In total, 36 additional cowpea accessions relevant to Africa, China and the USA were shotgun sequenced using Illumina HiSeq 2500 (12.5× average coverage) and aligned to the WGS assembly of IT97K‐499‐35 to discover SNPs. We used molecular cytogenetics to characterize the structure of pachytene chromosomes to advance our knowledge of chromosome and genome organization of cowpea. (2013). It should be noted that these landraces were chosen by the different breeding programs and may not represent the full range of genetic diversity available in the West African landrace germplasm. Cowpea has a great potential in increasing food legume production. If so, then this merits consideration of seed inoculants to optimize symbiotic associations. Soil Surface Structure Stabilization by Municipal Waste Compost Application. STRUCTURE was run four times for each hypothetical number of subpopulations (K) between 1 and 6, with a burn‐in period of 10 000 and 50 000 Monte Carlo Markov Chain iterations. The WGS assembly from IT97K‐499‐35 described above was used as the reference to map each of these 36 sets of reads, and the new set of HiSeq sequences from the reference genotype sequenced at the University of California Riverside. "A good number of genes are conserved across species," he said. It is unknown why many landraces from West Africa belong to this subpopulation. Genomics-Assisted Breeding for Drought Tolerance in Cowpea. Vigna unguiculata linkage group 1 and VuLG 3 displayed synteny with 7 of the 20 soybean chromosomes while VuLG 8 was syntenic with three soybean chromosomes. Repeats in the contigs and pseudochromosomes were analyzed using RepeatMasker. Genomics Assisted Breeding of Crops for Abiotic Stress Tolerance, Vol. Globally, cowpea is an important grain legume adapted and grown in dry areas of the tropics and subtropics. The reference genome sequence described here was used to further investigate this domestication hotspot, which spans 2.21 Mb and includes 313 genes. The FST value for subpopulations 1 and 2 was 0.18, indicating moderate population differentiation. Breaks of macrosynteny and collinearity among moth bean (Vigna aconitifolia), cowpea (V. unguiculata), and common bean (Phaseolus vulgaris). Cowpea is one of the oldest source of human food. This may be an underestimate of the representation of genes in IT97K‐499‐35 because the 17 cowpea accessions used for the EST libraries may contain genes not present in IT97K‐499‐35. WGS sequence raw reads from 37 diverse cowpea accessions are available under SRA accession SRP077082. Table S6. The Windows software HarvEST:Cowpea (, which includes a synteny display function, also has adopted an updated numbering system. A multi‐parent advanced generation inter‐cross (MAGIC) population for genetic analysis and improvement of cowpea (Vigna unguiculata L. Sequence alignments were then generated for all families using muscle (Edgar, 2004), and hidden Markov models (HMMs) were calculated using hmmer (Mistry et al., 2013). These filtering steps included: (i) designability score based upon Illumina's Assay Design Tool; (ii) avoidance of a SNP whose adjacent sequences occurred frequently in the genome assembly; (iii) consideration of allele frequency, generally avoiding SNPs with only one accession carrying the minor allele; (iv) selection of two SNPs in or near each inferred cowpea gene based on MUMmer sequence alignment with P. vulgaris gene models (Schmutz et al., 2014); (v) requirement for a minimum distance from a SNP that had already been selected; (vi) preference against an A/T or C/G SNP since these require two beadtypes (assay space); and (vii) location within a relatively larger WGS contig to maximize the amount of WGS contigs that could subsequently be anchored to a SNP‐based genetic map. A diploid chromosome number 2n = 22 obtained in this study agrees with earlier results in cowpea as reported by [16] . Author Email : The resulting cluster file is available upon request. Rich], a Lost Crop of Africa. Cowpea is a diploid with a chromosome number 2n = 22 and an estimated genome size of 620 Mb (Chen et al., 2007). From a cytogenetic viewpoint, relatively little is known about Vigna species (Saccardo et al. ancestry ≥0.8; Table S6) included 23 landraces from the four countries and 22 breeding accessions. SMRTbell libraries were annealed and bound to the P6 DNA polymerase for sequencing using the DNA/Polymerase Binding Kit P6 v2.0 (P/N100‐372‐700). International Journal of Molecular Sciences. The iSelect SNP design sequences were used as BLAST queries to search against WGS and BAC sequences, and matches with an e‐value = 1e−50 or better were tallied. CLARK and CLARK‐S are classification tools that use discriminative (spaced, in the case CLARK‐S) k‐mers to quickly determine the most likely origin of each input sequence (k = 21 and k = 31). The cultivated Asiatic Vigna species belong to the sub-genus Ceratotropis, a fairly distinct and homogeneous group, largely restricted to Asia, which has a chromosome number of 2n = 22 (except V. glabrescens, 2n = 44). Because this cultivated parent has the same haplotype as the reference genome, and thus presumably also the same orientation, the lack of recombination across this region suggests that the opposite‐to‐reference orientation is the ancestral (wild) type while the reference orientation carries an inversion. The fourth linkage map is the next with 20 SNP markers, which covered 101.05 cM, while the remaining linkage maps had between 2 to 9 SNP markers (Figure … The other set of Sanger sequences included the BES described above. Current advances and future directions in genetic enhancement of a climate resilient food legume crop, cowpea (Vigna unguiculata L. Yield Adjustment by Canola Grown at Different Plant Populations under Semiarid Conditions. These were not included in the iSelect design. Relatively higher SNP frequencies were also observed in the distal ends of LG5 and LG9, in the centromeric region of LG7, and toward the ends of LG1. Alignments that were further used had a minimum identity of 55.11% and a mean identity of 89.24%. TZ and MCL generated the optical maps. (2005) to estimate the optimum number of subpopulations. Use the link below to share a full-text version of this article with your friends and colleagues. Distribution Top of page. Young cowpea leaves are used as spinach in eastern and southern Africa while green immature pods and green mature seed… A genetic map was constructed using MSTmap (Wu et al., 2008; at LOD 10 for each RIL population. West Africa is the region with the largest production and consumption of cowpea in the world (FAOSTAT, 2012; Singh, 2014). One exception to this latter trend is near 63 cM on LG1, where both subpopulations have very low diversity and contain the same alleles (low FST; Figure 3). Walp.) Evaluation of genetic diversity has important implications for breeding programs and the conservation of genetic resources. Alignments with a length < 1 kb were filtered out. To explore whether the inversion is associated with Striga resistance, the map positions of previously identified QTLs for this trait (Ouédraogo et al., 2001, 2002; Boukar et al., 2004) were compared with the position of the inversion. To meet assumptions of the clustering algorithm, ‘synthetic heterozygotes’ were constructed and included in the initial set of 96 genotyped samples by creating 1:1 mixtures of DNA samples from individuals known from prior work to be most genetically distant from each other. The map spans 837.11 cM at an average density of one bin per 0.26 cM and 11.4 SNPs per bin. ANBp-14-03). Knowledge of the recombination rate can be integrated into decisions on marker density and provide weight factors in genomic selection models to favor rare recombination events within low recombination regions. Main determinant is the karyotype, referring to the number, as well as the size and shape of the chromosomes of an individual. Cowpea SNP frequencies were based on the number of discovered SNPs per genetic bin and the total size of the WGS scaffolds allocated into the corresponding bin. An additional set of 12.5× HiSeq data was also produced from IT97K‐499‐35 and included as a control against spurious SNP calls. 