Genetic improvement of drought tolerance in cowpea (Vigna unguiculata): Integrating physiology, genomics, and breeding

Introduction

Cowpea (Vigna unguiculata (L.) Walp.) is a major grain legume of the tropics and subtropics, particularly in sub-Saharan Africa and South Asia, where it supports food, nutritional, and income security of millions of smallholder farmers. Cowpea grains contain 20-25% protein along with essential amino acids, vitamins, and minerals, while crop residues serve as valuable livestock fodder, making the crop integral to mixed farming systems. In addition, cowpea improves soil fertility through biological nitrogen fixation and performs well in low-input systems with limited fertilizer access (Namatsheve et al., 2020) [34] .

Genetic gains have therefore been modest, particularly under terminal and reproductive-stage drought, and are further constrained by interacting stresses such as heat, pests, and diseases (Carvalho et al., 2017) [7] . Although cowpea possesses adaptive traits such as deep rooting and early maturity, current breeding progress is insufficient to counter climate-driven yield instability. Consequently, recent studies emphasize integrating multi-environment testing, traitbased phenotyping, and molecular and genomic approaches including QTL mapping, GWAS, genomic selection, and marker-assisted breeding to elucidate the genetic basis of drought tolerance and develop high-yielding, climate-resilient cowpea cultivars for drought-prone agro-ecologies (Agbicodo et al., 2009) [7] .

Types of droughts in cowpea production systems

Drought occurs at different growth stages in cowpea, and its timing strongly determines physiological responses and yield loss; therefore, early-season, mid-season, and terminal drought are now distinguished for targeted phenotyping and breeding (Deffo et al., 2024; Ezin et al., 2021; Hall, 2012) [11,13,18] .

Early-season drought

Early-season drought limits crop establishment, canopy expansion, and root and nodule development, reducing stomatal conductance, biomass accumulation, and nitrogen fixation, and thereby lowering yield potential even after stress relief (Olorunwa et al., 2021; Melo et al., 2025; Poudel et al., 2025) [33, 26, 37] . Screening at this stage targets rooting vigor, root:shoot ratio, stem greenness, and maintenance of gas exchange (Santos et al., 2020; Olorunwa et al., 2021) [41,33] .

Mid-season drought

Mid-season drought, often caused by rainfall breaks, reduces leaf area, photosynthesis, and assimilate supply to developing reproductive organs. Although partial acclimation and improved water-use efficiency may occur, flower production and pod set are commonly reduced, lowering yield (Hall, 2012; Carvalho et al., 2019; Olorunwa et al., 2021) [18,33,6] . Key traits include delayed senescence, water-use efficiency, and deeper rooting (Carvalho et al., 2019) [8] .

Terminal drought

Terminal drought, from flowering to pod filling, is the most damaging, causing declines in plant water status, chlorophyll, and photochemical efficiency, leading to flower and pod abortion, reduced seed size, and low harvest index (Deffo et al., 2024; Ezin et al., 2021; Nunes et al., 2022) [11,13,32] . Even small genotypic differences in stomatal control and stay-green behavior can result in large yield differences under terminal stress (Moreira et al., 2023) [29] . Phenological escape, reproductive-stage tolerance, and stress priming are therefore major breeding targets (Hall, 2012; Tankari et al., 2021) [18,49] . Across stages, drought effects are stage-dependent: early stress reshapes root and nodule development, mid-season stress limits assimilate supply, and terminal stress directly restricts pod set and grain filling, causing the greatest yield losses. Multi-stage screening is thus essential to identify genotypes combining early resilience, reproductive-stage tolerance, and stable yield under variable rainfall (Deffo et al., 2024; Ezin et al., 2021; Hall, 2012) [11,13,18] .

Sensitive growth stages of cowpea to drought stress 1.2.1 Germination and seedling stage

Germination and early seedling growth are highly droughtsensitive, with reduced imbibition, germination percentage, and seedling vigor, leading to poor crop establishment (Carvalho et al., 2017; Esan et al., 2021; Tanasale et al., 2025) [7,12,48] . PEGinduced stress consistently reduces germination, radicle and plumule length, and seedling biomass across cowpea genotypes (Carvalho et al., 2017; Tanasale et al., 2025) [7,48] . Early drought strongly restricts root and shoot growth, leaf area, and nodulation, thereby lowering early source capacity and later yield potential (Nkomo et al., 2022; Poudel et al., 2025; Ritte et al., 2025) [31,37,40] . Physiologically, stress rapidly decreases stomatal conductance, increases canopy temperature, and suppresses photosynthesis and chlorophyll fluorescence, and although partial recovery after rewatering may occur, early stress often leaves persistent reductions in canopy size and yield (Nkomo et al., 2022; Olorunwa et al., 2021) [31,33] .

