The pattern of genetic variability in a core collection of 2,021 cowpea accessions.

Cowpea is a highly drought-adapted leguminous crop with great promise for improving agricultural sustainability and food security. Here, we report analyses derived from array-based genotyping of 2,021 accessions constituting a core subset of the world's largest cowpea collection, held at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. We used this dataset to examine genetic variation and population structure in worldwide cowpea. We confirm that the primary pattern of population structure is two geographically defined subpopulations originating in West and East Africa, respectively, and that population structure is associated with shifts in phenotypic distribution. Furthermore, we establish the cowpea core collection as a resource for genome-wide association studies by mapping the genetic basis of several phenotypes, with a focus on seed coat pigmentation patterning and color. We anticipate that the genotyped IITA Cowpea Core Collection will serve as a powerful tool for mapping complex traits, facilitating the acceleration of breeding programs to enhance the resilience of this crop in the face of rapid global climate change.

Urine and Dried Blood Spots From Children and Pregnant Women Reveal Phytochemicals, Amino Acids, and Carnitine Metabolites as Cowpea Consumption Biomarkers.

SCOPE: Legumes consumption has been proven to promote health across the lifespan; cowpeas have demonstrated efficacy in combating childhood malnutrition and growth faltering, with an estimated malnutrition prevalence of 35.6% of children in Ghana. This cowpea feeding study aimed to identify a suite of metabolic consumption biomarkers in children and adults. METHODS AND RESULTS: Urine and dried blood spots (DBS) from 24 children (9-21 months) and 21 pregnant women (>18 years) in Northern Ghana are collected before and after dose-escalated consumption of four cowpea varieties for 15 days. Untargeted metabolomics identified significant increases in amino acids, phytochemicals, and lipids. The carnitine metabolism pathway is represented by 137 urine and 43 DBS metabolites, with significant changes to tiglylcarnitine and acetylcarnitine. Additional noteworthy candidate biomarkers are mansouramycin C, N-acetylalliin, proline betaine, N2, N5-diacetylornithine, S-methylcysteine, S-methylcysteine sulfoxide, and cis-urocanate. S-methylcysteine and S-methylcysteine sulfoxide are targeted and quantified in urine. CONCLUSION: This feeding study for cowpea biomarkers supports the utility of a suite of key metabolites classified as amino acids, lipids, and phytochemicals for dietary legume and cowpea-specific food exposures of global health importance.

A view of the pan-genome of domesticated Cowpea (Vigna unguiculata [L.] Walp.).

Cowpea, Vigna unguiculata L. Walp., is a diploid warm-season legume of critical importance as both food and fodder in sub-Saharan Africa. This species is also grown in Northern Africa, Europe, Latin America, North America, and East to Southeast Asia. To capture the genomic diversity of domesticates of this important legume, de novo genome assemblies were produced for representatives of six subpopulations of cultivated cowpea identified previously from genotyping of several hundred diverse accessions. In the most complete assembly (IT97K-499-35), 26,026 core and 4963 noncore genes were identified, with 35,436 pan genes when considering all seven accessions. GO terms associated with response to stress and defense response were highly enriched among the noncore genes, while core genes were enriched in terms related to transcription factor activity, and transport and metabolic processes. Over 5 million single nucleotide polymorphisms (SNPs) relative to each assembly and over 40 structural variants >1 Mb in size were identified by comparing genomes. Vu10 was the chromosome with the highest frequency of SNPs, and Vu04 had the most structural variants. Noncore genes harbor a larger proportion of potentially disruptive variants than core genes, including missense, stop gain, and frameshift mutations; this suggests that noncore genes substantially contribute to diversity within domesticated cowpea.

Corrigendum: Seed coat pattern QTL and development in cowpea (Vigna unguiculata [L.] Walp.).

[This corrects the article DOI: 10.3389/fpls.2019.01346.].

Non-targeted metabolomics of cooked cowpea (Vigna unguiculata) and pigeon pea (Cajanus cajan) from Ghana using two distinct and complementary analytical platforms.

