiments and disease severity scores taken from two greenhouse experiments. Based on annotated genic SNPs as well as synteny with soybean (Glycine max) and Medicago truncatula, candidate resistance genes were found within mapped QTL intervals. QTL Mac-2 explained the largest percent R2 and was identified in three field and one greenhouse experiments where the QTL peak co-located with a SNP marker derived from a pectin esterase inhibitor encoding gene. Maturity effects on the expression of resistance were indicated by the co-location of Mac-6 and Mac-7 QTLs with maturity-related senescence QTLs Mat-2 and Mat-1, respectively. Homologs of the ELF4 and FLK flowering genes were found in corresponding syntenic soybean regions. Only three Macrophomina resistance QTLs co-located with delayed drought-induced premature senescence QTLs previously mapped in the same population, suggesting that largely different genetic mechanisms mediate cowpea response to drought stress and Macrophomina infection.ConclusionEffective sources of host resistance were identified in this study. QTL mapping and synteny analysis identified genomic loci harboring resistance factors and revealed candidate genes with potential for further functional genomics analysis.

Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites with a broad host range which includes economically important crop species. Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume grown in many regions where root-knot nematodes are a major problem in production fields. Several sources of resistance to root-knot nematode have been identified in cowpea, including the widely used Rk gene. As part of a study to elucidate the mechanism of Rk-mediated resistance, the histological response to avirulent M. incognita feeding of a resistant cowpea cultivar CB46 was compared with a susceptible near-isogenic line (in CB46 background). Most root-knot nematode resistance mechanisms in host plants that have been examined induced a hypersensitive response (HR). However, there was no typical HR in resistant cowpea roots and nematodes were able to develop normal feeding sites similar to those in susceptible roots up to 9-14 d post inoculation (dpi). From 14-21 dpi giant cell deterioration was observed and the female nematodes showed arrested development and deterioration. Nematodes failed to reach maturity and did not initiate egg laying in resistant roots. These results confirmed that the induction of resistance is relatively late in this system. Typically in pathogen resistance HR is closely associated with an oxidative burst (OB) in infected tissue. The level of reactive oxygen species release in both compatible and incompatible reactions during early and late stages of infection was also quantified. Following a basal OB during early infection in both susceptible and resistant roots, which was also observed in mechanically wounded root tissues, no significant OB was detected up to 14 dpi, a profile consistent with the histological observations of a delayed resistance response. These results will be useful to design gene expression experiments to dissect Rk-mediated resistance at the molecular level.

Heat tolerance of contrasting cowpea lines in short and long days

High-temperature damage to reproductive processes of cowpea (Vigna unguiculata L. Walp.) occurs when minimum night air temperatures are greater than 20°C. The extent of damage is strongly influenced by photoperiod. Genes conferring heat tolerance under hot long-day environments have been identified in cowpea, and breeding lines with this trait have been developed. Genetic variability within cowpea germplasm for heat tolerance has not been assessed in short days. The objectives of this study were to determine if the heat-tolerant accessions and breeding lines developed from them under hot long-day conditions also exhibit heat tolerance under hot short-day conditions. In addition, we evaluated the heat tolerance of 48 lines that do not flower in hot long-day conditions because of sensitivity to heat or photoperiod. Three short-day experiments and one long-day experiment were conducted in greenhouses with average daily minimum and maximum air temperatures of 27 and 36°C, respectively. Simultaneously, moderate-temperature short-day experiments were conducted in nearby greenhouses with average daily minimum and maximum air temperatures of 18 and 32°C, respectively. Days to first flower, pods per peduncle, total number of pods, and grain yield were determined in all experiments. Individual seed weight and seeds per pod were determined for two of the three hot short-day and moderate-temperature short-day experiments. Heat-tolerant lines that had been selected under hot long-day conditions had larger than average grain yield (i.e. heat tolerance) under hot short days. Several lines bred for high grain yield under hot tropical conditions had heat tolerance under short days but not under long days, whereas many cultivars developed under cooler tropical conditions did not exhibit heat tolerance. In hot short-day environments, grain yield was positively correlated with pods per peduncle and number of pods per plant, indicating the importance of these traits for conferring heat tolerance in these lines. Most lines experienced substantial heat-induced reductions in number of seeds per pod, even though there were no strong indications of carbohydrate source limitations. Two of the heat-tolerant lines, B89-600 and TN88-63, did not exhibit heat-induced reductions in number of seeds per pod in hot short days, however, and represent additional sources of heat tolerance that would further enhance grain yield in hot production zones.

Lower soil moisture threshold for transpiration decline under water deficit correlates with lower canopy conductance and higher transpiration efficiency in drought-tolerant cowpea

As water availability is critical for reproduction, terminal drought tolerance may involve water-saving traits. Experiments were undertaken under different vapour pressure deficit (VPD) and water regimes (water stress (WS) and well watered (WW)) to test genotypic differences and trait relationships in the fraction of transpirable soil water (FTSW) at which transpiration declines, canopy conductance (proxied by transpiration rate (TR, g H2Ocm-2h-1)), canopy temperature depression (CTD, °C), transpiration efficiency (TE, gkg-1) and growth parameters, using 15 contrasting cowpea (Vigna unguiculata (L.) Walp.) genotypes. Under WW conditions at the vegetative and early podding stages, plant mass and leaf area were larger under low VPD, and was generally lower in tolerant than in sensitive genotypes. Several tolerant lines had lower TR under WW conditions and restricted TR more than sensitive lines under high VPD. Under WS conditions, transpiration declined at a lower FTSW in tolerant than in sensitive lines. Tolerant lines also maintained higher TR and CTD under severe stress. TE was higher in tolerant genotypes under WS conditions. Significant relationships were found between TR, and TE, CTD and FTSW under different water regimes. In summary, traits that condition how genotypes manage limited water resources discriminated between tolerant and sensitive lines. Arguably, a lower canopy conductance limits plant growth and plant water use, and allows tolerant lines to behave like unstressed plants until the soil is drier and to maintain a higher TR under severe stress, as lower TR at high VPD leads to higher TE.

Detection and validation of single feature polymorphisms in cowpea (Vigna unguiculata L. Walp) using a soybean genome array

BackgroundCowpea (Vigna unguiculata L. Walp) is an important food and fodder legume of the semiarid tropics and subtropics worldwide, especially in sub-Saharan Africa. High density genetic linkage maps are needed for marker assisted breeding but are not available for cowpea. A single feature polymorphism (SFP) is a microarray-based marker which can be used for high throughput genotyping and high density mapping.ResultsHere we report detection and validation of SFPs in cowpea using a readily available soybean (Glycine max) genome array. Robustified projection pursuit (RPP) was used for statistical analysis using RNA as a surrogate for DNA. Using a 15% outlying score cut-off, 1058 potential SFPs were enumerated between two parents of a recombinant inbred line (RIL) population segregating for several important traits including drought tolerance, Fusarium and brown blotch resistance, grain size and photoperiod sensitivity. Sequencing of 25 putative polymorphism-containing amplicons yielded a SFP probe set validation rate of 68%.ConclusionWe conclude that the Affymetrix soybean genome array is a satisfactory platform for identification of some 1000s of SFPs for cowpea. This study provides an example of extension of genomic resources from a well supported species to an orphan crop. Presumably, other legume systems are similarly tractable to SFP marker development using existing legume array resources.