Genetic analysis of abiotic and biotic resistance in cowpea [Vigna unguiculata (L.) Walp.]

Cowpea [Vigna unguiculata (L.) Walp.] is a most versatile African crop, it feeds people, their livestock and because of its ability in nitrogen-fixation, it improves soil fertility, and consequently helps to increase the yields of cereal crops when grown in rotation and contributes to the sustainability of cropping systems. Because of its ability to tolerate some level of drought stress, cowpea is a crop of choice where > 10 million hectares are cultivated to cowpea in the semiarid Savanna and Sahelian zones of West and Central Africa. However due to the infrequent drought stress throughout cropping seasons over the last 3 decades, the crop suffers important yield reduction. Moreover, cowpea plants under water stress condition are more vulnerable to diseases, parasites and insect pests attacks. In this thesis we carried out genetic analyses of seedling and terminal drought tolerance and cowpea bacterial blight (CoBB) resistance. Two cowpea genotypes with contrasting reactions to drought and CoBB stresses, Danila (resistant) and TVu7778 (susceptible) were used to develop recombinant inbred lines (RILs) mapping population. The RILs and parents were phenotyped for CoBB resistance and for physiological drought tolerance traits and productivity traits under different water regimes in multiple environments at seedling and adult plant stages. A genetic linkage map of 282 single nucleotide polymorphism (SNP) loci covering a map distance of 633 cM distributed over 11 linkage groups (LG) from the same RILs was used for quantitative traits loci (QTL) analyses. Moisture stress significantly affected RILs performances with number of pods per plant as the yield component most adversely affected by water stress. Correlation and path analyses revealed that grain yield components (mainly number of pods per plant) and plant biomass had the largest direct effects on grain yield under moisture stress and irrigation. Stem greenness was an excellent predictor of seedling survival to drought (r2 = 0.91). Two QTLs were identified for each of the three traits scored under greenhouse, drought-induced trifoliate senescence (DTS), stem greenness (Stg) and survival (Sur) on LG3 and LG7. For all traits measured under field trials, a total of 42 QTLs were detected, 4 for stomatal conductance (Gs), 6 for delayed leaf senescence (DLS), 5 for flowering time and 16 for grain yield components (pod number/plant, seed number/pod, seed weight), 6 for grain yield and 5 for fodder yield. Three QTLs were detected for CoBB resistance, with two major ones (named CoBB-1 and CoBB-2 confirmed over two experiments) on LG3 and LG5 and one minor QTL (CoBB-3 only for experiment 1) on LG9. Although number of QTLs detected seems to be important, association between QTLs of different traits sharing a common genomic region was observed on LG3, LG5, LG7 and LG8 where QTLs for Gs, DLS, and flowering time co-localized with QTLs for yield parameters, DLS and DTS shared common chromosomal regions with CoBB resistance, on LG3 and LG5. These results suggest that common genes might mediate CoBB resistance and DLS. Two QTLs detected for DLS in the field, co-localized with QTLs under greenhouse for seedling drought tolerance traits DTS, Stg and Sur on LG7. This indicates the presence of potential loci controlling senescence in this genomic region. Moreover, this genomic region represents a syntenic genomic region between cowpea, soybean and Medicago. Our findings provide evidence for QTLs mediating seedling and terminal drought tolerance and CoBB resistance in cowpea. QTL and phenotypic analysis revealed that it should be possible to pyramid CoBB resistance with seedling and terminal drought tolerance. The fact that the genetic map of Danila x TVu7778 is integrated in a consensus map of cowpea of 6 RIL populations will permit comparative genomic studies which will enhance the discovery of functional markers for MAS of seedling and terminal drought tolerance and CoBB resistance in cowpea. <br/

Breeding drought tolerant cowpea: constraints, accomplishments, and future prospects

This review presents an overview of accomplishments on different aspects of cowpea breeding for drought tolerance. Furthermore it provides options to enhance the genetic potential of the crop by minimizing yield loss due to drought stress. Recent efforts have focused on the genetic dissection of drought tolerance through identification of markers defining quantitative trait loci (QTL) with effects on specific traits related to drought tolerance. Others have studied the relationship of the drought response and yield components, morphological traits and physiological parameters. To our knowledge, QTLs with effects on drought tolerance have not yet been identified in cowpea. The main reason is that very few researchers are working on drought tolerance in cowpea. Some other reasons might be related to the complex nature of the drought stress response, and partly to the difficulties associated with reliable and reproducible measurements of a single trait linked to specific molecular markers to be used for marker assisted breeding. Despite the fact that extensive research has been conducted on the screening aspects for drought tolerance in cowpea only very few¿like the `wooden box¿ technique¿have been successfully used to select parental genotypes exhibiting different mechanisms of drought tolerance. Field and pot testing of these genotypes demonstrated a close correspondence between drought tolerance at seedling and reproductive stages. Some researchers selected a variety of candidate genes and used differential screening methods to identify cDNAs from genes that may underlie different drought tolerance pathways in cowpea. Reverse genetic analysis still needs to be done to confirm the functions of these genes in cowpea. Understanding the genetics of drought tolerance and identification of DNA markers linked to QTLs, with a clear path towards localizing chromosomal regions or candidate genes involved in drought tolerance will help cowpea breeders to develop improved varieties that combine drought tolerance with other desired traits using marker assisted selection

