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Heterosis is defined as increased performance of the F1 hybrid relative to its parents. In the current study, a cohort of populations and parents were created to evaluate and understand heterosis across generations (i.e., F1 to F3) in lentil, a self-pollinated annual diploid (2n = 2× = 14) crop species. Lentil plants were evaluated for heterotic traits in terms of plant height, biomass fresh weight, seed number, yield per plant and 100 grain weight. A total of 47 selected lentil genotypes were cross hybridized to generate 72 F1 hybrids. The F1 hybrids from the top five crosses exhibited between 31%-62% heterosis for seed number with reference to the better parent. The five best performing heterotic crosses were selected with a negative control for evaluation at the subsequent F2 generation and only the tails of the distribution taken forward to be assessed in the F3 generation as a sub selection. Overall, heterosis decreases across the subsequent generations for all traits studied. However, some individual genotypes were identified at the F2 and sub-selected F3 generations with higher levels of heterosis than the best F1 mean value (hybrid mimics). The phenotypic data for the selected F2 and sub selected F3 hybrids were analysed, and the study suggested that 100 grain weight was the biggest driver of yield followed by seed number. A genetic diversity analysis of all the F1 parents failed to correlate genetic distance and divergence among parents with heterotic F1's. Therefore, genetic distance was not a key factor to determine heterosis in lentil. The study highlights the challenges associated with different breeding systems for heterosis (i.e., F1 hybrid-based breeding systems and/or via hybrid mimics) but demonstrates the potential significant gains that could be achieved in lentil productivity.

Soil salinity is a major abiotic stress, limiting lentil productivity worldwide. Understanding the genetic basis of salt tolerance is vital to develop tolerant varieties. A diversity panel consisting of 276 lentil accessions was screened in a previous study through traditional and image-based approaches to quantify growth under salt stress. Genotyping was performed using two contrasting methods, targeted (tGBS) and transcriptome (GBS-t) genotyping-by-sequencing, to evaluate the most appropriate methodology. tGBS revealed the highest number of single-base variants (SNPs) (c. 56,349), and markers were more evenly distributed across the genome compared to GBS-t. A genome-wide association study (GWAS) was conducted using a mixed linear model. Significant marker-trait associations were observed on Chromosome 2 as well as Chromosome 4, and a range of candidate genes was identified from the reference genome, the most plausible being potassium transporters, which are known to be involved in salt tolerance in related species. Detailed mineral composition performed on salt-treated and control plant tissues revealed the salt tolerance mechanism in lentil, in which tolerant accessions do not transport Na+ ions around the plant instead localize within the root tissues. The pedigree analysis identified two parental accessions that could have been the key sources of tolerance in this dataset.

Intensive breeding of cultivated lentil has resulted in a relatively narrow genetic base, which limits the options to increase crop productivity through selection. Assessment of genetic diversity in the wild gene pool of lentil, as well as characterization of useful and novel alleles/genes that can be introgressed into elite germplasm, presents new opportunities and pathways for germplasm enhancement, followed by successful crop improvement. In the current study, a lentil collection consisting of 467 wild and cultivated accessions that originated from 10 diverse geographical regions was assessed, to understand genetic relationships among different lentil species/subspecies. A total of 422,101 high-confidence SNP markers were identified against the reference lentil genome (cv. CDC Redberry). Phylogenetic analysis clustered the germplasm collection into four groups, namely, Lens culinaris/Lens orientalis, Lens lamottei/Lens odemensis, Lens ervoides, and Lens nigricans. A weak correlation was observed between geographical origin and genetic relationship, except for some accessions of L. culinaris and L. ervoides. Genetic distance matrices revealed a comparable level of variation within the gene pools of L. culinaris (Nei's coefficient 0.01468-0.71163), L. ervoides (Nei's coefficient 0.01807-0.71877), and L. nigricans (Nei's coefficient 0.02188-1.2219). In order to understand any genic differences at species/subspecies level, allele frequencies were calculated from a subset of 263 lentil accessions. Among all cultivated and wild lentil species, L. nigricans exhibited the greatest allelic differentiation across the genome compared to all other species/subspecies. Major differences were observed on six genomic regions with the largest being on Chromosome 1 (c. 1 Mbp). These results indicate that L. nigricans is the most distantly related to L. culinaris and additional structural variations are likely to be identified from genome sequencing studies. This would provide furt

Soil salinity is a major abiotic stress in Australian lentil-producing areas. It is therefore imperative to identify genetic variation for salt tolerance in order to develop lentil varieties suitable for saline soils. Conventional screening methods include the manual assessment of stress symptoms, which can be very laborious, time-consuming, and error-prone. Recent advances in image-based high-throughput phenotyping (HTP) technologies have provided unparalleled opportunities to screen plants for a range of stresses, such as salt toxicity. The current study describes the development and application of an HTP method for salt toxicity screening in lentils. In a pilot study, six lentil genotypes were evaluated to determine the optimal salt level and the growth stage for distinguishing lentil genotypes using red–green–blue (RGB) images on a LemnaTec Scanalyzer 3D phenomics platform. The optimized protocol was then applied to screen 276 accessions that were also assessed earlier in a conventional phenotypic screen. Detailed phenotypic trait assessments, including plant growth and green/non-green color pixels, were made and correlated to the conventional screen (r = 0.55; p < 0.0001). These findings demonstrated the improved efficacy of an image-based phenotyping approach that is high-throughput, efficient, and better suited to modern breeding programs.

