Chromosome 2H Research Articles

Genome-Wide Association Study of Leaf Rust Resistance at Seedling and Adult Plant Stages in a Global Barley Panel

Barley leaf rust caused by Puccinia hordei (Ph) is one of the major limiting biotic stresses of barley production worldwide and causes yield losses of up to 60%. A diversity panel of 316 barley genotypes (AM2017) composed of released cultivars, advanced breeding lines and landraces was screened for Ph resistance at the seedling stage using two isolates (SRT-SAT and SRT-MRC), while the adult plant stage resistance screening was conducted at the disease hotspot location of Sidi Allal Tazi (SAT) for the cropping seasons of 2017 and 2019. The phenotypic responses were combined with 36,793 single nucleotide polymorphism (SNP) markers in a genome-wide association study (GWAS) using the general linear model (GLM), mixed linear model (MLM), settlement of MLM under progressively exclusive relationship (SUPER), multiple-locus MLM (MLMM), fixed and random model circulating probability unification (FarmCPU), and Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) in GAPIT3, and MLM (K+Q), MLM (K+PCA), and GLM (Q) models in TASSEL to identify genomic regions linked to Ph resistance. Fourteen barley genotypes were resistant (R) at the seedling stage to both Ph isolates, SRT-SAT and SRT-MRC, and twelve genotypes were either resistant (R) or moderately resistant (MR) at the adult plant stage, whereas only one genotype was resistant at the seedling stage, and moderately resistant at the adult plant stage. The genome scan revealed 58 significant marker trait associations (MTA) among which 34 were associated with seedling resistance (SR) and 24 with adult plant resistance (APR). Common genomic regions conferring resistance to Ph were identified at both stages on chromosome 2H (106.53 cM and at 107.37 cM), and on chromosome 7H (126.7 cM). Among the 58 MTA identified, 26 loci had been reported in previous studies, while the remaining 32 loci were regarded as novel. Furthermore, the functional annotation of candidate genes (CGs) adjacent to 36 SNP markers with proteins involved in disease resistance further confirms that some of the SNP markers from our study could be associated with Ph resistance in barley. The resistant barley genotypes and some of the SNP markers from this study with high R2 and additive effects can be converted into high-throughput functional markers for accelerated selection and pyramiding of leaf rust resistance genes in North African barley germplasm.

Role of reactive oxygen species in lesion mimic formation and conferred basal resistance to Fusarium graminearum in barley lesion mimic mutant 5386.

This study investigated the barley lesion mimic mutant (LMM) 5386, evidenced by a leaf brown spot phenotype localized on the chromosome 3H, and its conferred basal resistance to Fusarium graminearum. RNA-seq analysis identified 1453 genes that were differentially expressed in LMM 5386 compared to those in the wild type. GO and KEGG functional annotations suggested that lesion mimic formation was mediated by pathways involving oxidation reduction and glutathione metabolism. Additionally, reactive oxygen species (ROS) accumulation in brown spots was substantially higher in LMM 5386 than in the wild-type plant; therefore, antioxidant competence, which is indicated by ROS accumulation, was significantly lower in LMM 5386. Furthermore, the reduction of glycine in LMM 5386 inhibited glutathione biosynthesis. These results suggest that the decrease in antioxidant competence and glutathione biosynthesis caused considerable ROS accumulation, leading to programmed cell death, which eventually reduced the yield components in LMM 5386.

Characterization of Glossy Spike Mutants and Identification of Candidate Genes Regulating Cuticular Wax Synthesis in Barley (Hordeum vulgare L.).

