CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes.

High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods because of the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ∼40% of reads mapping to repeats, increasing those mapping to single-copy regions by ∼2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ∼10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ∼12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed because of low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely related species (L. orientalis). Our results showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

The INCREASE project: Intelligent Collections of food-legume genetic resources for European agrofood systems.

Food legumes are crucial for all agriculture-related societal challenges, including climate change mitigation, agrobiodiversity conservation, sustainable agriculture, food security and human health. The transition to plant-based diets, largely based on food legumes, could present major opportunities for adaptation and mitigation, generating significant co-benefits for human health. The characterization, maintenance and exploitation of food-legume genetic resources, to date largely unexploited, form the core development of both sustainable agriculture and a healthy food system. INCREASE will implement, on chickpea (Cicer arietinum), common bean (Phaseolus vulgaris), lentil (Lens culinaris) and lupin (Lupinus albus and L. mutabilis), a new approach to conserve, manage and characterize genetic resources. Intelligent Collections, consisting of nested core collections composed of single-seed descent-purified accessions (i.e., inbred lines), will be developed, exploiting germplasm available both from genebanks and on-farm and subjected to different levels of genotypic and phenotypic characterization. Phenotyping and gene discovery activities will meet, via a participatory approach, the needs of various actors, including breeders, scientists, farmers and agri-food and non-food industries, exploiting also the power of massive metabolomics and transcriptomics and of artificial intelligence and smart tools. Moreover, INCREASE will test, with a citizen science experiment, an innovative system of conservation and use of genetic resources based on a decentralized approach for data management and dynamic conservation. By promoting the use of food legumes, improving their quality, adaptation and yield and boosting the competitiveness of the agriculture and food sector, the INCREASE strategy will have a major impact on economy and society and represents a case study of integrative and participatory approaches towards conservation and exploitation of crop genetic resources.

Genetic analysis of early phenology in lentil identifies distinct loci controlling component traits.

Modern-day domesticated lentil germplasm is generally considered to form three broad adaptation groups: Mediterranean, South Asian, and northern temperate, which correspond to the major global production environments. Reproductive phenology plays a key role in lentil adaptation to this diverse ecogeographic variation. Here, we dissect the characteristic earliness of the pilosae ecotype, suited to the typically short cropping season of South Asian environments. We identified two loci, DTF6a and DTF6b, at which dominant alleles confer early flowering, and we show that DTF6a alone is sufficient to confer early flowering under extremely short photoperiods. Genomic synteny confirmed the presence of a conserved cluster of three florigen (FT) gene orthologues among potential candidate genes, and expression analysis in near-isogenic material showed that the early allele is associated with a strong derepression of the FTa1 gene in particular. Sequence analysis revealed a 7.4 kb deletion in the FTa1-FTa2 intergenic region in the pilosae parent, and a wide survey of >350 accessions with diverse origin showed that the dtf6a allele is predominant in South Asian material. Collectively, these results contribute to understanding the molecular basis of global adaptation in lentil, and further emphasize the importance of this conserved genomic region for adaptation in temperate legumes generally.

Genetic and gene expression analysis of flowering time regulation by light quality in lentil.

BACKGROUND AND AIMS: Flowering time is important due to its roles in plant adaptation to different environments and subsequent formation of crop yield. Changes in light quality affect a range of developmental processes including flowering time, but little is known about light quality-induced flowering time control in lentil. This study aims to investigate the genetic basis for differences in flowering response to light quality in lentil. METHODS: We explored variation in flowering time caused by changes in red/far-red-related light quality environments of a lentil interspecific recombinant inbred line (RIL) population developed from a cross between Lens culinaris cv. Lupa and L. orientalis accession BGE 016880. A genetic linkage map was constructed and then used for identifying quantitative trait loci (QTLs) associated with flowering time regulation under different light quality environments. Differential gene expression analysis through transcriptomic study and RT-qPCR were used to identify potential candidate genes. KEY RESULTS: QTL mapping located 13 QTLs controlling flower time under different light quality environments, with phenotypic variance explained ranging from 1.7 to 62.9 %. Transcriptomic profiling and gene expression analysis for both parents of this interspecific RIL population identified flowering-related genes showing environment-specific differential expression (flowering DEGs). One of these, a member of the florigen gene family FTa1 (LcFTa1), was located close to three major QTLs. Furthermore, gene expression results suggested that two other florigen genes (LcFTb1 and LcFTb2), MADS-box transcription factors such as LcAGL6/13d, LcSVPb, LcSOC1b and LcFULb, as well as bHLH transcription factor LcPIF6 and Gibberellin 20 oxidase LcGA20oxC,G may also be involved in the light quality response. CONCLUSIONS: Our results show that a major component of flowering time sensitivity to light quality is tightly linked to LcFTa1 and associated with changes in its expression. This work provides a foundation for crop improvement of lentil with better adaptation to variable light environments.

Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis.

BACKGROUND: Postharvest seed coat darkening in pinto bean is an undesirable trait resulting in a loss in the economic value of the crop. The extent of darkening varies between the bean cultivars and their storage conditions. RESULTS: Metabolite analysis revealed that the majority of flavonoids including proanthocyanidin monomer catechin accumulated at higher level in a regular darkening (RD) pinto line CDC Pintium than in a slow darkening (SD) line 1533-15. A transcriptome analysis was conducted to compare gene expression between CDC Pintium and 1533-15 and identify the gene (s) that may play a role in slow darkening processes in 1533-15 pinto. RNAseq against total RNA from RD and SD cultivars found several phenylpropanoid genes, metabolite transporter genes and genes involved in gene regulation or modification to be differentially expressed between CDC Pintium and 1533-15. CONCLUSION: RNAseq analysis and metabolite data of seed coat tissue from CDC Pintium and 1533-15 revealed that the whole proanthocyanidin biosynthetic pathway was downregulated in 1533-15. Additionally, genes that encode for putative transporter proteins were also downregulated in 1533-15 suggesting both synthesis and accumulation of proanthocyanidin is reduced in SD pintos.

White seed color in common bean (Phaseolus vulgaris) results from convergent evolution in the P (pigment) gene.

The presence of seed color in common bean (Phaseolus vulgaris) requires the dominant-acting P (pigment) gene, and white seed is a recessive phenotype in all domesticated races of the species. P was classically associated with seed size, thus describing it as the first genetic marker for a quantitative trait. The molecular structure of P was characterized to understand the selection of white seeds during bean diversification and the relationship of P to seed weight. P was identified by homology searches, a genome-wide association study (GWAS) and gene remodeling, and confirmed by gene silencing. Allelic variation was assessed by a combination of resequencing and marker development, and the relationship between P and seed weight was assessed by a GWAS study. P is a member of clade B of subclass IIIf of plant basic helix-loop-helix (bHLH) proteins. Ten race-specific P alleles conditioned the white seed phenotype, and each causative mutation affected at least one bHLH domain required for color expression. GWAS analysis confirmed the classic association of P with seed weight. In common bean, white seeds are the result of convergent evolution and, among plant species, orthologous convergence on a single transcription factor gene was observed.

Gene-based SNP discovery in tepary bean (Phaseolus acutifolius) and common bean (P. vulgaris) for diversity analysis and comparative mapping.

BACKGROUND: Common bean (Phaseolus vulgaris) is an important grain legume and there has been a recent resurgence in interest in its relative, tepary bean (P. acutifolius), owing to this species' ability to better withstand abiotic stresses. Genomic resources are scarce for this minor crop species and a better knowledge of the genome-level relationship between these two species would facilitate improvement in both. High-throughput genotyping has facilitated large-scale single nucleotide polymorphism (SNP) identification leading to the development of molecular markers with associated sequence information that can be used to place them in the context of a full genome assembly. RESULTS: Transcript-based SNPs were identified from six common bean and two tepary bean accessions and a subset were used to generate a 768-SNP Illumina GoldenGate assay for each species. The tepary bean assay was used to assess diversity in wild and cultivated tepary bean and to generate the first gene-based map of the tepary bean genome. Genotypic analyses of the diversity panel showed a clear separation between domesticated and cultivated tepary beans, two distinct groups within the domesticated types, and P. parvifolius was confirmed to be distinct. The genetic map of tepary bean was compared to the common bean genome assembly to demonstrate high levels of collinearity between the two species with differences limited to a few intra-chromosomal rearrangements. CONCLUSIONS: The development of the first set of genomic resources specifically for tepary bean has allowed for greater insight into the structure of this species and its relationship to its agriculturally more prominent relative, common bean. These resources will be helpful in the development of efficient breeding strategies for both species and will facilitate the introgression of agriculturally important traits from one crop into the other.