Genetic variability of cultivated cowpea in Benin assessed by random amplified polymorphic DNA

Characterization of genetic diversity among cultivated cowpea [Vigna unguiculata (L.) Walp.] varieties is important to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity in 70 cowpea accessions collected throughout Benin. Nine random primers were screened on 24 accessions to assess their ability to reveal polymorphisms in cowpea and four of them were selected for use in characterizing the total sample. A total of 32 amplified bands were generated by the four primers. The number of loci detected varied from 5 to 11. RAPD profiles were analysed and amplified polymorphic DNA fragments were used to construct a dendrogram, clustering the accessions into nine groups at a similarity index of 71% based on the Unweighted Pair-Group Method using Arithmetic Averages. The genetic diversity among the cowpea cultivars investigated was large and the RAPD proved to be a useful technique to characterise it. Based on the molecular variance, the fixation index suggests a large differentiation of cowpea cultivars in Benin

Identification of markers associated with bacterial blight resistance loci in cowpea (Vigna unguiculata (L.) Walp.)

Cowpea bacterial blight (CoBB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is a worldwide major disease of cowpea [Vigna unguiculata (L.) Walp.]. Among different strategies to control the disease including cultural practices, intercropping, application of chemicals, and sowing pathogen-free seeds, planting of cowpea genotypes with resistance to the pathogen would be the most attractive option to the resource poor cowpea farmers in sub-Saharan Africa. Breeding resistance cultivars would be facilitated by marker-assisted selection (MAS). In order to identify loci with effects on resistance to this pathogen and map QTLs controlling resistance to CoBB, eleven cowpea genotypes were screened for resistance to bacterial blight using 2 virulent Xav18 and Xav19 strains isolated from Kano (Nigeria). Two cowpea genotypes Danila and Tvu7778 were identified to contrast in their responses to foliar disease expression following leaf infection with pathogen. A set of recombinant inbred lines (RILs) comprising 113 individuals derived from Danila (resistant parent) and Tvu7778 (susceptible parent) were infected with CoBB using leaf inoculation method. The experiments were conducted under greenhouse conditions (2007 and 2008) and disease severity was visually assessed using a scale where 0 = no disease and 4 = maximum susceptibility with leaf drop. A single nucleotide polymorphism (SNP) genetic map with 282 SNP markers constructed from the same RIL population was used to perform QTL analysis. Using Kruskall-Wallis and Multiple-QTL model of MapQTL 5, three QTLs, CoBB-1, CoBB-2 and CoBB-3 were identified on linkage group LG3, LG5 and LG9 respectively showing that potential resistance candidate genes cosegregated with CoBB resistance phenotypes. Two of the QTLs CoBB-1, CoBB-2 were consistently confirmed in the two experiments accounting for up to 22.1 and to 17.4% respectively for the first and second experiments. Whereas CoBB-3 was only discovered for the first experiment (2007) with less phenotypic variation explained of about 10%. Our results represent a resource for molecular marker development that can be used for marker assisted selection of bacterial blight resistance in cowpe

Genetic variability of cultivated cowpea in Benin assessed by random amplified polymorphic DNA

Characterization of genetic diversity among cultivated cowpea [Vigna unguiculata (L.) Walp.] varieties is important to optimize the use of available genetic resources by farmers, local communities, researchersand breeders. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity in 70 cowpea accessions collected throughout Benin. Nine random primers were screened on24 accessions to assess their ability to reveal polymorphisms in cowpea and four of them were selected for use in characterizing the total sample. A total of 32 amplified bands were generated by the fourprimers. The number of loci detected varied from 5 to 11. RAPD profiles were analysed and amplified polymorphic DNA fragments were used to construct a dendrogram, clustering the accessions into ninegroups at a similarity index of 71% based on the Unweighted Pair-Group Method using Arithmetic Averages. The genetic diversity among the cowpea cultivars investigated was large and the RAPDproved to be a useful technique to characterise it. Based on the molecular variance, the fixation index suggests a large differentiation of cowpea cultivars in Benin

Genetic variability in yam cultivars from the Guinea- Sudan zone of Benin assessed by random amplified polymorphic DNA