Cuticular waxes comprise the hydrophobic layer that protects crops against nonstomatal water loss and biotic and abiotic stresses. Expanding on our current knowledge of the genes that are involved in cuticular wax biosynthesis and regulation plays an important role in dissecting the processes of cuticular wax metabolism. In this study, we identified the Cer-GN1 barley (Hordeum vulgare L.) mutant that is generated by ethyl methanesulfonate mutagenesis with a glossy spike phenotype that is controlled by a single recessive nuclear gene. A physiological analysis showed that the total cuticular wax loads of Cer-GN1 were one-third that of the progenitor wild-type (WT), and its water loss rate was significantly accelerated (p < 0.05). In addition, Cer-GN1 was defective in the glume's cuticle according to the toluidine blue dye test, and it was deficient in the tubule-shaped crystals which were observed on the glume surfaces by scanning electron microscopy. Using metabolomics and transcriptomics, we investigated the impacts of cuticular wax composition and waxy regulatory genes on the loss of the glaucous wax in the spikes of Cer-GN1. Among the differential metabolites, we found that 16-hydroxyhexadecanoic acid, which is one of the predominant C16 and C18 fatty acid-derived cutin monomers, was significantly downregulated in Cer-GN1 when it was compared to that of WT. We identified two novel genes that are located on chromosome 4H and are downregulated in Cer-GN1 (HvMSTRG.29184 and HvMSTRG.29185) that encode long-chain fatty acid omega-monooxygenase CYP704B1, which regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid. A quantitative real-time PCR revealed that the expression levels of HvMSTRG.29184 and HvMSTRG.29185 were downregulated at 1, 4, 8, 12, and 16 days after the heading stage in Cer-GN1 when it was compared to those of WT. These results suggested that HvMSTRG.29184 and HvMSTRG.29185 have CYP704B1 activity, which could regulate the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley. Their downregulation in Cer-GN1 reduced the synthesis of the cuticular wax components and ultimately caused the loss of the glaucous wax in the spikes. It is necessary to verify whether HvMSTRG.29184 and HvMSTRG.29185 truly encode a CYP704B1 that regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley.

A novel locus conferring resistance to Puccinia hordei maps to the genomic region corresponding to Rph14 on barley chromosome 2HS.

Barley leaf rust (BLR), caused by Puccinia hordei, is best controlled through genetic resistance. An efficient resistance breeding program prioritizes the need to identify, characterize, and map new sources of resistance as well as understanding the effectiveness, structure, and function of resistance genes. In this study, three mapping populations were developed by crossing Israelian barley lines “AGG-396,” “AGG-397,” and “AGG-403” (carrying unknown leaf rust resistance) with a susceptible variety “Gus” to characterize and map resistance. Genetic analysis of phenotypic data from rust testing F3s with a P. hordei pathotype 5457 P+ revealed monogenic inheritance in all three populations. Targeted genotyping-by-sequencing of the three populations detected marker trait associations in the same genomic region on the short arm of chromosome 2H between 39 and 57 Mb (AGG-396/Gus), 44 and 64 Mb (AGG-397/Gus), and 31 and 58 Mb (AGG-403/Gus), suggesting that the resistance in all three lines is likely conferred by the same locus (tentatively designated RphAGG396). Two Kompetitive allele-specific PCR (KASP) markers, HvGBSv2-902 and HvGBSv2-932, defined a genetic distance of 3.8 cM proximal and 7.1 cM distal to RphAGG396, respectively. To increase the marker density at the RphAGG396 locus, 75 CAPS markers were designed between two flanking markers. Integration of marker data resulted in the identification of two critical recombinants and mapping RphAGG396 between markers- Mloc-28 (40.75 Mb) and Mloc-41 (41.92 Mb) narrowing the physical window to 1.17 Mb based on the Morex v2.0 reference genome assembly. To enhance map resolution, 600 F2s were genotyped with markers- Mloc-28 and Mloc-41 and nine recombinants were identified, placing the gene at a genetic distance of 0.5 and 0.2 cM between the two markers, respectively. Two annotated NLR (nucleotide-binding domain leucine-rich repeat) genes (r2.2HG0093020 and r2.2HG0093030) were identified as the best candidates for RphAGG396. A closely linked marker was developed for RphAGG396 that can be used for marker-assisted selection.

Genome-Wide Association Study of Salt Tolerance-Related Traits during Germination and Seedling Development in an Intermedium-Spike Barley Collection.