Flowering stage

Flowering is consistently identified as the most droughtsensitive stage in cowpea (Deffo et al., 2024; Ezin et al., 2021; Tankari et al., 2021) [11,13,49] . Water deficit at flowering causes greater yield penalties than vegetative stress by reducing plant water status, chlorophyll content, and photochemical efficiency and by inducing extensive flower and pod abortion (Gnankambary et al., 2020; Nunes et al., 2022) [15,32] . Elevated canopy temperature and vapor pressure deficit further impair pollen function and fertilization, sharply reducing pod number the primary determinant of grain yield (Carvalho, Castro, et al., 2019; Ezin et al., 2021; Deffo et al., 2024) [6,8,13,11] .

Pod filling stage

Terminal drought during pod filling shortens the grain-filling period, accelerates senescence, and restricts assimilate and nitrogen remobilization, resulting in smaller seeds, reduced 100-seed weight, and lower harvest index (Ezin et al., 2021; Muhammed et al., 2024; Tankari et al., 2021) [13,30,49] . Stress imposed at early seed filling markedly reduces pod weight, seed number, and total grain yield, and under severe terminal drought, premature pod abortion and incomplete seed development can lead to drastic yield losses or near crop failure (Ezin et al., 2021; Muhammed et al., 2024) [13,30] .

Impact of drought on cowpea growth and yield 1.3.1 Germination and establishment

Low water potential reduces imbibition and delays or suppresses germination, causing poor and nonuniform emergence (Carvalho et al., 2019; Esan et al., 2021; Tanasale et al., 2025) [6,8,12,48] . PEG-induced stress consistently lowers germination percentage, radicle growth, and seedling vigor, although tolerant genotypes maintain relatively higher establishment (Carvalho et al., 2019) [19] . Poor establishment reduces stand density, early biomass, nodulation, and radiation interception, constraining nitrogen fixation and season-long yield potential (Esan et al., 2021; Olorunwa et al., 2021) [12,33] .

Vegetative growth

Vegetative-stage drought reduces leaf area, plant height, and shoot biomass (often by >50%) and alters biomass partitioning, increasing root:shoot ratio under moderate stress (Olorunwa et al., 2021; Poudel et al., 2025; Santos et al., 2020) [33,37,41] . Declines in stomatal conductance, transpiration, and photosynthesis, coupled with elevated canopy temperature and oxidative stress, reduce chlorophyll content and photochemical efficiency (Carvalho, Castro, et al., 2019; Nunes et al., 2022; Olorunwa et al., 2021) [6,8,32,33] . Severe or prolonged stress restricts both lateral and deep root development, impairing water and nutrient uptake and limiting source capacity for reproductive growth (Poudel et al., 2025; Santos et al., 2020) [37, 41] .

Flower drop, pod abortion, and seed filling

Reproductive stages are the most drought-sensitive. Water deficit at flowering or early pod set sharply increases flower and pod abortion, reduces pod number, seeds per pod, and seed weight, and can decrease yield by 50-90% (Deffo et [11, 13, 30, 49] . Terminal drought shortens the grain-filling period, accelerates senescence, and restricts assimilate and nitrogen remobilization, producing small, shriveled, low-quality seeds and a reduced harvest index (Ezin et al., 2021; Muhammed et al., 2024) [13,30] .

Effects on yield components

Across environments, drought consistently reduces pods per plant, grains per plant, seed size, biomass, and harvest index, with pod number emerging as the most sensitive and yielddetermining component under reproductive-stage stress (Deffo et al., 2024; Ezin et al., 2021) [11,13] . [25,41,23] . Early-stage screening shows that tolerant lines combine greater total root length, surface area, and fine-root proliferation with better shoot performance, reflecting efficient soil exploration rather than increased root mass alone (Poudel et al., 2025) [37] . Drought commonly increases root:shoot ratio and induces architectural plasticity, with genotypes maintaining shoot growth also showing stronger root investment (Santos et al., 2020) [41] . Highly resilient accessions further exhibit anatomical plasticity that supports efficient water uptake across contrasting moisture regimes (Iseki & Olaleye, 2025a) [19] .

