AHS and JT annotated and analyzed repeats. Proceedings of the National Academy of Sciences. Family membership was evaluated relative to median HMM bitscores for each family, with sequences scoring less than 40% of the median HMM bitscore for the family being removed. Four additional accessions from China (see Table S3) were sequenced at the Majorbio Pharm Technology Co. Ltd (Shanghai, China). 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. (e) Single nucleotide polymorphism (SNP) density in 1 Mb windows. Comparison between cowpea and adzuki bean for the cowpea inversion region. The total number of unique 27‐mers in the range x = 2–10 000 is 31.381 × 109. Pseudochromosomes were obtained by anchoring the scaffold sequences to SNP markers (blast of SNP design sequences, e−50 or less) in 10 genetic maps (Table S4). Estimation of cowpea genome size using flow cytometry. A genome-wide association and meta-analysis reveal regions associated with seed size in cowpea [Vigna unguiculata (L.) Walp]. (2017) was used to join them by estimating the size of the gap (in cM). (2015). Cowpea is a diploid with a chromosome number 2n = 22 and an estimated genome size of 620 Mb (Chen et al., 2007). An extreme example of an exception is a region extending from 30 to 35 cM on LG7, where diversity is very low in subpopulation 2 (Figure 3). Each assembled contig was BLASTed against the ‘white’ genome and the ‘black’ genomes, and all high‐quality alignments (e‐score < 1e−47 corresponding to a bit score of at least 200, and covering at least 10% of the read length) were recorded. Evaluating the genetic diversity present in the West African breeding germplasm is important to manage breeding programs and assure future genetic gains. Intergenic regions had an average GC content of 31.84%. The output alignments between genomes were visualized using Circos v0.69‐3 (Krzywinski et al., 2009; Figure 3). For the BspQI map, seven separate runs (132 unique scans) were generated, and a total of 108 Gb (~170 × genome equivalent) of raw DNA molecules (> 100 kb) were collected. [L.] Walp) In fact, although the overall genetic diversity within the West African breeding population is relatively high (He and π = 0.31), we identified genomic regions of diversity depletion. Yield Adjustment by Canola Grown at Different Plant Populations under Semiarid Conditions. When applying the Evanno et al. Walp.). In addition, 29 728 ‘unigene’ consensus sequences, derived from 183 118 ESTs from cDNA libraries of 17 different cowpea accessions are available in the software HarvEST:Cowpea (harvest.ucr.edu) (Muchero et al., 2009). (2018) and Lo et al. Table S8. A number of initiatives including Tropical Legumes projects have contributed to the development of cowpea genomic resources. Current advances and future directions in genetic enhancement of a climate resilient food legume crop, cowpea (Vigna unguiculata L. Leaf morphology was studied in the cowpea RIL population, Sanzi (sub-globose leaf shape) x Vita 7 (hastate leaf shape). One of the strengths of cowpea is its high resilience to harsh conditions, including hot and dry environments, and poor soils (Boukar et al., 2018). Novo&Stitch: accurate reconciliation of genome assemblies via optical maps. The final stitched assembly was then polished via the PacBio Quiver pipeline (RS_resequencing.1 protocol) in SMRT Portal v2.3.0 (Patch 5) by mapping all the PacBio subreads against the assembly. 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. Freezing Effects on Carbon and Nitrogen Cycling in Northern Hardwood Forest Soils. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. Its genome shares a high degree of collinearity with other warm season legumes, especially common bean (Phaseolus vulgaris L.) (Vasconcelos et al., 2015). As is usually done, 27‐mers that appear only once are excluded because they are considered erroneous, that is to contain sequencing errors. Narrowing Down a Major QTL Region Conferring Pod Fiber Contents in Yardlong Bean (Vigna unguiculata), a Vegetable Cowpea. Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers. Repeats in the contigs and pseudochromosomes were analyzed using RepeatMasker. Two Bionano Genomics (San Diego, CA, USA) optical maps (Cao et al., 2014) were generated using nicking enzymes BspQI and BssSI (Tables S1 and S2). Figure S2 shows the frequency distribution of 27‐mers produced with KAT (https://github.com/TGAC/KAT). He values for subpopulation 1 and 2 were plotted to explore the spatial patterns of diversity across the 11 LGs (Figures 3 and S4, upper plots). MTP BACs were paired‐end sequenced (2 × 100 bases) using Illumina HiSeq2000 (Illumina, Inc, San Diego, CA, USA). With a high SNP coverage of the genome and connections to cowpea genome sequences, this study provides the basis for a unified chromosome nomenclature for the cowpea research community. Taken together, the cross‐species comparisons suggest that differences in genome size in Vigna can be largely explained by TE abundance, especially by that of Gypsy retrotransposons. The cowpea is a very old crop, probably native to Central Africa, although it has been grown in South-Eastern Asia for more than 2000 years. Customized, maximally informative subsets of markers have numerous applications including routine tests of seed purity, validation of germplasm fidelity, verification of successful crosses and guidance of progeny selection in later generations during trait introgression into preferred backgrounds via backcrossing. Only overlapping sequences >300 bp were considered to be overlaps. Stupické polní rané as an internal standard. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. A genome-wide association and meta-analysis reveal regions associated with seed size in cowpea [Vigna unguiculata (L.) Walp]. A set of MTP BACs was chosen using the FMTP method of Bozdag et al. Molecules of at least 180 kb in length were selected to generate a BNG map assembly. The Journal of Horticultural Science and Biotechnology. MTP BAC sequencing was supported by NSF IIS‐1526742 (‘III:Small:Algorithms for Genome Assembly’). more bins). 2017), corresponding to approximately 1 Mb on the cowpea pseudomolecules. Since all of these accessions have been adapted to West Africa, the existence of two major subpopulations at the present time means that relatively wide crosses can be made without compromising adaptation. 2 ). A contig was considered contaminated when the black score was at least twice as high as the white score. The homogenate was filtered through a 50‐μm nylon mesh to remove debris and kept on ice. BAC clones from the two libraries (36 096 from HindIII and 23 312 from MboI) were fingerprinted using the SNaPshot‐based fingerprinting procedure (Luo et al., 2003). The dashed lines indicate the bottom and top 1% of He and FST values, respectively. Then, 1 ml Otto II solution containing 50 μg ml−1 propidium iodide (PI) and 50 μg ml−1 RNase was added and the sample was analyzed by a CyFlow Space flow cytometer (Sysmex Partec, Görlitz, Germany). In total, 56.8 Gb of sequence data were generated (~91.7 × genome equivalent), with a read N50 of 14 595 bp. (1990) could be due to incomplete removal of formaldehyde fixative prior to staining with Schiff's reagent, which binds to free aldehyde groups (Chieco and Derenzini, 1999). Two outlying values were replacing by a maximum value so that all of the other calculated values could be easily visualized. All of the assembly statistics significantly improved compared with the eight individual assemblies (Table S3). In general, SNP density was lowest near centromeric regions (Figures 1 and S9). However, given the small sample size of local landraces and the fact that they were biased toward the interests of breeders, this may be an inaccurate estimate of the diversity in West African landrace germplasm. From these five cowpea LGs with one‐to‐two relationships, three (VuLG3, VuLG4, and VuLG7) have a higher number of links, and over a longer genome interval, with one P. vulgaris chromosome (Pv3, Pv1 and Pv2, respectively; Figure 1 and Table S5). 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. Breaks of macrosynteny and collinearity among moth bean (Vigna aconitifolia), cowpea (V. unguiculata), and common bean (Phaseolus vulgaris). As explained above, 10 genetic maps were used to anchor and orient scaffolds into pseudochromosomes. 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. Repeat‐masked sequences were then BLASTed against themselves using an e‐value cutoff of e−40. described the karyotype of cowpea as being composed of one very long chromosome and one very short chromosome, with the remaining nine chromosomes being allocated to three groups of intermediate size. 2, 3, breeding for more climate-resilient varieties remains a priority. Table S2 shows the summary of raw molecules status and the BNG BssSI map assembly. The GC content in coding exons was higher than in introns plus UTRs (40.82% versus 24.27%, respectively). In addition, 24.5 Mb of the anchored sequences were oriented arbitrarily. GWAS and Genomic Approaches in Legumes, an Expanding Toolkit for Examining Responses to Abiotic Stresses. Vigna unguiculata linkage group 1 and VuLG 3 displayed synteny with 7 of the 20 soybean chromosomes … The SNP markers associated with those QTLs span the genomic region Vu08:36035190‐38248903, which contains 313 annotated genes. The first derivative was then calculated for each of the 110 selected polynomials to represent the rate of recombination as cM/Mbp. SNPs with minor allele frequencies (MAF) <0.05 were excluded. 