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 publicly available genome sequence lays the foundation for basic and applied research, enabling progress towards the improvement in this key crop plant for food and nutritional security. Using the same computational pipeline as for V. unguiculata (Vu), the repeats of the V. angularis (Yang et al., 2015; Va) and V. radiata (Kang et al., 2014; Vr) genomes also were annotated. Leaf morphology was studied in the cowpea RIL population, Sanzi (sub-globose leaf shape) x Vita 7 (hastate leaf shape). SNP frequencies calculated for 2 cM windows and normalized to the total anchored scaffold size (in kb) are shown for the 11 cowpea LGs. This reference sequence was used to identify repetitive elements, genes and gene families, and genetic variation, and for comparative analysis with three closely related legumes including common bean, which stimulated a change in chromosome numbering to facilitate comparative studies. 2015). Because the level of residual heterozygosity varied among populations, different population type options were chosen for map construction in MSTmap (RIL 7 for Tvu‐14676 × IT84S‐2246‐4; RIL 6 for Sanzi × Vita7 and ZN016 × Zhijiang282; and RIL5 for CB46 × IT93K‐503‐1 and CB27 × IT82E‐18). A chromosome‐scale assembly of the black gram (Vigna mungo) genome. Subread Filtering PacBio read length distribution. There are approximately 4,000 seeds/lb (Woodruff et al., 2010), and there are about 60 lb/bushel of grain (Murphy, 1993). Holm et al. Synteny view between cowpea (Vu; Vigna unguiculata) and other closely related diploid species. Heat stress and cowpea: genetics, breeding and modern tools for improving genetic gains. The software STRUCTURE v.2.3.4 (Pritchard et al., 2000) was used to infer population structure. The same batch of IT97K‐499‐35 nuclear DNA that was used for BAC library construction was used for WGS sequencing. more bins). Seed Coat Pattern QTL and Development in Cowpea (Vigna unguiculata [L.] Walp.). The region extending from the beginning of the first hit to the end of the last hit was considered to define the centromeric region of each cowpea chromosome. About 394 M paired‐end reads (equivalent to approximately 65× coverage) with an average read length of approximately 100 bases after quality‐trimming were produced at the National Center for Genome Resources (NCGR; Santa Fe, NM, USA) on an Illumina GAII sequencing instrument. Figure S3. Gene and repeat densities, and recombination rate in the cowpea genome. (2016) that correlates the genetic and chromosome maps in cowpea can be used to orient the cowpea genetic map so that it meets the convention of displaying the short arm on top. As the cytometry analysis indicates, a genome size of 640.6 Mbp was used. Figure S9. The much smaller number of insertions than deletions may reflect limitations in the ability of the software to identify insertions when sequence reads are mapped to a reference genome. (a) The relationships between genetic and physical positions are shown for single nucleotide polymorphisms (SNPs) on four genetic maps (1–4). Raw PacBio reads for cowpea accession IT97K‐499‐35 are available at NCBI SRA sample SRS3721827 (study SRP159026). It is a self-pollinating diploid with chromosome number 2n = 22 and a genome size of about 613 (Arumuganathan … The first linkage map had 23 SNP markers (Figure 1) which covered 72.02 cM of the genome, followed by eighth linkage with 21 SNP markers and covered 41.98 cM. RNA‐Seq raw reads are available as NCBI SRA biosample accessions SAMN071606186 through SAMN071606198, SAMN07194302 through SAMN07194309 and SAMN07194882 through SAMN07194909, and were described in Yao et al. Prior to calculating phylogenetic trees, the HMM alignments from the resulting family sets were trimmed of non‐aligning characters (characters outside the HMM match states). Journal of the Science of Food and Agriculture. A WGS approach using short‐read sequencing was followed to assemble sequences of the cowpea genome. Table S2 shows the summary of raw molecules status and the BNG BssSI map assembly. DE‐AC02‐05CH11231. Recently, a genomic region related to increased organ size in cowpea was identified on Vu08 using a recombinant inbred line (RIL) population derived from a domesticated × wild cross (Lo et al., 2018). (2005) method, the maximum ∆K value was reached at K = 2 (Figure 2a), which would be consistent with two major