Physiological and morphological

Shoot-based physiological traits associated with drought tolerance

Shoot traits provide integrative, relatively high-throughput indicators of drought tolerance and often show strong associations with yield-based indices. Droughttolerant cowpea genotypes consistently maintain higher relative water content (RWC), reflecting superior water uptake, osmotic adjustment, and membrane stability, and this trait is repeatedly linked to improved recovery and yield under stress (Shardendu et al., 2011) [24] . Canopy temperature serves as a robust proxy for stomatal conductance and root function, with tolerant lines maintaining cooler canopies and thermal imaging platforms showing strong correlations with biomass and yield under drought (Kim et al., 2020; Nunes et al., 2022) [22,37] . Variation in stomatal sensitivity to soil drying underpins contrasting drought strategies in cowpea; optimized regulation rather than extreme closure supports carbon assimilation while conserving water, and high-throughput phenotyping combined with GWAS has identified genomic regions controlling stomatal dynamics (Shardendu et al., 2011) [38] . Maintenance of chlorophyll content and stay-green behavior reflects protection of the photosynthetic apparatus and delayed senescence, with tolerant genotypes showing smaller SPAD declines, sustained photosynthesis, and improved pod and grain set under terminal drought. Drought-tolerant lines also retain higher photosynthetic efficiency (Fv/Fm, ETR), improved intrinsic water-use efficiency, and stronger post-stress recovery of gas exchange and fluorescence traits, indicating reduced irreversible damage and greater physiological resilience (Nunes et al., 2022; Lima et al., 2025b) [32,37] .

Biochemical and metabolic bases of cowpea drought tolerance

Drought tolerance in cowpea involves strong osmotic adjustment, driven by accumulation of proline, soluble sugars, galactinol, organic acids, phenylpropanoids, and inorganic ions, many of which correlate positively with yield under stress (Goufo et al., 2017; Ozturk et al., 2021) [16,35] .

Tolerant genotypes display more efficient antioxidant systems, with higher activities of SOD, CAT, APX, and GR and lower H₂O₂ and MDA, conferring improved membrane stability and recovery (Miri et al., 2022; Ribeiro et al., 2023) [27,39] . Proteomic studies consistently identify oxidoreductases as central components of drought adaptation (Ribeiro et al., 2023) [39] . At the regulatory level, rapid ABA biosynthesis and signaling drive early stomatal control and maintenance of plant water status, while drought-responsive miRNAs and hormone crosstalk (ABA, JA, SA, ethylene, cytokinins, brassinosteroids) coordinate osmolyte accumulation, antioxidant capacity, and stress-responsive growth modulation (Mishra et [28,50,43,45] .

Breeding approaches for genetic improvement of drought tolerance in cowpea 3.1 Major germplasm sources and their roles 3.1.1 Global collections and genebanks

Cowpea drought improvement is fundamentally supported by the extensive diversity conserved in global genebanks, particularly at IITA, which maintains >15, 000 cultivated accessions and >1, 500 wild Vigna accessions capturing wide variation in phenology, architecture, root traits, and abiotic stress adaptation (Boukar et al., 2020 [59,60,61] . Large diversity panels (≈99 to >1, 288 accessions) evaluated under managed drought have enabled the identification of stay-green, early-maturing, and drought-tolerant donors now widely used in climate-resilient breeding (Boukar et al., 2020) [19] .

Landraces and wild relatives

Landraces from Southern Africa, Mozambique, and the Sahel harbor high adaptive diversity and frequently outperform modern cultivars under terminal and intermittent drought (Gomes et al., 2021; Macharia et al., 2025) [62,63] . Wild Vigna relatives provide additional drought-adaptive alleles, particularly for root plasticity (stele proportion, aerenchyma, fine-root proliferation) and dual tolerance to water deficit and excess moisture (Boukar et al., 2020) [19] , and are increasingly targeted for pre-breeding and introgression.

Mini-core collections and MAGIC resources 3.2.1 Mini-core panels

IITA mini-core collections (~300 accessions) capture most global SNP diversity and are central to drought phenotyping and GWAS (C. Fatokun et al., 2018) [19] . These panels have facilitated the discovery of loci controlling flowering time, stay-green behavior, root traits, and yield stability, thereby accelerating parental selection and gene discovery.