Increased salinity is one of the major consequences of climatic change affecting global crop production. The early stages in the barley (Hordeum vulgare L.) life cycle are considered the most critical phases due to their contributions to final crop yield. Particularly, the germination and seedling development are sensitive to numerous environmental stresses, especially soil salinity. In this study, we aimed to identify SNP markers linked with germination and seedling development at 150 mM NaCl as a salinity treatment. We performed a genome-wide association study (GWAS) using a panel of 208 intermedium-spike barley (H. vulgare convar. intermedium (Körn.) Mansf.) accessions and their genotype data (i.e., 10,323 SNPs) using the genome reference sequence of “Morex”. The phenotypic results showed that the 150 mM NaCl salinity treatment significantly reduced all recorded germination and seedling-related traits compared to the control treatment. Furthermore, six accessions (HOR 11747, HOR 11718, HOR 11640, HOR 11256, HOR 11275 and HOR 11291) were identified as the most salinity tolerant from the intermedium-spike barley collection. GWAS analysis indicated that a total of 38 highly significantly associated SNP markers under control and/or salinity traits were identified. Of these, two SNP markers on chromosome (chr) 1H, two on chr 3H, and one on chr 4H were significantly linked to seedling fresh and dry weight under salinity stress treatment. In addition, two SNP markers on chr 7H were also significantly associated with seedling fresh and dry weight but under control condition. Under salinity stress, one SNP marker on chr 1H, 5H and 7H were detected for more than one phenotypic trait. We found that in most of the accessions exhibiting the highest salinity tolerance, most of the salinity-related QTLs were presented. These results form the basis for detailed studies, leading to improved salt tolerance breeding programs in barley.

Genome-Wide Association Study of Agronomic Traits in European Spring Barley from Polish Gene Bank

The barley old cultivars can be used as a source of genetic diversity to breed new varieties well adapted to different environmental conditions. In this study, 431 European barley accessions were evaluated phenotypically across 2 years under field conditions and genotypically using DArTseq to gain insight into the genetic architecture of phenology, biomass, yield components, and seed yield traits. Accessions were grouped into sub-collections by cultivation period (group A—cultivated prior to 1985, B—cultivated after 1985, and C—Polish landraces), and by European country of origin or European region, to compare their phenological and agronomic value for 16 traits such as: days to heading (DH), days to milk-waxy stage (DMW), days to maturity (DM), days to harvest (DPH), plant height (PH), lodging (LT), row number (RN), spike density (SD), spike length (SL), grain per spike (NGS), glume colour (GC1), grain awn type (GAT), grain covering (GT), grain pericarp colour (GPC), and 1000—grain weight (TGW), and to indicate marker-trait associations (MTAs) with these traits. Based on the results, phenotypic plants per se under field conditions and spikes, or seeds under laboratory conditions it was possible to cluster the collection. DH negatively correlated with PH and important yield components, such TGW. Overall, the genome-wide association study (GWAS) analysis identified 143 MTAs associated with these traits. Twenty-three MTAs were associated with plant phenological stages: 5 MTAs with DH, 6 MTAs with DMW, 5 MTAs with DM, and 9 MTAs with DPH. Eighty-nine SMTAs for plant phenotypic traits were identified: 1 for LT and 88 MTAs for PH. Thirty-one markers were identified for agronomic yield traits: 16 MTAs for SD, 11 MTAs for NGS, and 4 for TGW. One association result, 7241263-17 on chromosome 2H, corresponded to the genomic region mapped for DM, DPH, and SD. Marker 3258999-37-C/T on chromosome 2H, significant for NGS, was closely located to results 3263044-31-G/T for SD and 3263989-64-A/C for DMW. On chromosome 6H, closely located were markers significant for SD (3255466-35-C/G) and for NGS (3259102-57-C/A). Moreover, on chromosome 6H, closely located were markers significant for SD (3918801-14-G/A), for NGS (3666407-49-T/G), and for DMW (3663162-62-A/C). The well-characterized barley collection and identified MTAs markers will be used to create a Polish Genebank platform and will serve as a valuable resource for precise breeding programs.