Legumes are global staple foods with multiple human health properties that merit detailed composition analysis in cooked forms. This study analyzed cowpea [Vigna unguiculata] (three varieties: Dagbantuya, Sangyi, and Tukara), pigeon pea [Cajanus cajan], and common bean [Phaseolus vulgaris] using two distinct ultra-performance liquid chromatography mass spectrometry (UPLC-MS) platforms and analytical workflows. Comparisons between cowpea and pigeon pea consumed in Ghana, and common bean (navy bean) from USA, revealed 75 metabolites that differentiated cowpeas. Metabolite fold-change comparisons resulted in 142 metabolites with significantly higher abundance in cowpea, and 154 higher in abundance from pigeon pea. 3-(all-trans-nonaprenyl)benzene-1,2-diol, N-tetracosanoylphytosphingosine, and sitoindoside II are novel identifications in cowpea, with notably higher abundance than other legumes tested. Cowpea variety specific markers were tonkinelin (Dagbantuya), pheophytin A (Sangyi), and linoleoyl ethanolamide (Tukara). This study identified novel cowpea and pigeon pea food metabolites that warrant continued investigation as bioactive food components following consumption in people.

Genome-Wide Association Study Reveals Candidate Genes for Flowering Time in Cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp., diploid, 2n = 22) is a major crop used as a protein source for human consumption as well as a quality feed for livestock. It is drought and heat tolerant and has been bred to develop varieties that are resilient to changing climates. Plant adaptation to new climates and their yield are strongly affected by flowering time. Therefore, understanding the genetic basis of flowering time is critical to advance cowpea breeding. The aim of this study was to perform genome-wide association studies (GWAS) to identify marker trait associations for flowering time in cowpea using single nucleotide polymorphism (SNP) markers. A total of 368 accessions from a cowpea mini-core collection were evaluated in Ft. Collins, CO in 2019 and 2020, and 292 accessions were evaluated in Citra, FL in 2018. These accessions were genotyped using the Cowpea iSelect Consortium Array that contained 51,128 SNPs. GWAS revealed seven reliable SNPs for flowering time that explained 8-12% of the phenotypic variance. Candidate genes including FT, GI, CRY2, LSH3, UGT87A2, LIF2, and HTA9 that are associated with flowering time were identified for the significant SNP markers. Further efforts to validate these loci will help to understand their role in flowering time in cowpea, and it could facilitate the transfer of some of this knowledge to other closely related legume species.

Genetic, anatomical, and environmental patterns related to pod shattering resistance in domesticated cowpea [Vigna unguiculata (L.) Walp].

Pod shattering, which causes the explosive release of seeds from the pod, is one of the main sources of yield losses in cowpea in arid and semi-arid areas. Reduction of shattering has therefore been a primary target for selection during domestication and improvement of cowpea, among other species. Using a mini-core diversity panel of 368 cowpea accessions, four regions with a statistically significant association with pod shattering were identified. Two genes (Vigun03g321100 and Vigun11g100600), involved in cell wall biosynthesis, were identified as strong candidates for pod shattering. Microscopic analysis was conducted on a subset of accessions representing the full spectrum of shattering phenotypes. This analysis indicated that the extent of wall fiber deposition was highly correlated with shattering. The results from this study also demonstrate that pod shattering in cowpea is exacerbated by arid environmental conditions. Finally, using a subset of West African landraces, patterns of historical selection for shattering resistance related to precipitation in the environment of origin were identified. Together, these results shed light on sources of resistance to pod shattering, which will, in turn, improve climate resilience of a major global nutritional staple.

BAC- and oligo-FISH mapping reveals chromosome evolution among Vigna angularis, V. unguiculata, and Phaseolus vulgaris.

Cytogenomic resources have accelerated synteny and chromosome evolution studies in plant species, including legumes. Here, we established the first cytogenetic map of V. angularis (Va, subgenus Ceratotropis) and compared this new map with those of V. unguiculata (Vu, subgenus Vigna) and P. vulgaris (Pv) by BAC-FISH and oligopainting approaches. We mapped 19 Vu BACs and 35S rDNA probes to the 11 chromosome pairs of Va, Vu, and Pv. Vigna angularis shared a high degree of macrosynteny with Vu and Pv, with five conserved syntenic chromosomes. Additionally, we developed two oligo probes (Pv2 and Pv3) used to paint Vigna orthologous chromosomes. We confirmed two reciprocal translocations (chromosomes 2 and 3 and 1 and 8) that have occurred after the Vigna and Phaseolus divergence (~9.7 Mya). Besides, two inversions (2 and 4) and one translocation (1 and 5) have occurred after Vigna and Ceratotropis subgenera separation (~3.6 Mya). We also observed distinct oligopainting patterns for chromosomes 2 and 3 of Vigna species. Both Vigna species shared similar major rearrangements compared to Pv: one translocation (2 and 3) and one inversion (chromosome 3). The sequence synteny identified additional inversions and/or intrachromosomal translocations involving pericentromeric regions of both orthologous chromosomes. We propose chromosomes 2 and 3 as hotspots for chromosomal rearrangements and de novo centromere formation within and between Vigna and Phaseolus. Our BAC- and oligo-FISH mapping contributed to physically trace the chromosome evolution of Vigna and Phaseolus and its application in further studies of both genera.