MAGIC populations

The eight-parent cowpea MAGIC population (~305 F₈ RILs) integrates diverse drought-tolerance, disease-resistance, and agronomic alleles into highly recombined, breeding-ready backgrounds (B. L. [64,65,66] . GWAS within this population has resolved SNPs for drought indices, phenology, seed weight, and grain yield, positioning MAGIC lines as dual-purpose resources for high-resolution mapping and rapid breeding deployment (Ravelombola et al., 2021) [66] . [6,8,11,13,15] .

Managed

Stage-specific and multi-environment screening.

Vegetative-stage screening targets seedling vigor, nodulation, and root development, while reproductive-stage screening emphasizes flower retention, pod set, and harvest index (Alidu et al., 2019; Ezin et al., 2021) [13,37] . Sequential filtering across growth stages and locations improves selection efficiency and retention of broadly resilient genotypes (Alidu et al., 2019) [11] .

Semi-controlled and controlled platforms

Screenhouse, pot, lysimeter, rhizotron, and PEG-based systems enable precise moisture control and detailed measurement of physiological and root traits, including transpiration efficiency, rooting depth, and post-stress recovery (Nkomo et [31,33,37] .

High-throughput phenotyping integration

Infrared thermography, multispectral indices (NDVI, PRI), and chlorophyll fluorescence provide rapid, non-destructive indicators of canopy temperature, photosynthetic performance, and stress progression, strongly associated with drought tolerance and root traits (Carvalho, Castro, et [6,33,37] . These phenomic traits are increasingly integrated with GWAS and genomic selection to enable predictive drought breeding pipelines (Nkomo et al., 2022; Poudel et al., 2025) [31,37] .

Drought tolerance indices and selection metrics in cowpea 3.4.1 Behavior, heritability, and G×E of yield-based indices

Under managed drought and non-stress environments, most yield-based drought indices in cowpea show significant genetic variation and strong genotype, environment, and G×E effects, closely paralleling grain yield responses and confirming their biological relevance (Singini et al., 2024) [67] . Across studies, STI, GMP, MP, HM, YI, YSI, DRI, and modified indices (MST1, MST2) consistently emerge as the most reliable for identifying genotypes combining high yield and drought tolerance (Ajayi, 2024) [11] . These indices generally exhibit moderate to high broad-sense heritability (≈30-60% or higher) and substantial genetic advance, supporting their use for indirect selection (Ajayi, 2020, 2024) [2] . MP, GMP, HM, YI, and STI are repeatedly reported as highly correlated with yield under both stressed and non-stressed conditions, making them particularly suitable for selecting broadly adapted, highyielding genotypes (Singini et al., 2024) [67] . Comparable trends across legumes and cereals further validate STI-, GMP-, HM-, MP-, YSI-, YI-, and DRI-based selection frameworks (Sánchez-Reinoso et al., 2020) [42] . By contrast, TOL, SSI, YR, and DSI mainly quantify yield loss and, while useful for identifying susceptible genotypes, show weaker associations with stressed yield and may misclassify high-yielding but drought-sensitive lines when used alone (Ajayi, 2020; Sánchez-Reinoso et al., 2020) [2,42] .

Functional grouping and rank-based selection

Multivariate grouping approaches (Fernandez grouping, PCA, clustering, and rank-sum methods) are widely used to translate multiple indices into breeding decisions. Greenhouse and field studies consistently place STI, GMP, MP, HM, YI, and REI on the primary productivity-tolerance axis, efficiently distinguishing high-yielding tolerant genotypes (Group A), while SSI and TOL reliably flag unstable, drought-susceptible materials (Deffo et al., 2024) [11] . Rank-sum selection across MP, GMP, HM, YI, and STI has repeatedly identified a small, elite subset of drought-tolerant parents for arid and semi-arid environments (Singini et al., 2024) [67] . Multi-trait base indices and biplots further partition genotypes into functional classes (high-yielding/tolerant, high-yielding/susceptible, lowyielding/tolerant, low-yielding/susceptible), facilitating earlygeneration advancement.

Multivariate and stability analyses

Across cowpea panels, PCA typically explains ~70-80% of total variation within the first two components, with PC1 dominated by STI, GMP, MP, HM, YI, YSI, DRI, and stressed yield, defining a composite axis of productivity, drought tolerance, and stability (Ajayi, 2020, 2024; Deffo et al., 2024) [2, 11] . Cluster analysis and GT/GYT biplots simplify simultaneous selection across indices (Deffo et al., 2024) [11] . For multi-environment performance, AMMI and GGE biplots effectively discriminate unstable high-yielding genotypes from those combining yield and wide adaptation, supporting targeted cultivar recommendation (Mekonnen et al., 2022) [68] .