Host and pathogen genetics reveal an inverse gene-for-gene association in the P. teres f. maculata-barley pathosystem.

Pathogen and host genetics were used to uncover an inverse gene-for-gene interaction where virulence genes from the pathogen Pyrenophora teres f. maculata target barley susceptibility genes, resulting in disease. Although models have been proposed to broadly explain how plants and pathogens interact and coevolve, each interaction evolves independently, resulting in various scenarios of host manipulation and plant defense. Spot form net blotch is a foliar disease of barley caused by Pyrenophora teres f. maculata. We developed a barley population (Hockett × PI 67381) segregating for resistance to a diverse set of P. teres f. maculata isolates. Quantitative trait locus analysis identified major loci on barley chromosomes (Chr) 2H and 7H associated with resistance/susceptibility. Subsequently, we used avirulent and virulent P. teres f. maculata isolates to develop a pathogen population, identifying two major virulence loci located on Chr1 and Chr2. To further characterize this host-pathogen interaction, progeny from the pathogen population harboring virulence alleles at either the Chr1 or Chr2 locus was phenotyped on the Hockett × PI 67381 population. Progeny harboring only the Chr1 virulence allele lost the barley Chr7H association but maintained the 2H association. Conversely, isolates harboring only the Chr2 virulence allele lost the barley Chr2H association but maintained the 7H association. Hockett × PI 67381 F2 individuals showed susceptible/resistant ratios not significantly different than 15:1 and results from F2 inoculations using the single virulence genotypes were not significantly different from a 3:1 (S:R) ratio, indicating two dominant susceptibility genes. Collectively, this work shows that P. teres f. maculata virulence alleles at the Chr1 and Chr2 loci are targeting the barley 2H and 7H susceptibility alleles in an inverse gene-for-gene manner to facilitate colonization.

Genome wide association study and haplotype analysis reveals the role of HvHKT1;5 in potassium retention but not Na+ exclusion in barley (Hordeum vulgare L.)

Salinity stress is one of the major abiotic stresses which affect grain yield and quality in crops. Identifying and exploring the QTL/genes and elite alleles that control salt tolerance is crucial for breeding salt-tolerant crop varieties. In this study, a diverse panel of 214 barley ( Hordeum vulgare L.) accessions were used to identify quantitative trait loci (QTL) for salt tolerance and Na + and K + content in leaves under salt stress by MLM model and MapQTL6.0 software. Significant correlations between leaf ion content and salt tolerance were observed. One major QTL, Q.SL.2H, was identified on chromosome 2H and was responsible for the overall salinity tolerance through regulating Na + exclusion under salt stress. The other major QTL was detected on chromosome 4H that overlapped with HvHKT1;5 gene and was responsible for K + retention in leaves. Several other minor QTL were detected by MapQTL that could help in selecting salt-tolerant varieties. The haplotype analysis showed that amino acid substitutions of HvHKT1;5 in exon2 at 416 and 438 may have contributed to K + retention under salinity stress. These findings indicate the existence of two major mechanisms for salinity tolerance determined by two major QTL and challenge the accepted views on the role of HvHKT1;5 transporters in Na + exclusion in barley adaptive response to salinity stress. • A significant correlation between leaf Na + content and salt tolerance was observed. • A major QTL for salinity tolerance on 2H, conferring salinity tolerance by regulating Na + exclusion under salt stress. • Substitutions occurred at 416 and 438 of HvHKT1;5 may have significant influences on K + retention under salinity stress. • These findings challenge the accepted views on the role of HvHKT1;5 transporters in Na + exclusion in barley adaptive response to salinity stress.

Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites.

Genome-wide marker effect heterogeneity is associated with a large effect dormancy locus in winter malting barley.

