Philip A. Roberts

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.

A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): Evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters.

The root lesion nematodes of the genus Pratylenchus Filipjev, 1936 are migratory endoparasites of plant roots, considered among the most widespread and important nematode parasites in a variety of crops. We obtained gene sequences from the D2 and D3 expansion segments of 28S rRNA partial and 18S rRNA from 31 populations belonging to 11 valid and two unidentified species of root lesion nematodes and five outgroup taxa. These datasets were analyzed using maximum parsimony and Bayesian inference. The alignments were generated using the secondary structure models for these molecules and analyzed with Bayesian inference under the standard models and the complex model, considering helices under the doublet model and loops and bulges under the general time reversible model. The phylogenetic informativeness of morphological characters is tested by reconstruction of their histories on rRNA based trees using parallel parsimony and Bayesian approaches. Phylogenetic and sequence analyses of the 28S D2-D3 dataset with 145 accessions for 28 species and 18S dataset with 68 accessions for 15 species confirmed among large numbers of geographical diverse isolates that most classical morphospecies are monophyletic. Phylogenetic analyses revealed at least six distinct major clades of examined Pratylenchus species and these clades are generally congruent with those defined by characters derived from lip patterns, numbers of lip annules, and spermatheca shape. Morphological results suggest the need for sophisticated character discovery and analysis for morphology based phylogenetics in nematodes.

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.

Cowpea–Soybean Synteny Clarified through an Improved Genetic Map

Linkage mapping is relevant to modern plant biology and provides a framework for downstream analyses including quantitative trait loci identification, map‐based cloning, assessment of diversity, association mapping, and molecular breeding. Here, we report a consensus genetic map of cowpea [Vigna unguiculata (L.) Walp.] and synteny to other legumes based on expressed sequence tag (EST)‐derived single nucleotide polymorphisms (SNPs). In total, 1293 individuals representing 13 mapping populations were genotyped using an Illumina 1536 GoldenGate Assay. A consensus map containing 1107 EST‐derived SNP markers (856 bins) on 11 linkage groups (680 cM) was constructed from 13 population‐specific maps. This effort combined six new population‐specific maps and seven revised population‐specific maps to construct an improved consensus map with 33% more bins, 19% more markers, and improved marker order when compared to the previous cowpea SNP consensus map. Comparative and whole genome visualizations are presented as a framework for discussing map quality and synteny with soybean [Glycine max (L.) Merr.].

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.

The Mi-9 Gene from Solanum arcanum Conferring Heat-Stable Resistance to Root-Knot Nematodes Is a Homolog of Mi-1

Resistance conferred by the Mi-1 gene from Solanum peruvianum is effective and widely used for limiting root-knot nematode (Meloidogyne spp.) yield loss in tomato (Solanum lycopersicum), but the resistance is ineffective at soil temperatures above 28°C. Previously, we mapped the heat-stable resistance gene Mi-9 in Solanum arcanum accession LA2157 to the short arm of chromosome 6, in a genetic interval as Mi-1 and the Cladosporium fulvum resistance gene Cf2. We developed a fine map of the Mi-9 region by resistance and marker screening of an F2 population and derived F3 families from resistant LA2157 × susceptible LA392. Mi-1 intron 1 flanking primers were designed to amplify intron 1 and fingerprint Mi-1 homologs. Using these primers, we identified seven Mi-1 homologs in the mapping parents. Cf-2 and Mi-1 homologs were mapped on chromosome 6 using a subset of the F2. Cf-2 homologs did not segregate with Mi-9 resistance, but three Mi-1 homologs (RH1, RH2, and RH4) from LA2157 and one (SH1) from LA392 colocalized to the Mi-9 region. Reverse transcriptase-polymerase chain reaction analysis indicated that six Mi-1 homologs are expressed in LA2157 roots. We targeted transcripts of Mi-1 homologs for degradation with tobacco (Nicotiana tabacum) rattle virus (TRV)-based virus-induced gene silencing using Agrobacterium infiltration with a TRV-Mi construct. In most LA2157 plants infiltrated with the TRV-Mi construct, Mi-9-meditated heat-stable root-knot nematode resistance was compromised at 32°C, indicating that the heat-stable resistance is mediated by a homolog of Mi-1.

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.

Relationships between Meloidogyne incognita resistance genes in Lycopersicon peruvianum differentiated by heat sensitivity and nematode virulence.

Resistance to Meloidogyne incognita (Kofoid and White) Chitwood in clones of Lycopersicon peruvianum (L.) Mill. PI 126443-1MH, 270435-2R2 and 2704353MH, their F1, a field-produced F2, and their test-cross (TC1) populations, was evaluated based on egg masses and eggs produced on root systems. Reactions to M. incognita isolates differing in virulence to gene Mi were determined at 25°C (Mi expressed) and 32°C (Mi not expressed). PI 126443-1MH, 270435-2R2, 270435-3MH, and their F1 progenies were resistant to Mi-virulent and Mi-avirulent isolates. At 32°C with a Mi-avirulent isolate and at 25°C with a Mi-virulent isolate, four TC1 generations segregated into resistant: susceptible (R∶S) ratios close to 3∶1. These results indicated resistance to Mi-(a)virulent M. incognita isolates is conferred by different non-allelic dominant genes in PI 126443-1MH, 270435-2R2 and 270435-3MH. The F2 progeny of PI 126443-1MH x EPP-1, challenged with Mi-avirulent M. incognita at 32°C and with Mi-virulent M. incognita at both 25°C and 32°C, segregated with a ratio of 3∶1 (R∶S), indicating expression of a single dominant resistance gene in PI 126443-1MH in each case. In dual screenings on clones of the same individual plants from the TC1 and F2 segregating populations, some individual plants were susceptible at 32°C to a Mi-avirulent isolate but resistant to the Mi-virulent isolate, and vice versa, suggesting that different but linked genes confer heat-stable resistance to Mi-avirulent M. incognita and resistance to Mi-virulent M. incognita. We propose the symbol Mi-5 for the gene in PI 126443 clone 1MH and the symbol Mi-6 for the gene in PI 270435 clone 3MH which both confer resistance to Mi-avirulent M. incognita isolates at high temperature. We propose the symbol Mi-7 for the gene in PI 270435 clone 3MH and the symbol Mi-8 for the gene in PI 270435 clone 2R2 that both confer resistance to the Mi-virulent M. incognita isolate 557R at moderate (25°C) temperature. The novel resistance genes are linked and reside in a genomic region in each parental clone that is independent from the Mi locus.