A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns.

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.

Population Structure and Genetic Diversity in Korean Cowpea Germplasm Based on SNP Markers.

Cowpea is one of the most essential legume crops providing inexpensive dietary protein and nutrients. The aim of this study was to understand the genetic diversity and population structure of global and Korean cowpea germplasms. A total of 384 cowpea accessions from 21 countries were genotyped with the Cowpea iSelect Consortium Array containing 51,128 single-nucleotide polymorphisms (SNPs). After SNP filtering, a genetic diversity study was carried out using 35,116 SNPs within 376 cowpea accessions, including 229 Korean accessions. Based on structure and principal component analysis, a total of 376 global accessions were divided into four major populations. Accessions in group 1 were from Asia and Europe, those in groups 2 and 4 were from Korea, and those in group 3 were from West Africa. In addition, 229 Korean accessions were divided into three major populations (Q1, Jeonra province; Q2, Gangwon province; Q3, a mixture of provinces). Additionally, the neighbor-joining tree indicated similar results. Further genetic diversity analysis within the global and Korean population groups indicated low heterozygosity, a low polymorphism information content, and a high inbreeding coefficient in the Korean cowpea accessions. The population structure analysis will provide useful knowledge to support the genetic potential of the cowpea breeding program, especially in Korea.

Breaks of macrosynteny and collinearity among moth bean (Vigna aconitifolia), cowpea (V. unguiculata), and common bean (Phaseolus vulgaris).

Comparative cytogenetic mapping is a powerful approach to gain insights into genome organization of orphan crops, lacking a whole sequenced genome. To investigate the cytogenomic evolution of important Vigna and Phaseolus beans, we built a BAC-FISH (fluorescent in situ hybridization of bacterial artificial chromosome) map of Vigna aconitifolia (Vac, subgenus Ceratotropis), species with no sequenced genome, and compared with V. unguiculata (Vu, subgenus Vigna) and Phaseolus vulgaris (Pv) maps. Seventeen Pv BACs, eight Vu BACs, and 5S and 35S rDNA probes were hybridized in situ on the 11 Vac chromosome pairs. Five Vac chromosomes (Vac6, Vac7, Vac9, Vac10, and Vac11) showed conserved macrosynteny and collinearity between V. unguiculata and P. vulgaris. On the other hand, we observed collinearity breaks, identified by pericentric inversions involving Vac2 (Vu2), Vac4 (Vu4), and Vac3 (Pv3). We also detected macrosynteny breaks of translocation type involving chromosomes 1 and 8 of V. aconitifolia and P. vulgaris; 2 and 3 of V. aconitifolia and P. vulgaris; and 1 and 5 of V. aconitifolia and V. unguiculata. Considering our data and previous BAC-FISH studies, six chromosomes (1, 2, 3, 4, 5, and 8) are involved in major karyotype divergences between genera and five (1, 2, 3, 4, and 5) between Vigna subgenera, including mechanisms such as duplications, inversions, and translocations. Macrosynteny breaks between Vigna and Phaseolus suggest that the major chromosomal rearrangements have occurred within the Vigna clade. Our cytogenomic comparisons bring new light on the degree of shared macrosynteny and mechanisms of karyotype diversification during Vigna and Phaseolus evolution.

Identification of QTL for perenniality and floral scent in cowpea (Vigna unguiculata [L.] Walp.).

Perennial habit and floral scent are major traits that distinguish domesticated cowpeas from their wild relatives. However, the genetic basis of these two important traits remains largely unknown in cowpea. Plant longevity, a perenniality-related trait, and floral scent, an outcrossing trait, were investigated using a RIL population derived from a cross between a domesticated and a wild cowpea. QTL analysis revealed three significant loci, one on chromosome 8 associated with plant longevity and two, on chromosomes 1 and 11, for floral scent. Genes within the QTL regions were identified. Genes encoding an F-box protein (Vigun08g215300) and two kinases (Vigun08g217000, Vigun08g217800), and involved in physiological processes including regulation of flowering time and plant longevity, were identified within the perenniality QTL region. A cluster of O-methyltransferase genes (Vigun11g096800, Vigun11g096900, Vigun11g097000, Vigun11g097600, and Vigun11g097800) was identified within the floral scent QTL region. These O-methyltransferase cowpea genes are orthologs of the Arabidopsis N-acetylserotonin O-methyltransferase (ASMT) gene, which is involved in the biosynthesis of melatonin. Melatonin is an indole derivative, which is an essential molecule for plant interactions with pollinators. These findings lay the foundation for further exploration of the genetic mechanisms of perenniality and floral scent in cowpea. Knowledge from this study can help in the development of new extended-growth cycle lines with increased yield or lines with increased outcrossing for population breeding.