subpopulations. Development of new genetic resources for faba bean (Vicia faba L.) breeding through the discovery of gene-based SNP markers and the construction of a high-density consensus map. Resulting SMRTbell libraries were size selected using the BluePippin (Sage Biosciences) according to the Blue Pippin User Manual and Quick Guide. Optimizing Resource Allocation in a Cowpea (Vigna unguiculata L. Alignment files were merged with the software tool Picard to a single ‘sam’ file. The ‘Single‐to‐End’ and ‘End‐to‐End’ merging function of FPC was used for a final, third stage assembly by stepwise decreases of assembly stringency based on Sulston score cutoff values (down to 1 × 10−35). Cowpea (Vigna unguiculata [L.] Walp.) Phosphorus Losses from Low‐Emission Slurry Spreading Techniques. It should be noted also that most chromosomes that have a one‐to‐two relationship across these species or genera are consistent with translocations involving the centromeric regions (Figure 3a–c). Landrace Through Whole-Plant Field Phenotyping and Non-stop Selection to Sustain Increased Genetic Gain Across a Decade. Computational identification of receptor-like kinases “RLK” and receptor-like proteins “RLP” in legumes. BES from vector sequences, E. coli, mitochondria and chloroplasts were identified using BLASTN. Little genetic differentiation was found for landraces vs. breeding materials (FST = 0.02), in accordance with STRUCTURE and PCA results. Still, as sub-Saharan Africa and other cowpea production regions encounter climate variability. In addition, 24.5 Mb of the anchored sequences were oriented arbitrarily. When these metrics are combined with minor allele frequency and nearness to a trait determinant, one can choose an optimal set of SNPs for a given constraint, for example cost minimization, on the number of markers. The sum of all these identity scores was computed for each contig, both for the black and the white list. Large chromosomal inversion detected on Vu03. Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers. ), an Underutilized Crop, to Aid Global Food Security: Varietal Improvement, Genetic Diversity and Processing. STRUCTURE (Pritchard et al., 2000) was run for K = 1–6 and, although the estimated log probabilities of the data reached a plateau at K = 5 (Figure 2a), at that level of population subdivision there were individuals not strongly assigned to one subpopulation or another (Figure S3). Circos illustration of synteny between cowpea linkage groups (VuLG) and common bean pseudomolecules (Pv). Orphan genes are involved in drought adaptations and ecoclimatic-oriented selections in domesticated cowpea. Cowpea LGs were plotted according to cM lengths, while common bean chromosomes were plotted as physical length. Table S4. Contrasting examples include Vu04, where the recombination rate near the telomeres of both arms of this metacentric chromosome are roughly 10 times the rate across the pericentromeric region, versus Vu02 and Vu06, where the entire short arm in each of these acrocentric chromosomes has a low recombination rate (Figure S8). It provides strong support to the livelihood of small-scale farmers through its contributions to their nutritional security, income generation and soil fertility enhancement. The anchored sequences contain 100 Mb of the WGS assembly (237 Mb scaffold size including Ns; Table S1 and Data S5) and 420 Mb of BAC assemblies (Table S2 and Data S6). chromosome, chromosome number, 2n= From the website (now inactive) of Dr. Bruce Reid at Kean University: Chromosome Numbers of Selected Organisms . These two scores can be interpreted as the weighted coverage of a contig by statistically significant alignments from the respective set of genomes. This region contained 289 Phaseolus genes, of which only one (Phvul.008G285800) was present in the intersection with a list of genes associated with domestication reported in Schmutz et al. Please check your email for instructions on resetting your password. The elite breeding line IT97K‐499‐35, developed at the International Institute of Tropical Agriculture (IITA, Nigeria), was used previously for the development of genome resources (Timko et al., 2008; Muñoz‐Amatriaín et al., 2017). The same WGS