Exploiting genetic and genomic resources to enhance productivity and abiotic stress adaptation of underutilized pulses.

Underutilized pulses and their wild relatives are typically stress tolerant and their seeds are packed with protein, fibers, minerals, vitamins, and phytochemicals. The consumption of such nutritionally dense legumes together with cereal-based food may promote global food and nutritional security. However, such species are deficient in a few or several desirable domestication traits thereby reducing their agronomic value, requiring further genetic enhancement for developing productive, nutritionally dense, and climate resilient cultivars. This review article considers 13 underutilized pulses and focuses on their germplasm holdings, diversity, crop-wild-crop gene flow, genome sequencing, syntenic relationships, the potential for breeding and transgenic manipulation, and the genetics of agronomic and stress tolerance traits. Recent progress has shown the potential for crop improvement and food security, for example, the genetic basis of stem determinacy and fragrance in moth bean and rice bean, multiple abiotic stress tolerant traits in horse gram and tepary bean, bruchid resistance in lima bean, low neurotoxin in grass pea, and photoperiod induced flowering and anthocyanin accumulation in adzuki bean have been investigated. Advances in introgression breeding to develop elite genetic stocks of grass pea with low ß-ODAP (neurotoxin compound), resistance to Mungbean yellow mosaic India virus in black gram using rice bean, and abiotic stress adaptation in common bean, using genes from tepary bean have been carried out. This highlights their potential in wider breeding programs to introduce such traits in locally adapted cultivars. The potential of de-domestication or feralization in the evolution of new variants in these crops are also highlighted.

Genome-Wide association analysis of phenotypic traits in Bambara groundnut under drought-stressed and non-stressed conditions based on DArTseq SNP.

Introduction: Bambara groundnut (BG) (Vigna subterranea [L.] Verdc) is an indigenous, resilient, but underutilized leguminous crop that occurs mostly as genetically heterogeneous landraces with limited information on the drought tolerant attributes. This study elucidates the associations between sequencing-based diversity array technology (DArTseq) and phenotypic character as well as differing indices related to drought tolerance in one hundred accessions of Bambara groundnut. Methods: The field experiments were conducted at IITA research stations in Kano and Ibadan between 2016 and 2018 planting seasons. The experiments were arranged in randomised complete block design with three replications, under the different water regimes. The phenotypic traits evaluated was further to construct the dendrogram. Genome-wide association mapping was conducted based on 5927 DArTs loci with < 20% missing data. Results and Discussions: The genome wide association study predicted drought tolerance in Bambara accessions for geometric mean productivity (GMP) and stress tolerance index (STI). TVSu-423 had the highest GMP and STI values (28.50, 2.40), while TVSu-2017 had the lowest at GMP (1.74) and STI (0.01) respectively. The relative water content (%) was significantly higher for accessions; TVSu-266 (60.35, 61.49), TVSu-2 (58.29, 53.94), and TVSu-411 (55.17, 58.92) in 2016/2017 and 2017/2018, respectively. The phenotypic characters studied delineated the accessions into two major clusters and five distinct sub-clusters, indicating variations across all the geographical locations. The 5,927 DArTseq genomic markers in association with STI further grouped the 100 accessions into two main clusters. TVSu-1897 from Botswana (Southern Africa) was in the first cluster, while the remaining 99 accessions from Western, Central, and Eastern Africa made up the second cluster. The eight significant Quantitative Trait Loci (QTLs) (24346377|F|0-22:A>G-22:A>G, 24384105|F|0-56:A>G33 :A> G, 24385643|F|0-53:G>C-53:G>C, 24385696|F|0-43:A>G-43:A>G, 4177257|F|0-44:A>T-44:A>T, 4182070|F|0-66:G>A-66:G>A, 4183483|F|0-24:G>A-24:G>A, 4183904|F|0-11:C>T-11:C>T) identified with Bonferroni threshold was in association with STI, indicative of variations under the drought-stressed condition. The observation of consistent SNPs in the 2016 and 2017 planting seasons, as well as in combination with the 2016 and 2017 planting seasons, led to the designation of these QTLs as significant. The drought selected accessions could form basis for hybridization breeding. The identified quantitative trait loci could be useful in marker-assisted selection in drought molecular breeding programs.

Genome-Wide Association Study Revealed SNP Alleles Associated with Seed Size Traits in African Yam Bean (Sphenostylis stenocarpa (Hochst ex. A. Rich.) Harms).