3.4.4

Physiology-integrated and multiple-indicator frameworks Integration of physiological traits strengthens index-based selection. Canopy temperature, stomatal conductance, chlorophyll-based indices (e.g., PRI), and root system traits are strongly associated with drought response and biomass production, highlighting vigorous rooting as a key donor trait (Poudel et al., 2025) [37] . Photosynthetic rate, transpiration, water-use efficiency, chlorophyll fluorescence, and staygreen behavior consistently differentiate tolerant and susceptible genotypes (Nunes et al., 2022) [33] . Composite frameworks integrating yield indices with physiological and root traits have successfully identified accessions with dual tolerance to drought and excess moisture (Isek & Olaleye, 2025a), consistent with evidence from other legumes supporting multiindicator selection for climate resilience (Abderemane et al., 2025) [1] .

Genomic integration of drought indices

Drought indices are increasingly used as quantitative phenotypes for genetic analysis. GWAS in a 305-line cowpea MAGIC population detected numerous SNPs and interaction networks associated with yield-based drought indices (Ravelombola et al., 2021) [66] . Although prediction accuracies are generally low to moderate, they support marker-assisted and genomic selection directly targeting integrated drought response phenotypes rather than stressed yield alone (Ravelombola et al., 2021) [66] .

Trait-Based Selection in Cowpea for Drought Tolerance

Trait-based selection targets heritable component traits underpinning drought avoidance, tolerance, and recovery. These traits now function not only as phenotypic selection criteria but also as quantitative targets in QTL mapping, GWAS, and genomic selection, enabling their systematic integration into modern breeding pipelines (Agbicodo et al., 2009; Jalal et al., 2023) [51,56] .

Phenology and development

Early flowering and maturity consistently confer advantage under terminal drought, though excessively early genotypes may compromise yield potential, necessitating multienvironment evaluation (Agbicodo et al., 2009) [13] . Significant genetic variation and moderate-to-high heritability for flowering and maturity enable reliable classification for environment specific breeding (Ezin et al., 2021) [13] . Drought indices for phenology and associated SNPs identified in MAGIC populations further validate phenology as a priority genomic target (Ravelombola et al., 2021) [66] .

Root system architecture and water acquisition

Drought-tolerant genotypes typically express greater root biomass, higher root:shoot ratios, and deeper, denser root systems, which are associated with improved shoot growth and yield stability (Poudel et al., 2025; Santos et al., 2020) [37,41] . Early root vigor, surface area, and tap-root diameter correlate strongly with biomass and are increasingly dissected through GWAS, with major loci identified particularly on chromosome 5 supporting marker-assisted selection for root-mediated drought adaptation (Poudel et al., 2025; Zaffar et al., 2024) [37,77] .

Physiological traits and plant water relations

Tolerant genotypes maintain higher relative water content, lower canopy temperature, and superior photosynthetic performance (ΦPSII, ETR), coupled with sensitive stomatal regulation under soil drying (Poudel et al., 2025; Ravelombola et al., 2020) [37,56] . High-throughput phenotyping of transpiration dynamics (θcri, K) reveals substantial genetic variation and SNPs governing stomata responsiveness (Wu et al., 2021a) [56] . Chlorophyll fluorescence, SPAD, PRI, and NDVI are strongly associated with drought injury and grain yield, establishing them as powerful non-destructive screening and GWAS tools (Poudel et al., 2025) [37] . Biochemica markers, including osmolytes, antioxidant capacity, and membrane stability, further differentiate tolerant cultivars (Jalal et al., 2023; Ritte et al., 2025) [51,40] .

Reproductive resilience and yield components

Under drought, tolerant landraces and improved lines maintain higher pod number, seed weight, harvest index, and yield despite reduced biomass (Ezin et al., 2021) [13] . Photosynthetic stability traits correlate with grain yield and map to genes involved in photoprotection and stress signaling, linking physiology with reproductive success (Sansa et al., 2024) [69] . Seedling greenness, chlorosis tolerance, and poststress recovery reliably predict survival and later field performance, supporting their use as early-generation filters (Ravelombola et al., 2020, 2025a; Ritte et al., 2025) [38,40] .

Integration with modern breeding strategies

Drought-related traits including phenology, root architecture, stomatal behavior, canopy temperature, greenness, and photosynthetic parameters are now routinely integrated into GWAS and MAGIC analyses, yielding numerous SNPs associated with drought indices and adaptive traits (Poudel et al., 2025; Ravelombola et al., 2021) [37,56] . Although genomic prediction accuracies are generally low to moderate, they support genomic selection when combined with robust phenotypic indices (Li et al., 2018; Ravelombola et al., 2021) [70, 66] .