Wild Help for Enhancing Genetic Resistance in Lentil Against Fungal Diseases.

Lentil (Lens culinaris) is one of the cool season grain legume crops and an important source of dietary proteins and fibre. Fungal diseases are main constraints to lentil production and account for significant yield and quality losses. Lentil has a narrow genetic base presumably due to a bottleneck during domestication and as a result, any resistance to fungal diseases in the cultivated genepool is gradually eroded and overcome by pathogens. New sources of resistance have been identified in wild lentil (Lens ervoides). This article provides an overview of harnessing resistance potential of wild germplasm to enhance genetic resistance in lentil cultivars using next-generation sequencing-based genotyping, comparative genomics and marker-assisted selection breeding.

Genome wide SNP identification in chickpea for use in development of a high density genetic map and improvement of chickpea reference genome assembly.

BACKGROUND: In the whole genome sequencing, genetic map provides an essential framework for accurate and efficient genome assembly and validation. The main objectives of this study were to develop a high-density genetic map using RAD-Seq (Restriction-site Associated DNA Sequencing) genotyping-by-sequencing (RAD-Seq GBS) and Illumina GoldenGate assays, and to examine the alignment of the current map with the kabuli chickpea genome assembly. RESULTS: Genic single nucleotide polymorphisms (SNPs) totaling 51,632 SNPs were identified by 454 transcriptome sequencing of Cicer arietinum and Cicer reticulatum genotypes. Subsequently, an Illumina GoldenGate assay for 1,536 SNPs was developed. A total of 1,519 SNPs were successfully assayed across 92 recombinant inbred lines (RILs), of which 761 SNPs were polymorphic between the two parents. In addition, the next generation sequencing (NGS)-based GBS was applied to the same population generating 29,464 high quality SNPs. These SNPs were clustered into 626 recombination bins based on common segregation patterns. Data from the two approaches were used for the construction of a genetic map using a population derived from an intraspecific cross. The map consisted of 1,336 SNPs including 604 RAD recombination bins and 732 SNPs from Illumina GoldenGate assay. The map covered 653 cM of the chickpea genome with an average distance between adjacent markers of 0.5 cM. To date, this is the most extensive genetic map of chickpea using an intraspecific population. The alignment of the map with the CDC Frontier genome assembly revealed an overall conserved marker order; however, a few local inconsistencies within the Cicer arietinum pseudochromosome 1 (Ca1), Ca5 and Ca8 were detected. The map enabled the alignment of 215 unplaced scaffolds from the CDC Frontier draft genome assembly. The alignment also revealed varying degrees of recombination rates and hotspots across the chickpea genome. CONCLUSIONS: A high-density genetic map using RAD-Seq GBS and Illumina GoldenGate assay was developed and aligned with the existing kabuli chickpea draft genome sequence. The analysis revealed an overall conserved marker order, although some localized inversions between draft genome assembly and the genetic map were detected. The current analysis provides an insight of the recombination rates and hotspots across the chickpea genome.

Gene-based SNP discovery and genetic mapping in pea.

KEY MESSAGE: Gene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance. Pea (Pisum sativum L.) is one of the world's oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.

Genetic diversity and association mapping of iron and zinc concentrations in chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is the world's second most important pulse crop after common bean. Chickpea has historically been an important daily staple in the diet of millions of people, especially in the developing countries. Current chickpea breeding programs have mainly been directed toward high yield, biotic and abiotic stress resilience that has increased global production, but less attention has been directed toward improving micronutrient concentrations in seeds. In an effort to develop micronutrient-dense chickpea lines, a study to examine the variability and to identify SNP alleles associated with seed iron and zinc concentrations was conducted using 94 diverse accessions of chickpea. The results indicated that there is substantial variability present in chickpea germplasm for seed iron and zinc concentrations. In the current set of germplasm, zinc is negatively correlated with grain yield across all locations and years; whereas the negative correlation between iron and grain yield was only significant at the Elrose locality. Eight SNP loci associated with iron and (or) zinc concentrations in chickpea seeds were identified. One SNP located on chromosome 1 (chr1) is associated with both iron and zinc concentrations. On chr4, three SNPs associated with zinc concentration and two SNPs for iron concentration were identified. Two additional SNP loci, one on chr6 and the other on chr7, were also found to be associated with iron and zinc concentrations, respectively. The results show potential opportunity for molecular breeding for improvement of seed iron and zinc concentrations in chickpea.