Perspectives on Low Temperature Tolerance and Vernalization Sensitivity in Barley: Prospects for Facultative Growth Habit.

One option to achieving greater resiliency for barley production in the face of climate change is to explore the potential of winter and facultative growth habits: for both types, low temperature tolerance (LTT) and vernalization sensitivity are key traits. Sensitivity to short-day photoperiod is a desirable attribute for facultative types. In order to broaden our understanding of the genetics of these phenotypes, we mapped quantitative trait loci (QTLs) and identified candidate genes using a genome-wide association studies (GWAS) panel composed of 882 barley accessions that was genotyped with the Illumina 9K single-nucleotide polymorphism (SNP) chip. Fifteen loci including 5 known and 10 novel QTL/genes were identified for LTT-assessed as winter survival in 10 field tests and mapped using a GWAS meta-analysis. FR-H1, FR-H2, and FR-H3 were major drivers of LTT, and candidate genes were identified for FR-H3. The principal determinants of vernalization sensitivity were VRN-H1, VRN-H2, and PPD-H1. VRN-H2 deletions conferred insensitive or intermediate sensitivity to vernalization. A subset of accessions with maximum LTT were identified as a resource for allele mining and further characterization. Facultative types comprised a small portion of the GWAS panel but may be useful for developing germplasm with this growth habit.

Seed Coat Pattern QTL and Development in Cowpea (Vigna unguiculata [L.] Walp.).

The appearance of the seed is an important aspect of consumer preference for cowpea (Vigna unguiculata [L.] Walp.). Seed coat pattern in cowpea has been a subject of study for over a century. This study makes use of newly available resources, including mapping populations, a reference genome and additional genome assemblies, and a high-density single nucleotide polymorphism genotyping platform, to map various seed coat pattern traits to three loci, concurrent with the Color Factor (C), Watson (W), and Holstein (H) factors identified previously. Several gene models encoding proteins involved in regulating the later stages of the flavonoid biosynthesis pathway have been identified as candidate genes, including a basic helix-loop-helix gene (Vigun07g110700) for the C locus, a WD-repeat gene (Vigun09g139900) for the W locus and an E3 ubiquitin ligase gene (Vigun10g163900) for the H locus. A model of seed coat development, consisting of six distinct stages, is described to explain some of the observed pattern phenotypes.

A genome-wide association and meta-analysis reveal regions associated with seed size in cowpea [Vigna unguiculata (L.) Walp].

KEY MESSAGE: This paper combined GWAS, meta-analysis and sequence homology comparison with common bean to identify regions associated with seed size variation in domesticated cowpea. Seed size is an important trait for yield and commercial value in dry-grain cowpea. Seed size varies widely among different cowpea accessions, and the genetic basis of such variation is not yet well understood. To better decipher the genetic basis of seed size, a genome-wide association study (GWAS) and meta-analysis were conducted on a panel of 368 cowpea diverse accessions from 51 countries. Four traits, including seed weight, length, width and density were evaluated across three locations. Using 51,128 single nucleotide polymorphisms covering the cowpea genome, 17 loci were identified for these traits. One locus was common to weight, width and length, suggesting pleiotropy. By integrating synteny-based analysis with common bean, six candidate genes (Vigun05g036000, Vigun05g039600, Vigun05g204200, Vigun08g217000, Vigun11g187000, and Vigun11g191300) which are implicated in multiple functional categories related to seed size such as endosperm development, embryo development, and cell elongation were identified. These results suggest that a combination of GWAS meta-analysis with synteny comparison in a related plant is an efficient approach to identify candidate gene (s) for complex traits in cowpea. The identified loci and candidate genes provide useful information for improving cowpea varieties and for molecular investigation of seed size.

The genome of cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

Orphan genes are involved in drought adaptations and ecoclimatic-oriented selections in domesticated cowpea.