Seed size is an important yield and quality-determining trait in higher plants and is also crucial to their evolutionary fitness. In African yam bean (AYB), seed size varies widely among different accessions. However, the genetic basis of such variation has not been adequately documented. A genome-wide marker-trait association study was conducted to identify genomic regions associated with four seed size traits (seed length, seed width, seed thickness, and 100-seed weight) in a panel of 195 AYB accessions. A total of 5416 SNP markers were generated from the diversity array technology sequence (DArTseq) genotype-by-sequencing (GBS)- approach, in which 2491 SNPs were retained after SNP quality control and used for marker-trait association analysis. Significant phenotypic variation was observed for the traits. Broad-sense heritability ranged from 50.0% (seed width) to 66.4% (seed length). The relationships among the traits were positive and significant. Genome-wide association study (GWAS) using the general linear model (GLM) and the mixed linear model (MLM) approaches identified 12 SNP markers significantly associated with seed size traits across the six test environments. The 12 makers explained 6.5-10.8% of the phenotypic variation. Two markers (29420334|F|0-52:C>G-52:C>G and 29420736|F|0-57:G>T-57:G>T) with pleiotropic effects associated with seed width and seed thickness were found. A candidate gene search identified five significant markers (100026424|F|0-37:C>T-37:C>T, 100041049|F|0-42:G>C-42:G>C, 100034480|F|0-31:C>A-31:C>A, 29420365|F|0-55:C>G-55:C>G, and 29420736|F|0-57:G>T-57:G>T) located close to 43 putative genes whose encoding protein products are known to regulate seed size traits. This study revealed significant makers not previously reported for seed size in AYB and could provide useful information for genomic-assisted breeding in AYB.

A review of biotechnological approaches towards crop improvement in African yam bean (Sphenostylis stenocarpa Hochst. Ex A. Rich.).

Globally, climate change is a major factor that contributes significantly to food and nutrition insecurity, limiting crop yield and availability. Although efforts are being made to curb food insecurity, millions of people still suffer from malnutrition. For the United Nations (UN) Sustainable Development Goal of Food Security to be achieved, diverse cropping systems must be developed instead of relying mainly on a few staple crops. Many orphan legumes have untapped potential that can be of significance for developing improved cultivars with enhanced tolerance to changing climatic conditions. One typical example of such an orphan crop is Sphenostylis stenocarpa Hochst. Ex A. Rich. Harms, popularly known as African yam bean (AYB). The crop is an underutilised tropical legume that is climate-resilient and has excellent potential for smallholder agriculture in sub-Saharan Africa (SSA). Studies on AYB have featured morphological characterisation, assessment of genetic diversity using various molecular markers, and the development of tissue culture protocols for rapidly multiplying propagules. However, these have not translated into varietal development, and low yields remain a challenge. The application of suitable biotechnologies to improve AYB is imperative for increased yield, sustainable utilisation and conservation. This review discusses biotechnological strategies with prospective applications for AYB improvement. The potential risks of these strategies are also highlighted.

Effect-directed profiling and identification of bioactive metabolites from field, in vitro-grown and acclimatized Musa spp. accessions using high-performance thin-layer chromatography-mass spectrometry.

Bananas and plantains (Musa spp.) are used as nutritious foods, and at the same time, are a source of phytoconstituents for the pharmaceutical industry. As biological activities of especially the pulp and peel of Musa spp. have been documented, this study investigated the variation in the secondary metabolite profiles of the leaves from field, in vitro-grown and acclimatized accessions. The genetic fidelity of the diverse accessions was assessed using diversity array technology sequencing. It showed that the in vitro-grown accessions were true-to-type with the field samples. The antioxidant and anticholinesterase activities of the samples from different culture systems (field and in vitro) were evaluated by UV-spectrophotometry and compared to high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA). The latter was applied for the first time for effect-directed profiling of the polar and medium polar sample components via different biochemical and biological assays. Compound zones showed acetyl-/butylrylcholinesterase inhibition (zones 1-4), α-/ß-glucosidase inhibition (zones 1 and 2) as well as antioxidative (zones 1-3) and antimicrobial (zone 4) activities. Structures were preliminary assigned by HPTLC-HRMS. The HPTLC was effective for bioactivity-guided characterization of the bioactive constituents in Musa spp. accessions. Accumulation of useful metabolites, especially compounds with antioxidant and anticholinesterase properties, was higher in samples from in vitro system. This validated the use of plant tissue culturing as an alternative method for large scale production of plant material and supply of bioactive constituents.

Further Evidence That the Genebank Standards for Drying Orthodox Seeds May Not Be Optimal for Subsequent Seed Longevity.