Understanding genome structure facilitates the use of wild lentil germplasm for breeding: A case study with shattering loci.

Plant breeders are generally reluctant to cross elite crop cultivars with their wild relatives to introgress novel desirable traits due to associated negative traits such as pod shattering. This results in a genetic bottleneck that could be reduced through better understanding of the genomic locations of the gene(s) controlling this trait. We integrated information on parental genomes, pod shattering data from multiple environments, and high-density genetic linkage maps to identify pod shattering quantitative trait loci (QTLs) in three lentil interspecific recombinant inbred line populations. The broad-sense heritability on a multi-environment basis varied from 0.46 (in LR-70, Lens culinaris × Lens odemensis) to 0.77 (in LR-68, Lens orientalis × L. culinaris). Genetic linkage maps of the interspecific populations revealed reciprocal translocations of chromosomal segments that differed among the populations, and which were associated with reduced recombination. LR-68 had a 2-5 translocation, LR-70 had 1-5, 2-6, and 2-7 translocations, and LR-86 had a 2-7 translocation in one parent relative to the other. Segregation distortion was also observed for clusters of single nucleotide polymorphisms on multiple chromosomes per population, further affecting introgression. Two major QTL, on chromosomes 4 and 7, were repeatedly detected in the three populations and contain several candidate genes. These findings will be of significant value for lentil breeders to strategically access novel superior alleles while minimizing the genetic impact of pod shattering from wild parents.

Ancient orphan crop joins modern era: gene-based SNP discovery and mapping in lentil.

BACKGROUND: The genus Lens comprises a range of closely related species within the galegoid clade of the Papilionoideae family. The clade includes other important crops (e.g. chickpea and pea) as well as a sequenced model legume (Medicago truncatula). Lentil is a global food crop increasing in importance in the Indian sub-continent and elsewhere due to its nutritional value and quick cooking time. Despite this importance there has been a dearth of genetic and genomic resources for the crop and this has limited the application of marker-assisted selection strategies in breeding. RESULTS: We describe here the development of a deep and diverse transcriptome resource for lentil using next generation sequencing technology. The generation of data in multiple cultivated (L. culinaris) and wild (L. ervoides) genotypes together with the utilization of a bioinformatics workflow enabled the identification of a large collection of SNPs and the subsequent development of a genotyping platform that was used to establish the first comprehensive genetic map of the L. culinaris genome. Extensive collinearity with M. truncatula was evident on the basis of sequence homology between mapped markers and the model genome and large translocations and inversions relative to M. truncatula were identified. An estimate for the time divergence of L. culinaris from L. ervoides and of both from M. truncatula was also calculated. CONCLUSIONS: The availability of the genomic and derived molecular marker resources presented here will help change lentil breeding strategies and lead to increased genetic gain in the future.

Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum.

BACKGROUND: Anthracnose of lentil, caused by the hemibiotrophic fungal pathogen Colletotrichum truncatum is a serious threat to lentil production in western Canada. Colletotrichum truncatum employs a bi-phasic infection strategy characterized by initial symptomless biotrophic and subsequent destructive necrotrophic colonization of its host. The transition from biotrophy to necrotrophy (known as the biotrophy-necrotrophy switch [BNS]) is critical in anthracnose development. Understanding plant responses during the BNS is the key to designing a strategy for incorporating resistance against hemibiotrophic pathogens either via introgression of resistance genes or quantitative trait loci contributing to host defense into elite cultivars, or via incorporation of resistance by biotechnological means. RESULTS: The in planta BNS of C. truncatum was determined by histochemical analysis of infected lentil leaf tissues in time-course experiments. A total of 2852 lentil expressed sequence tags (ESTs) derived from C. truncatum-infected leaf tissues were analyzed to catalogue defense related genes. These ESTs could be assembled into 1682 unigenes. Of these, 101 unigenes encoded membrane and transport associated proteins, 159 encoded proteins implicated in signal transduction and 387 were predicted to be stress and defense related proteins (GenBank accessions: JG293480 to JG293479). The most abundant class of defense related proteins contained pathogenesis related proteins (encoded by 125 ESTs) followed by heat shock proteins, glutathione S-transferase, protein kinases, protein phosphatase, zinc finger proteins, peroxidase, GTP binding proteins, resistance proteins and syringolide-induced proteins. Quantitative RT-PCR was conducted to compare the expression of two resistance genes of the NBS-LRR class in susceptible and partially resistant genotypes. One (contig186) was induced 6 days post-inoculation (dpi) in a susceptible host genotype (Eston) whereas the mRNA level of another ( LT21-1990) peaked 4 dpi in a partially resistant host genotype (Robin), suggesting roles in conditioning the susceptibility and conferring tolerance to the pathogen, respectively. CONCLUSIONS: Data obtained in this study suggest that lentil cells recognize C. truncatum at the BNS and in response, mount an inducible defense as evident by a high number of transcripts (23% of the total pathogen-responsive lentil transcriptome) encoding defense related proteins. Temporal expression polymorphism of defense related genes could be used to distinguish the response of a lentil genotype as susceptible or resistant.