Orphan genes (OGs) are genes that are restricted to a single species or a particular taxonomic group. To date, little is known about the functions of OGs in domesticated crops. Here, we report our findings on the relationships between OGs and environmental adaptation in cowpea (Vigna unguiculata). We identified 578 expressed OGs, of which 73.2% were predicted to be non-coding. Transcriptomic analyses revealed a high rate of OGs that were drought inducible in roots when compared with conserved genes. Co-expression analysis further revealed the possible involvement of OGs in stress response pathways. Overexpression of UP12_8740, a drought-inducible OG, conferred enhanced tolerance to osmotic stresses and soil drought. By combining Capture-Seq and fluorescence-based Kompetitive allele-specific PCR (KASP), we efficiently genotyped single nucleotide polymorphisms (SNPs) on OGs across a 223 accession cowpea germplasm collection. Population genomic parameters, including polymorphism information content (PIC), expected heterozygosity (He), nucleotide diversity (π), and Tajima's D statistics, that were calculated based on these SNPs, showed distinct signatures between the grain- and vegetable-type subpopulations of cowpea. This study reinforces the idea that OGs are a valuable resource for identifying new genes related to species-specific environmental adaptations and fosters new insights that artificial selection on OGs might have contributed to balancing the adaptive and agronomic traits in domesticated crops in various ecoclimatic conditions.

A Novel Root-Knot Nematode Resistance QTL on Chromosome Vu01 in Cowpea.

The root-knot nematode (RKN) species Meloidogyne incognita and M. javanica cause substantial root system damage and suppress yield of susceptible cowpea cultivars. The narrow-based genetic resistance conferred by the Rk gene, present in some commercial cultivars, is not effective against Rk-virulent populations found in several cowpea production areas. The dynamics of virulence within RKN populations require a broadening of the genetic base of resistance in elite cowpea cultivars. As part of this goal, F1 and F2 populations from the cross CB46-Null (susceptible) x FN-2-9-04 (resistant) were phenotyped for M. javanica induced root-galling (RG) and egg-mass production (EM) in controlled growth chamber and greenhouse infection assays. In addition, F[Formula: see text] families of the same cross were phenotyped for RG on field sites infested with Rk-avirulent M. incognita and M. javanica The response of F1 to RG and EM indicated that resistance to RKN in FN-2-9-04 is partially dominant, as supported by the degree of dominance in the F2 and F[Formula: see text] populations. Two QTL associated with both RG and EM resistance were detected on chromosomes Vu01 and Vu04. The QTL on Vu01 was most effective against aggressive M. javanica, whereas both QTL were effective against avirulent M. incognita Allelism tests with CB46 x FN-2-9-04 progeny indicated that these parents share the same RKN resistance locus on Vu04, but the strong, broad-based resistance in FN-2-9-04 is conferred by the additive effect of the novel resistance QTL on Vu01. This novel resistance in FN-2-9-04 is an important resource for broadening RKN resistance in elite cowpea cultivars.

Identification of Candidate Genes Controlling Black Seed Coat and Pod Tip Color in Cowpea (Vigna unguiculata [L.] Walp).

Seed coat color is an important part of consumer preferences for cowpea (Vigna unguiculata [L.] Walp). Color has been studied in numerous crop species and has often been linked to loci controlling the anthocyanin biosynthesis pathway. This study makes use of available resources, including mapping populations, a reference genome, and a high-density single nucleotide polymorphism genotyping platform, to map the black seed coat and purple pod tip color traits, with the gene symbol Bl, in cowpea. Several gene models encoding MYB domain protein 113 were identified as candidate genes. MYB domain proteins have been shown in other species to control expression of genes encoding enzymes for the final steps in the anthocyanin biosynthesis pathway. PCR analysis indicated that a presence/absence variation of one or more MYB113 genes may control the presence or absence of black pigment. A PCR marker has been developed for the MYB113 gene Vigun05g039500, a candidate gene for black seed coat color in cowpea.

Identification of QTL controlling domestication-related traits in cowpea (Vigna unguiculata L. Walp).

Cowpea (Vigna unguiculata L. Walp) is a warm-season legume with a genetically diverse gene-pool composed of wild and cultivated forms. Cowpea domestication involved considerable phenotypic changes from the wild progenitor, including reduction of pod shattering, increased organ size, and changes in flowering time. Little is known about the genetic basis underlying these changes. In this study, 215 recombinant inbred lines derived from a cross between a cultivated and a wild cowpea accession were used to evaluate nine domestication-related traits (pod shattering, peduncle length, flower color, days to flowering, 100-seed weight, pod length, leaf length, leaf width and seed number per pod). A high-density genetic map containing 17,739 single nucleotide polymorphisms was constructed and used to identify 16 quantitative trait loci (QTL) for these nine traits. Based on annotations of the cowpea reference genome, genes within these regions are reported. Four regions with clusters of QTL were identified, including one on chromosome 8 related to increased organ size. This study provides new knowledge of the genomic regions controlling domestication-related traits in cowpea as well as candidate genes underlying those QTL. This information can help to exploit wild relatives in cowpea breeding programs.