Modern cowpea drought-breeding pipelines increasingly

Screen early generations under controlled stress for seedling survival, greenness, root vigor, transpiration dynamics, and canopy temperature (Li et al., 2018; Poudel et al., 2025; Ravelombola et al., 2020, 2025a; Santos et al., 2020; Wu et al., 2021a) [38,37,41,56] ; advance selected lines to managed-stress field trials integrating drought indices with phenology, physiology, and reproductive traits (Ezin et al., 2021; Sansa et al., 2025) [13,69] ; and apply GWAS and genomic selection to pyramid complementary alleles for phenology, root systems, stomatal control, photosynthesis, and reproductive resilience into elite backgrounds (Ravelombola et al., 2021; Sansa et al., 2024; Wu et al., 2021a; Zaffar et al., 2024) [38,51,56,77] .

Conventional vs. modern breeding strategies for drought tolerance in cowpea

Cowpea drought breeding is shifting from purely phenotypic, field-based selection toward integrated pipelines combining managed stress screening, trait-based phenotyping, and molecular tools. Because drought tolerance is complex and polygenic, no single strategy is sufficient; genetic gain increasingly depends on the strategic integration of conventional and modern approaches.

Conventional selection and pedigree breeding

Early cowpea improvement relied on mass and pedigree selection for yield and survival under drought, sometimes supported by seedling screening systems (e.g., wooden box methods) that showed correspondence with adult-stage tolerance (Agbicodo et al., 2009; Mofokeng & Mashingaidze, 2019; Nkomo et al., 2020) [58,71,72] . While this approach delivered adapted cultivars, progress has been slow due to low heritability, strong environmental effects, and large G×E interactions (Agbicodo et al., 2009; Mofokeng & Mashingaidze, 2019) [58,71] . Consequently, multi-environment testing with AMMI and GGE biplots is now routine to identify stable, high-yielding parents and advanced lines for droughtprone regions (Sansa et al., 2025; Singini et al., 2024) [67,69] .

Marker-assisted selection and QTL introgression

Early cowpea improvement relied on mass and pedigree selection for yield and survival under drought, sometimes supported by seedling screening systems (e.g., wooden box methods) that showed correspondence with adult-stage tolerance (Agbicodo et al., 2009; Mofokeng & Mashingaidze, 2019; Nkomo et al., 2020) [58,71,72] . While this approach delivered adapted cultivars, progress has been slow due to low heritability, strong environmental effects, and large G×E interactions (Agbicodo et al., 2009; Mofokeng & Mashingaidze, 2019) [58,71] . Consequently, multi-environment testing with AMMI and GGE biplots is now routine to identify stable, high-yielding parents and advanced lines for droughtprone regions (Sansa et al., 2025; Singini et al., 2024) [69,67] .

GWAS-enabled breeding

GWAS using GBS, resequencing, and MAGIC populations has greatly expanded marker density and mapping resolution, identifying loci controlling chlorosis tolerance, greenness, yield components, phenology, and drought indices, with strong signals on chromosomes 1, 3, 5, and 11 and sets of diagnostic SNPs suitable for marker development (Ravelombola et al., 2021, 2025b) [38] . These resources provide a foundation for candidate gene discovery, marker pyramiding, and low-cost diagnostic panels.

Genomic selection for drought tolerance

Genomic prediction for phenology, yield traits, and drought indices in cowpea shows low-to-moderate accuracy, with higher performance for simpler traits (Ravelombola et al., 2021; Rosero et al., 2020) [66,73] . Despite this, both empirical and simulation studies indicate that genomic selection can significantly increase genetic gain per unit time when combined with phenotypic selection, making it a promising complement for improving complex, polygenic drought tolerance.

Speed breeding and rapid-cycling pipelines

Although formal speed-breeding systems are less developed in cowpea, accelerated generation advance through off-season nurseries, controlled environments, and early-generation phenotypic and molecula screening is increasingly emphasized (Mofokeng & Mashingaidze, 2019; Nkomo et al., 2021) [71] . Seedling drought screening combined with marker filtering enables rapid elimination of inferior lines before resourceintensive field testing (Nkomo et al., 2020; Ravelombola et al., 2020) [72,74] .