Genotypic abundance of carotenoids and polyphenolics in the hull of field pea (Pisum sativum L.).

BACKGROUND: Consumption of pulse crops, including field pea, is considered effective for a healthy diet. Hulls (seed coats) play an important role for protection of the cotyledon and embryo, but also as mediating positive effects on health outcomes. The biochemical attributes of field pea hulls were thus assessed to determine the occurrence of specific phytochemicals and their genotypic variability. RESULTS: Sequestered bioproducts in mature hulls predominantly consisted of trans-lutein and chlorophylls a and b. Trace amounts of other carotenoid and pheophytin metabolites were identified. In developing hulls, violaxanthin, neoxanthin, lutein, zeaxanthin, chlorophylls a and b and ß-carotene were detected. Genotypic differences in the accumulation of lutein and chlorophylls a and b were observed over years and locations. Polyphenolics and hydroxybenzoic acids were detected in the 'dun' and 'maple' field pea types-the only genotypes to have pigmented hulls. Unextractable patches of condensed tannin influenced the visual uniformity of the maple and dun genotypes, CDC Rocket and CDC Dundurn. CONCLUSIONS: Within the yellow and green market classes, carotenoid and chlorophyll accumulation was consistent. Green cotyledon varieties sequestered higher concentrations of lutein than the yellow cotyledon varieties. Maple and dun types were more variable, reflective of different selection criteria. The occurrence of flavonoid-related compounds was correlated only with pigmented seed coat genotypes. The dietary potential of the chlorophylls and carotenoids that accumulated in the hulls split from the green and yellow field pea types is discussed as a value-added prospect in food supplements.

Focusing the GWAS Lens on days to flower using latent variable phenotypes derived from global multienvironment trials.

Adaptation constraints within crop species have resulted in limited genetic diversity in some breeding programs and areas where new crops have been introduced, for example, for lentil (Lens culinaris Medik.) in North America. An improved understanding of the underlying genetics involved in phenology-related traits is valuable knowledge to aid breeders in overcoming limitations associated with unadapted germplasm and expanding their genetic diversity by introducing new, exotic material. We used a large, 18 site-year, multienvironment dataset phenotyped for phenology-related traits across nine locations and over 3 yr along with accompanying latent variable phenotypes derived from a photothermal model and principal component analysis (PCA) of days from sowing to flower (DTF) data for a lentil diversity panel (324 accessions), which has also been genotyped with an exome capture array. Genome-wide association studies (GWAS) on DTF across multiple environments helped confirm associations with known flowering-time genes and identify new quantitative trait loci (QTL), which may contain previously unknown flowering time genes. Additionally, the use of latent variable phenotypes, which can incorporate environmental data such as temperature and photoperiod as both GWAS traits and as covariates, strengthened associations, revealed additional hidden associations, and alluded to potential roles of the associated QTL. Our approach can be replicated with other crop species, and the results from our GWAS serve as a resource for further exploration into the complex nature of phenology-related traits across the major growing environments for cultivated lentil.

RNA-Seq and Gene Ontology Analysis Reveal Differences Associated With Low R/FR-Induced Shade Responses in Cultivated Lentil and a Wild Relative.