Genomic selection accelerates genetic progress in crop breeding through the prediction of future phenotypes of selection candidates based on only their genomic information. Here we report genetic correlations and genomic prediction accuracies in 22 agronomic, disease, and seed quality traits measured across multiple years (2015-2017) in replicated trials under rain-fed and irrigated conditions in Victoria, Australia. Two hundred and two spring canola lines were genotyped for 62,082 Single Nucleotide Polymorphisms (SNPs) using transcriptomic genotype-by-sequencing (GBSt). Traits were evaluated in single trait and bivariate genomic best linear unbiased prediction (GBLUP) models and cross-validation. GBLUP were also expanded to include genotype-by-environment G × E interactions. Genomic heritability varied from 0.31to 0.66. Genetic correlations were highly positive within traits across locations and years. Oil content was positively correlated with most agronomic traits. Strong, not previously documented, negative correlations were observed between average internal infection (a measure of blackleg disease) and arachidic and stearic acids. The genetic correlations between fatty acid traits followed the expected patterns based on oil biosynthesis pathways. Genomic prediction accuracy ranged from 0.29 for emergence count to 0.69 for seed yield. The incorporation of G × E translates into improved prediction accuracy by up to 6%. The genomic prediction accuracies achieved indicate that genomic selection is ready for application in canola breeding.

Food security is one of major concerns for the growing global population. Modern agricultural biotechnologies, such as genetic modification, are a possible solution through enabling an increase of production, more efficient use of natural resources, and reduced environmental impacts. However, new crop varieties with altered genetic materials may be subjected to safety assessments to fulfil the regulatory requirements, prior to marketing. The aim of the assessment is to evaluate the impact of products from the new crop variety on human, animal, and the environmental health. Although, many studies on the risk assessment of genetically modified (GM) food have been published, little consideration to GM feedstuff has been given, despite that between 70 to 90% of all GM crops and their biomass are used as animal feed. In addition, in some GM plants such as forages that are only used for animal feeds, the assessment of the genetic modification may be of relevance only to livestock feeding. In this article, the regulatory framework of GM crops intended for animal feed is reviewed using the available information on GM food as the baseline. Although, the majority of techniques used for the safety assessment of GM food can be used in GM feed, many plant parts used for livestock feeding are inedible to humans. Therefore, the concentration of novel proteins in different plant tissues and level of exposure to GM feedstuff in the diet of target animals should be considered. A further development of specific methodologies for the assessment of GM crops intended for animal consumption is required, in order to provide a more accurate and standardized assessment to the GM feed safety.

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+ (gene of interest positive) and GOI- (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+ and GOI- for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI- compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI-. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte-grass associations. Endophyte-host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.

Lentil (Lens culinaris Medik.) is a self-pollinating, diploid, annual, cool-season, food legume crop that is cultivated throughout the world. Ascochyta blight (AB), caused by Ascochyta lentis Vassilievsky, is an economically important and widespread disease of lentil. Development of cultivars with high levels of durable resistance provides an environmentally acceptable and economically feasible method for AB control. A detailed understanding of the genetic basis of AB resistance is hence highly desirable, in order to obtain insight into the number and influence of resistance genes. Genetic linkage maps based on single nucleotide polymorphisms (SNP) and simple sequence repeat (SSR) markers have been developed from three recombinant inbred line (RIL) populations. The IH × NF map contained 460 loci across 1461.6 cM, while the IH × DIG map contained 329 loci across 1302.5 cM and the third map, NF × DIG contained 330 loci across 1914.1 cM. Data from these maps were combined with a map from a previously published study through use of bridging markers to generate a consensus linkage map containing 689 loci distributed across seven linkage groups (LGs), with a cumulative length of 2429.61 cM at an average density of one marker per 3.5 cM. Trait dissection of AB resistance was performed for the RIL populations, identifying totals of two and three quantitative trait loci (QTLs) explaining 52 and 69% of phenotypic variation for resistance to infection in the IH × DIG and IH × NF populations, respectively. Presence of common markers in the vicinity of the AB_IH1- and AB_IH2.1/AB_IH2.2-containing regions on both maps supports the inference that a common genomic region is responsible for conferring resistance and is associated with the resistant parent, Indianhead. The third QTL was derived from Northfield. Evaluation of markers associated with AB resistance across a diverse lentil germplasm panel revealed that the identity of alleles associated with AB_IH1 predicted the phenotyp

Field pea (Pisum sativum L.) is an important grain legume consumed both as human food and animal feed. However, productivity in low rainfall regions can be significantly reduced by inferior soils containing high levels of boron and/or salinity. Furthermore, powdery mildew (PM) (Erysiphe pisi) disease also causes significant yield loss in warmer regions. Breeding for tolerance to these abiotic and biotic stresses are major aims for pea breeding programs and the application of molecular markers for these traits could greatly assist in developing improved germplasm at a faster rate. The current study reports the evaluation of a near diagnostic marker, PsMlo, associated with PM resistance and boron (B) tolerance as well as linked markers associated with salinity tolerance across a diverse set of pea germplasm. The PsMlo1 marker predicted the PM and B phenotypic responses with high levels of accuracy (>80%) across a wide range of field pea genotypes, hence offers the potential to be widely adapted in pea breeding programs. In contrast, linked markers for salinity tolerance were population specific; therefore, application of these markers would be suitable to relevant crosses within the program. Our results also suggest that there are possible new sources of salt tolerance present in field pea germplasm that could be further exploited.

BACKGROUND: Field pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs. RESULTS: In this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for

BACKGROUND: Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site--leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database. RESULTS: Amplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach. A total of 102 unique partial R genes were cloned, sequenced and functionally annotated. Analysis of motif structure and R gene phylogeny demonstrated that Lolium R genes cluster with putative ortholoci, and evolved from common ancestral origins. Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps. Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes. A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs). CONCLUSION: This study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass. SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics. In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-for-gene mechanisms for multiple host pathogen-interacti