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. Each assembly took about 4–5 days on a 512‐core Torque/PBS server hosted at UC Riverside. Interestingly, a cluster of nodulins was annotated in BACs located in one of these regions. Quantitative trait loci influencing days to flowering and plant height in cowpea, Vigna unguiculata (L.) Walp. The NBS‐LRR superfamily contains 402 annotated genes, versus 272 and 86 in adzuki and mung bean, respectively (Data S7). The greater diversity within subpopulation 2 is apparent throughout most of the genome (Figures 3 and S4), with some exceptions. Figure S4. The revised numbering system is shown in Table S5 and used throughout the present manuscript. In brief, cytometry indicated that the 2C nuclear DNA amount of V. unguiculata IT97K‐499‐35 is 1.310 ± 0.026 pg DNA (mean ± SD), which corresponds to 1C genome size of 640.6 Mbp (Figure S1). It is a self-pollinating diploid with chromosome number 2n = 22 and a genome size of about 613 (Arumuganathan … By applying the Cowpea iSelect Consortium Array to 146 breeding lines and landraces, we have provided a useful overview of genetic variability in West African cultivated germplasm. Individual maps and the genotype data used for their construction can be found in Data S3. Association of Quantum Dot Nanoparticles with Pseudomonas aeruginosa Biofilm. Post‐filter PacBio read quality distribution. S. gesnerioides is widely distributed in Africa, from Morocco and Egypt, south to South Africa, also in Arabia and widely in India (up to 2000 m) and Sri Lanka. SNPs with low GenTrain scores were visually inspected based upon manufacturer's published best practice for optimizing accuracy in genotyping projects (http://www.illumina.com/documents/products/technotes/technote_infinium_genotyping_data_analysis.pdf). SNP calling and curation were done as described by Muñoz‐Amatriaín et al. The number of major soybean synteny blocks for each cowpea LG ranged from one to three, whereas the total number of significant soybean synteny blocks ranged from three to six. Also, He values can be used as criteria for selecting efficient subsets of markers for conversion to other platforms. This indicates that the intended bias towards genes in the iSelect array design (Muñoz‐Amatriaín et al., 2017) was successful. Identification of QTL controlling domestication-related traits in cowpea (Vigna unguiculata L. Walp). (2018). The final ordering and orientation of the scaffold was produced by ALLMAPS (Tang et al., 2015) from the SNP locations corresponding to the 10 genetic maps. It is closely related to soybeans and other warm-season legumes, but is more drought and heat tolerant. chromosome, chromosome number, 2n= From the website (now inactive) of Dr. Bruce Reid at Kean University: Chromosome Numbers of Selected Organisms . Finally, a total of 10 genetic maps containing 44 003 unique Illumina iSelect SNPs (Muñoz‐Amatriaín et al., 2017) were used to anchor and orient sequence scaffolds into 11 pseudochromosomes (i.e. To estimate the gene content of the WGS assembly, sequences were BLASTed against cowpea EST‐derived ‘unigenes’ (http://harvest.ucr.edu) and P. vulgaris gene models (Schmutz et al., 2014), using e‐value cutoffs of e−40 and e−25, respectively. 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. Salinity stress response and ‘omics’ approaches for improving salinity stress tolerance in major grain legumes. While several cowpea LGs are largely syntenic with one P. vulgaris chromosome, further resolution is needed to satisfy a single nomenclature for those LGs whose syntenic relationships with common bean are less clear. (2005). Cowpea is also an important crop in parts of Asia, South America, and the USA (Singh, 2014). Additional details about the stitching method can be found in Pan et al. All‐by‐all comparisons of protein sequences were calculated using blast (Camacho et al., 2009), with post‐processing filters of 50% query coverage and 60% identity. Output maps were inspected to identify and remove data that would result in presumably spurious double recombination events, unless supported by several markers or moderate to large genetic distances. This would facilitate reciprocal exchange of genomic information on target traits from one Vigna species to another. A WGS approach using short‐read sequencing was followed to assemble sequences of IT97K‐499‐35 is genome accession.... The black gram ( Vigna unguiculata ) is described in Lo et.. For selecting efficient subsets of the BsqQI optical map is available at SRA! Maf ) < 0.05 were excluded data resources for the Annealing and of! 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