Lentil is an important pulse crop not only because of its high nutrient value but also because of its ecological advantage in a sustainable agricultural system. Our previous work showed that the cultivated lentil and wild lentil germplasm respond differently to light environments, especially to low R/FR-induced shade conditions. Little is known about how cultivated and wild lentils respond to shade at the level of gene expression and function. In this study, transcriptomic profiling of a cultivated lentil (Lupa, L. culinaris) and a wild lentil (BGE 016880, L. orientalis) at several growth stages is presented. De novo transcriptomes were assembled for both genotypes, and differential gene expression analysis and gene ontology enrichment analysis were performed. The transcriptomic resources generated in this study provide fundamental information regarding biological processes and genes associated with shade responses in lentils. BGE 016880 and Lupa shared a high similarity in their transcriptomes; however, differential gene expression profiles were not consistent between these two genotypes. The wild lentil BGE 016880 had more differentially expressed genes than the cultivated lentil Lupa. Upregulation of genes involved in gibberellin, brassinosteroid, and auxin synthesis and signaling pathways, as well as cell wall modification, in both genotypes explains their similarity in stem elongation response under the shade. Genes involved in jasmonic acid and flavonoid biosynthesis pathways were downregulated in BGE 016880 only, and biological processes involved in defense responses were significantly enriched in the wild lentil BGE 016880 only. Downregulation of WRKY and MYB transcription factors could contribute to the reduced defense response in BGE 016880 but not in Lupa under shade conditions. A better understanding of shade responses of pulse crop species and their wild relatives will play an important role in developing genetic strategies for crop improvement in response to changes in light environments.

An evolutionary look into the history of lentil reveals unexpected diversity.

The characterization and preservation of genetic variation in crops is critical to meeting the challenges of breeding in the face of changing climates and markets. In recent years, the use of single nucleotide polymorphisms (SNPs) has become routine, allowing us to understand the population structure, find divergent lines for crosses, and illuminate the origin of crops. However, the focus on SNPs overlooks other forms of variation, such as copy number variation (CNVs). Lentil is the third most important cold-season legume and was domesticated in the Fertile Crescent. We genotyped 324 accessions that represent its global diversity, and using both SNPs and CNVs, we dissected the population structure and genetic variation, and identified candidate genes. Eight clusters were detected, most of them located in or near the Fertile Crescent, even though different clusters were present in distinct regions. The cluster from South Asia was particularly differentiated and presented low diversity, contrasting with the clusters from the Mediterranean and the northern temperate. Accessions from North America were mainly assigned to one cluster and were highly diverse, reflecting the efforts of breeding programs to integrate variation. Thirty-three genes were identified as candidates under selection and among their functions were sporopollenin synthesis in pollen, a component of chlorophyll B reductase that partially determines the antenna size, and two genes related to the import system of chloroplasts. Eleven percent of all lentil genes and 21% of lentil disease resistance genes were affected by CNVs. The gene categories overrepresented in these genes were "enzymes," "Cell Wall Organization," and "external stimuli response." All the genes found in the latter were associated with pathogen response. CNVs provided information about population structure and might have played a role in adaptation. The incorporation of CNVs in diversity studies is needed for a broader understanding of how they evolve and contribute to domestication.

UAV Image-Based Crop Growth Analysis of 3D-Reconstructed Crop Canopies.

Plant growth rate is an essential phenotypic parameter for quantifying potential crop productivity. Under field conditions, manual measurement of plant growth rate is less accurate in most cases. Image-based high-throughput platforms offer great potential for rapid, non-destructive, and objective estimation of plant growth parameters. The aim of this study was to assess the potential for quantifying plant growth rate using UAV-based (unoccupied aerial vehicle) imagery collected multiple times throughout the growing season. In this study, six diverse lines of lentils were grown in three replicates of 1 m2 microplots with six biomass collection time-points throughout the growing season over five site-years. Aerial imagery was collected simultaneously with each manual measurement of the above-ground biomass time-point and was used to produce two-dimensional orthomosaics and three-dimensional point clouds. Non-linear logistic models were fit to multiple data collection points throughout the growing season. Overall, remotely detected vegetation area and crop volume were found to produce trends comparable to the accumulation of dry weight biomass throughout the growing season. The growth rate and G50 (days to 50% of maximum growth) parameters of the model effectively quantified lentil growth rate indicating significant potential for image-based tools to be used in plant breeding programs. Comparing image-based groundcover and vegetation volume estimates with manually measured above-ground biomass suggested strong correlations. Vegetation area measured from a UAV has utility in quantifying lentil biomass and is indicative of leaf area early in the growing season. For mid- to late-season biomass estimation, plot volume was determined to be a better estimator. Apart from traditional traits, the estimation and analysis of plant parameters not typically collected in traditional breeding programs are possible with image-based methods, and this can create new opportunities to improve breeding efficiency mainly by offering new phenotypes and affecting selection intensity.