Maximizing seed longevity is important for genebanks to efficiently manage their accessions, reducing the frequency of costly regeneration cycles and the loss of genetic integrity. Research on rice seeds has shown that subsequent longevity in air-dry storage can be improved by drying seeds, which are metabolically active at harvest (moisture contents above a critical value close to 16.5%), for an initial period at a higher temperature (40°C-60°C) than that currently recommended by the current genebank standards (5°C-20°C). The aim of this study was to test whether similar benefits could be achieved in two legume species-cowpea and soya bean-by drying freshly harvested seeds, from two separate harvests, at 40°C and 35% relative humidity, for up to 8 days before equilibrium drying in a drying room (17°C and 15% relative humidity). Improvements in longevity were observed in three of the four accessions of soya bean, with the greatest improvement generally occurring after the maximum duration (8 days) at the higher temperature. However, of the five accessions of cowpea, only seeds of TVu-9698 and TVu-13209 from the first harvest, and of TVu-13193 from the second harvest, showed an improvement in longevity compared with drying following the standard protocol. A negative effect of high-temperature drying was also observed in one accession of cowpea, TVu-11980, but only in seeds harvested later in the season, 13 weeks after planting. This research not only provides evidence of the potential benefits of drying orthodox seeds at an alternative, higher, temperature instead of at the conventional lower temperature, before long-term storage, but also raises awareness of how genebanks can improve the management of their accessions.

Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement.

Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection.

De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification.

BACKGROUND: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. RESULTS: Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. CONCLUSIONS: This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.

A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover.

BACKGROUND: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. RESULTS: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. CONCLUSIONS: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a "core set" of ancestrally derived homeolog specific variants in white clover.

The genetic location of the self-incompatibility locus in white clover (Trifolium repens L.).

White clover (Trifolium repens L.) is a forage legume of considerable economic importance in temperate agricultural systems. It has a strong self-incompatibility system. The molecular basis of self-incompatibility in T. repens is unknown, but it is under the control of a single locus, which is expressed gametophytically. To locate the self-incompatibility locus (S locus) in T. repens, we carried out cross-pollination experiments in an F(1) mapping population and constructed a genetic linkage map using amplified fragment length polymorphism and simple sequence repeat markers. As the first step in a map-based cloning strategy, we locate for the first time the S locus in T. repens on a genetic linkage map, on the homoeologous linkage group pair 1 (E), which is broadly syntenic to Medicago truncatula L. chromosome 1. On the basis of this syntenic relationship, the possibility that the S locus may or may not possess an S-RNase gene is discussed.

Construction, characterization, and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.).

White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37 248 clones with an average insert size of approximately 85 kb, representing an approximate 3-fold coverage of the white clover genome based on an estimated genome size of 960 Mb. The BAC library was pooled and screened by polymerase chain reaction (PCR) amplification using both white clover microsatellites and PCR-based markers derived from Medicago truncatula, resulting in an average of 6 hits per marker; this supports the estimated 3-fold genome coverage in this allotetraploid species. PCR-based screening of 766 clones with a multiplex set of chloroplast primers showed that only 0.5% of BAC clones contained chloroplast-derived inserts. The library was further evaluated by sequencing both ends of 724 of the clover BACs. These were analysed with respect to their sequence content and their homology to the contents of a range of plant gene, expressed sequence tag, and repeat element databases. Forty-three microsatellites were discovered in the BAC-end sequences (BESs) and investigated as potential genetic markers in white clover. The BESs were also compared with the partially sequenced genome of the model legume M. truncatula with the specific intention of identifying putative comparative-tile BACs, which represent potential regions of microsynteny between the 2 species; 14 such BACs were discovered. The results suggest that a large-scale BAC-end sequencing strategy has the potential to anchor a significant proportion of the genome of white clover onto the gene-space sequence of M. truncatula.

A simple model of feedback regulation for nitrate uptake and N2 fixation in contrasting phenotypes of white clover.

A simple three equation model is proposed for the feedback regulation of nitrate uptake and N2 fixation, based on the concentration of the organic N substrate pool within the plant and two parameters denoting the N substrate concentrations at which half-maximal inhibition occurs. This model simulated three contrasting phenotypes of white clover (Trifolium repens L.) inbred lines with (1) normal rates of nitrate uptake and N2 fixation (NNU); (2) low rates of nitrate uptake (LNU); and (3) very low rates of N2 fixation (VLF). The LNU phenotype was simulated by a decrease in the value of the inhibition parameter for nitrate uptake and the VLF phenotype was simulated by a decrease in the value of the N2 fixation inhibition parameter. The model was tested against nitrate uptake data obtained from white clover plants growing in flowing nutrient culture. There was an accurate prediction of the increase in nitrate uptake caused by N2 fixation activity of the NNU and LNU inbred lines being interrupted by a switch in gas phase from air to Ar : O2. The model was also tested against data for nitrate uptake, N2 fixation and %N from fixation for the three inbred clover lines grown in flowing nutrient culture at 0, 5 or 20 mmol m(-3) N(3-). Again there was accurate prediction of nitrate uptake, although simulated values for N2 fixation were more variable. The simple model has potential use as a sub-routine in larger models of legume growth under field conditions.