Integrated synthesis

Evidence across Sections 4.3-4.6 demonstrates that effective cowpea drought improvement now depends on integrated, multi-stage pipelines aligning managed stress environments, drought indices, and mechanistic traits with GWAS, MAS, and genomic selection. Managed screening increases selection accuracy, indices stabilize genotype ranking across environments, and physiological traits anchor selection in biological function. The convergence of high-throughput phenotyping, MAGIC populations, and genomic tools is enabling earlier, faster, and more predictive selection, shifting cowpea breeding from yield-only approaches toward physiology-guided, genomics-assisted frameworks.

Genomic and molecular approaches for drought tolerance in cowpea 4.1 Genomic resources and reference genomes

The development of high-quality reference genomes and pangenomes has transformed cowpea into a genomics-enabled crop for drought research. A chromosome-scale reference genome of IT97K-499-35 resolved the 11 chromosomes, revealed extensive synteny with common bean, and provided a foundation for QTL discovery and candidate gene identification (Muñoz-Amatriaín et al., 2018; Lonardi et al., 2017) [64,65] . Recent pan-genome analyses across cultivated accessions identified ~35, 000 pan-genes, including ~5, 000 non-core genes enriched for stress-and defense-related functions, highlighting presence/absence and structural variation as major reservoirs of drought-adaptive diversity (Liang et al., 2024) [84] . High-density SNP resources now support population-scale drought genomics, including the 51K iSelect array, GBS and DArTseq platforms, whole-genome resequencing panels, and breeder-friendly KASP markers (Boukar et al., 2016; Muñoz-Amatriaín et al., 2017) [75,64] . Global diversity panels and a cowpea MAGIC population provide structured germplasm for high-resolution GWAS and drought trait dissection (Ravelombola et al., 2021; Sansa et al., 2024) [66,69] . These resources have enabled precise mapping of loci controlling drought injury, photosynthetic resilience, stomatal behavior, and yield stability, consistently identifying hotspots on Vu01, Vu03, Vu05, and Vu11 and linking genome variation to ABA signaling, ROS detoxification, and photoprotection pathways.

QTL mapping for drought and adaptive traits in cowpea

Early biparental QTL studies established drought tolerance in cowpea as a polygenic and environment-sensitive trait. Mapping in RIL populations identified multiple loci for premature senescence, biomass recovery, stay-green phenotype, and yield under drought, with pleiotropic QTL linking delayed senescence to post-flowering drought tolerance (Muchero et al., 2009, 2013) [76] . Candidate-gene anchored QTL further implicated stress-responsive genes associated with photosynthesis and membrane protection. Although these studies provided foundational drought-linked regions and markers, their resolution and allelic diversity were limited. The expansion of genomic resources has shifted cowpea drought research toward GWAS-based, populationscale dissection of complex drought responses.

Genome-wide association studies (GWAS) for drought and adaptive traits in cowpea

GWAS using resequencing, GBS, DArTseq, and SNP arrays across diversity panels and MAGIC populations has revealed drought adaptation as highly polygenic, with loci distributed across all chromosomes (Ravelombola et al., 2021, 2025b; Sansa et al., 2024) [56,69] . Seedling-stage GWAS identified major loci for leaf chlorosis and greenness linked to ABA signaling, ROS detoxification, and chloroplast maintenance (Ravelombola et al., 2025b) [38] . Gravimetric GWAS uncovered loci controlling stomatal closure dynamics and water-use strategies (Wu et al., 2021b) [56] . Field GWAS connected chlorophyll fluorescence parameters and yield stability with photoprotective and redox-related genes (Sansa et al., 2024) [69] . Root-focused GWAS further identified loci for root biomass and architecture, particularly on Vu05, highlighting belowground adaptation mechanisms (Zaffar et al., 2024) [77] . Collectively, cowpea GWAS converge on recurrent genomic hotspots and core processes involving ABA signaling, stomatal regulation, photosynthetic resilience, and root system plasticity, delivering validated markers and candidate genes for genomicsassisted breeding.

