Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci

Barley (Hordeum vulgare) cv. ‘Golden Promise’ is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from ‘Haruna Nijo’ × ‘Golden Promise,’ as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross ‘Golden Promise’ × ‘Full Pint.’ Based on SNP genotype, we selected lines having ‘Golden Promise’ alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to ‘Golden Promise.’ The results validate that the genetic substitution of TFA alleles from ‘Golden Promise’ can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars

Application of Genomics to Understand Salt Tolerance in Lentil

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

Safety Assessment of Genetically Modified Feed: Is There Any Difference From Food?

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

Characterization of Genetic and Allelic Diversity Amongst Cultivated and Wild Lentil Accessions for Germplasm Enhancement

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 further insights into evolutionary relationships between cultivated and wild lentil germplasm, for germplasm improvement and introgression

SNP-Based Linkage Mapping for Validation of QTLs for Resistance to Ascochyta Blight in Lentil.

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 phenotypic responses with high levels of accuracy (~86%), and therefore have the potential to be widely adopted in lentil breeding programs. The availability of RIL-based maps, a consensus map, and validated markers linked to AB resistance provide important resources for lentil improvement

SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.)

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 selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance. CONCLUSION: The SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars

Development and Application of Image-Based High-Throughput Phenotyping Methodology for Salt Tolerance in Lentils

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

Validation of molecular markers associated with boron tolerance, powdery mildew resistance and salinity tolerance in field peas

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

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species