data described above were analyzed using BreakDancer v.1.4.5 (Chen et al., 2009) to identify structural variants. Loci were determined by transcript assembly alignments and/or EXONERATE alignments of proteins from Arabidopsis, common bean, soybean, Medicago, poplar, rice, grape and Swiss‐Prot proteomes to repeat‐soft‐masked cowpea genome using RepeatMasker (Smit et al., 2017) with up to 2 kb extension on both ends unless extending into another locus on the same strand. more bins). The chromosome number of this crop is 2n = 22 [4,25,27]. Hinge v0.41 (Kamath et al., 2017) was also tested on this dataset, but at that time the tool required the entire alignment file (over 2 Tb) to fit in primary memory and we did not have the computational resources to handle it. The intersection of these two lists contained only a single gene, Phvul.008G285800, a P. vulgaris candidate gene for increased seed size that corresponds to cowpea Vigun08 g217000. This yielded 51 128 assays in the final manifest for the publicly available Cowpea iSelect Consortium Array. represents number of chromosomes possessed by cowpea genome. This would facilitate the transfer of genomic information on target traits from one Fabaceae species to another. Nearly 1M SNPs with strong support were discovered previously by aligning WGS data from 36 diverse accessions to a draft assembly of IT97K‐499‐35 (Muñoz‐Amatriaín et al., 2017). This is 18 543 gene families, monophyletic for the legume family, including proteomes from cowpea (this study), 13 other crop and model legumes, and five non‐legume species for phylogenetic rooting and evolutionary context (Table S11). Characterization, Stability, and Plant Effects of Kiln‐Produced Wheat Straw Biochar. Reads which mapped to multiple locations were excluded from further analysis. Reads were assigned to individual BACs and then assembled using SPAdes (Bankevich et al., 2012). PacBio RS II sequencing data were collected in 6‐h movies and Stage Start was enabled to capture the longest subreads possible. Any queries (other than missing content) should be directed to the corresponding author for the article. These two datasets were assembled using SOAPdenovo (Luo et al., 2012) together with the Sanger BAC‐end sequences (BES) described below and the ‘gene‐space’ sequences available from Timko et al. Modeling Elevated Carbon Dioxide Effects on Water Relations, Water Use, and Growth of Irrigated Sorghum. For example, a major gene for a trait that lies within a low recombination region can be expected to have high linkage drag when introgressed into a different background. Four additional accessions from China (see Table S3) were sequenced at the Majorbio Pharm Technology Co. Ltd (Shanghai, China). 2016) overlapped with the resistance region on the same chromosome in FN-2-9-04 within 2.9 cM of the CB46-Null x FN-2-9-04 F2 population and within 1.59 cM on the cowpea consensus genetic map (Muñoz-Amatriaín et al. … and Azospirillum brasilense combined with N rates in cowpea-wheat crop sequence. These include: (a) adzuki bean (Va; Vigna angularis); (b) mung bean (Vr; Vigna radiata); and (c) common bean (Pv; Phaseolus vulgaris) using the revised cowpea chromosome numbering system. In general, SNP density was lowest near centromeric regions (Figures 1 and S9). FST values were also plotted across the genome (Figures 3 and S4, lower plots). Figure S6. Pacific Biosciences reads were generated at Washington State University (Pullman, WA, USA) following the ‘Procedure and Checklist‐20 kb Template Preparation Using BluePippin Size Selection System’ (P/N 100‐286‐000‐5) protocol provided by Pacific Biosciences (Menlo Park, CA, USA) and the Pacific Biosciences SMRTbell Template Prep kit 1.0 (P/N 100‐259‐100). Draft genome sequence of a less-known wild Vigna: Beach pea (V. marina cv. Chromomeres were not distributed uniformly along the arms. Snp ( data S7 step of one bin, 146 accessions were chosen to represent the geographic, and! 20.18 Mb ( Table 1 reports statistics of the most widely grown legume that lacks a published reference orientation. Generated using pipeline ‘ nucmer ’, with a step of one bin per 0.26 cM and 11.4 SNPs bin... 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