Multi-omics dissection of drought tolerance in cowpea

Transcriptomic, proteomic, and metabolomic studies demonstrate that cowpea drought tolerance is governed by coordinated regulatory networks integrating ABA signaling, transcription factor cascades, ROS detoxification, and osmotic adjustment (Goufo et al., 2017; Ribeiro et al., 2023b) [16,39] . ABA biosynthesis and signaling genes (e.g., VuNCED), WRKY, NAC, bZIP, and DREB transcription factors, aquaporins, and trehalose metabolism genes consistently emerge as central regulatory hubs (Fang et al., 2023; Matos et al., 2022) [55,78] . Proteomics shows that tolerant genotypes maintain photosynthetic and protective proteins while redirecting metabolism toward stress defense (Ribeiro et al. 2023b) [39] . Metabolomics highlights accumulation of osmoprotectants, amino acids, and flavonoids particularly in roots supporting a central role for root metabolic plasticity in drought sensing and signaling (Goufo et al., 2017) [16] . These convergent omics datasets provide functional bridges between GWAS/QTL signals and drought-responsive biological pathways.

Functional validation and candidate gene confirmation

Functional genomics has confirmed several cowpea drought regulators. Overexpression of VuNAC1/2 improved growth, yield components, and drought tolerance through enhanced photosynthesis, antioxidant activity, and water-use efficiency (Srivastava et al., 2022) [79] . miR408 was validated as a drought-regulatory hub controlling lignin remodeling, chlorophyll retention, and stomatal behavior (Mishra et al., 2021b) [28] . Heterologous studies further support roles for VuDREB and NAC factors in dehydration tolerance. CRISPR/Cas9 editing in hairy roots, cowpea protoplast systems, and agroinfiltration platforms now enable direct functional testing of candidate genes and promoters (Ji et al., 2019; Bridgeland et al., 2023) [80,81] . Integration of GWAS, omics, and in planta validation increasingly prioritizes regulatory network nodes controlling stomatal sensitivity, photosynthetic protection, and carbon remobilization. Functionally validated genes and biomarkers now support marker-assisted selection, biologically informed genomic selection, and emerging genome-editing strategies, positioning functional genomics as a critical bridge between association mapping and precision breeding of drought-resilient cowpea.

Future perspectives and research gaps

Despite major advances in genomics and functional studies, translating drought biology into climate-resilient cowpea cultivars remains constrained by environmental complexity, limited root integration, and slow breeding uptake. Future progress requires shifting from reductionist drought assays to integrative, climate-relevant, and breeder-oriented research frameworks. Climate-realistic phenotyping: Most studies rely on simplified drought screens that poorly represent farmer field conditions. There is a critical need for multi-season, multi-stress managed platforms with precise envirotyping to capture drought timing, intensity, recovery, and heat interactions, thereby improving the relevance of GWAS, genomic selection, and trait discovery. Root-centered drought biology: Cowpea drought research remains largely shoot-focused, despite the central role of root architecture, hydraulics, and rhizosphere interactions in drought avoidance. Field-scalable root phenotyping, genetic dissection of deep and plastic root systems, and integration of microbiome and symbiosis studies must become priority frontiers.  From loci to systems: Most drought-associated SNPs show small, environment-dependent effects. Future efforts should move toward systems-level drought genomics by integrating GWAS with regulatory networks, eQTL/pQTL mapping, pan-genomes, and machine-learning approaches to identify pathway hubs rather than isolated markers.  Closing the translational gap: Although many droughtresponsive genes have been identified, very few have been functionally validated or deployed. Priorities include scalable transformation and editing systems, validation of natural alleles, CRISPR-based allele mining, and early incorporation of functional markers into breeding pipelines.  Predictive and farmer-centered breeding: Nextgeneration cowpea improvement should integrate highthroughput phenomics, omics predictors, envirotyping, and crop modeling into AI-supported breeding pipelines. Equally important is aligning molecular drought tolerance with farmer-preferred traits through participatory breeding and on-farm evaluation to ensure adoption and impact.

Conclusion

Cowpea drought research is rapidly advancing through the integration of conventional breeding, physiological trait analysis, and genomics tools such as reference genomes, GWAS, MAGIC populations, and genomic selection. These efforts have clarified the complex, polygenic architecture of drought adaptation, highlighting key traits including root plasticity, stomatal regulation, and stay-green-mediated photosynthetic resilience. However, translation into farmerpreferred, climate-resilient cultivars remains limited, particularly under multi-stress field conditions. Critical gaps include underexplored root biology, limited functional validation of candidate genes, and weak integration of multiomics with predictive breeding. Future research should prioritize climate-realistic managed stress platforms, systemslevel genomics, and participatory breeding approaches to accelerate early-generation selection and environment-specific cultivar design. Such convergence offers a practical pathway to develop drought-resilient cowpea cultivars that sustain productivity, stabilize livelihoods, and enhance food security in drought-prone regions.

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