Yam (Dioscorea spp.) is an important food and cash crop in the Guinea-Sudan zone of Benin. The genetic diversity of about 70 cultivars of Dioscorea cayenensis/Dioscorea rotundata (Guinea yam) andabout 20 cultivars of Dioscorea alata (water yam) was analysed using random amplified polymorphic DNA (RAPD). The amplified bands revealed high polymorphism. These polymorphic DNA fragmentswere used to construct dendrograms, clustering all accessions into 18 groups: 12 for D. cayenensis/D. rotundata and six for D. alata. The analysis of molecular variance revealed highly significant variationamong species, among groups within species, and among cultivars within groups. The study showed that the genetic diversity changed along a spatial gradient. In general, there was a tendency that mostof the varieties from the north-east and north-west of the zone investigated appeared to be distinctive from the ones of the centre. However, few varieties were distributed randomly and did not reflect anyspecific relation to their zone of collection. The current study suggests that the Guinea-Sudan zone of Benin has a large gene pool of yam varieties. Yam farmers may have played a significant role in theenrichment and the maintenance of the genetic diversity of yam

Evaluating stress responses in cowpea under drought stress

Drought impact on plants is an increasing concern under the climate change scenario. Cowpea (Vigna unguiculata L. Walp.) is considered as one of the most tolerant legume crops to drought, being the search for the best well-adapted genotypes crucial to face the future challenges. Different approaches have been used for differentiating plant responses to drought stress. Plants of four cowpea genotypes were submitted to three watering regimens (a severe and moderate drought stress, and well-watered control) during 15 days, and several physiological, biochemical and molecular parameters were evaluated. Stressed plants revealed commonly-described drought stress characteristics, but not all assayed parameters were useful for discriminating plants with different drought severities or genotypes. The analyses which have contributed most to genotype discrimination were those related with stomatal function, and biochemical markers such as proline and anthocyanin contents. Antioxidant enzymes activities and related genes expression did not differed among genotypes or upon drought stress treatments, suggesting that scavenging enzymes are not involved in the differential ability of cowpea plants to survive under drought stress. This information will be useful to evaluate and use genetic resources, as well as design strategies for breeding cowpea resistance to drought stress.This study was supported by EUROLEGUME project. This project has received funding from: the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613781; National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019; and the project PEstOE/BIA/UI4046/2014; UID/MULTI/04046/2013

Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects

The global population is continuously increasing and is expected to reach nine billion by 2050. This huge population pressure will lead to severe shortage of food, natural resources and arable land. Such an alarming situation is most likely to arise in developing countries due to increase in the proportion of people suffering from protein and micronutrient malnutrition. Pulses being a primary and affordable source of proteins and minerals play a key role in alleviating the protein calorie malnutrition, micronutrient deficiencies and other undernourishment-related issues. Additionally, pulses are a vital source of livelihood generation for millions of resource-poor farmers practising agriculture in the semi-arid and sub-tropical regions. Limited success achieved through conventional breeding so far in most of the pulse crops will not be enough to feed the ever increasing population. In this context, genomics-assisted breeding (GAB) holds promise in enhancing the genetic gains. Though pulses have long been considered as orphan crops, recent advances in the area of pulse genomics are noteworthy, e.g. discovery of genome-wide genetic markers, high-throughput genotyping and sequencing platforms, high-density genetic linkage/QTL maps and, more importantly, the availability of whole-genome sequence. With genome sequence in hand, there is a great scope to apply genome-wide methods for trait mapping using association studies and to choose desirable genotypes via genomic selection. It is anticipated that GAB will speed up the progress of genetic improvement of pulses, leading to the rapid development of cultivars with higher yield, enhanced stress tolerance and wider adaptability

Priming of cowpea volatile emissions with defense inducers enhances the plant's attractiveness to parasitoids when attacked by caterpillars

BACKGROUND: The manipulation of herbivore-induced volatile organic compounds (HI-VOCs) via the application of the inducers BTH [benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester] and Laminarin [β-1,3-glucan] is known to enhance the attractiveness of caterpillar-damaged cotton and maize plants to parasitoids. To test if this is also the case for legumes, we treated cowpea with these inducers and studied the effects on HI-VOC emissions and the attraction of three generalist endoparasitoids. RESULTS: After applying the inducers and subjecting the plants to either real or mimicked herbivory by Spodoptera littoralis caterpillars, females of the parasitoids Campoletis sonorensis and Microplitis rufiventris showed a strong preference for BTH treated plants, whereas Cotesia marginiventris females were strongly attracted to both BTH and Laminarin treated plants with real or mimicked herbivory. Treated plants emitted more of certain HI-VOCs, but considerably less indole, linalool and several sesquiterpenes. Multivariate data analysis revealed that enhanced wasp attraction after treatment was correlated with high relative concentrations of nonanal, α-pinene, (E)-β-ocimene and DMNT, and with low relative concentrations of indole, (S)-linalool and (E)-β-farnesene. Inducer treatments had no significant effect on leaf consumption by the caterpillars. CONCLUSION: Our findings confirm that treating cowpea plants with inducers can enhance their attractiveness to biological control agents