Prediction of trait values in plant breeding populations typically relies on assumptions about marker effect homogeneity across populations. Evidence is presented for winter malting barley (Hordeum vulgare L.) germination traits that a single, causative, large-effect gene in the Seed dormancy 1 region on Chromosome 5H, HvAlaAT1 (Qsd1), leads to heterogeneous estimated marker effects genome wide between groups of otherwise related individuals carrying different Qsd1 alleles. This led to reduced prediction accuracy across alleles when a model was trained either on individuals carrying both alleles or one allele. Several genomic prediction models were tested to increase prediction accuracy within the Qsd1 allele groups. Small gains (5-12%) in prediction accuracy were realized using structured genomic best linear unbiased predictor models when information about the Qsd1 allele was used to stratify the population. We concluded that a single large-effect locus can lead to heterogeneous marker effects in the same breeding family. Variance partitioning based on large-effect loci can be used to inform best practices in designing genomic prediction models; however, there are likely few cases for which it may be practical to do this. For malting barley, if germination traits are highly associated with malting quality traits, then similar steps should be considered for malting quality trait prediction.

The evolutionary patterns of barley pericentromeric chromosome regions, as shaped by linkage disequilibrium and domestication.

SUMMARYThe distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene‐rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco‐geographical ranges. We defined and compared variant data across the pericentromeric and non‐pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single‐nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome‐wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two‐row and six‐row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non‐synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be ‘highly deleterious’. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.

Elucidating the genetic architecture controlling antioxidant status and ionic balance in barley under salt stress.

Association genetic analysis empowered us to identify candidate genes underlying natural variation of morpho-physiological, antioxidants, and grain yield-related traits in barley. Novel intriguing genomic regions were identified and dissected. Salinity stress is one of the abiotic stresses that influence the morpho-physiological, antioxidants, and yield-related traits in crop plants. The plants of a core set of 138 diverse barley accessions were analyzed after exposure to salt stress under field conditions during the reproductive phase. A genome-wide association scan (GWAS) was then conducted using 19,276 single nucleotide polymorphisms (SNPs) to uncover the genetic basis of morpho-physiological and grain-related traits. A wide range of responses to salt stress by the accessions was explored in the current study. GWAS detected 263 significantly associated SNPs with the antioxidants, K+/Na+ content ratio, and agronomic traits. Five genomic regions harbored interesting putative candidate genes within LD ± 1.2Mbp. Choromosome 2H harbored many candidate genes associated with the antioxidants ascorbic acid (AsA) and glutathione (GSH), such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), under salt stress. Markedly, an A:C SNP at 153,773,211bp on chromosome 7H is located inside the gene HORVU.MOREX.r3.7HG0676830 (153,772,300-153,774,057bp) that was annotated as L-gulonolactone oxidase, regulating the natural variation of SOD_S and APX_S. The allelic variation at this SNP reveals a negative selection of accessions carrying the C allele, predominantly found in six-rowed spring landraces originating from Far-, Near-East, and central Asia carrying photoperiod sensitive alleles having lower activity of enzymatic antioxidants. The SNP-trait associations detected in the current study constitute a benchmark for developing molecular selection tools for antioxidant compound selection in barley.

A new major QTL for flag leaf thickness in barley (Hordeum vulgare L.)

BackgroundCarbohydrate accumulation of photosynthetic organs, mainly leaves, are the primary sources of grain yield in cereals. The flag leaf plays a vital role in seed development, which is probably the most neglected morphological characteristic during traditional selection processes.ResultsIn this experiment, four flag leaf morphological traits and seven yield-related traits were investigated in a DH population derived from a cross between a wild barley and an Australian malting barley cultivar. Flag leaf thickness (FLT) showed significantly positive correlations with grain size. Four QTL, located on chromosomes 1H, 2H, 3H, and 5H, respectively, were identified for FLT. Among them, a major QTL was located on chromosome 3H with a LOD value of 18.4 and determined 32% of the phenotypic variation. This QTL showed close links but not pleiotropism to the previously reported semi-dwarf gene sdw1 from the cultivated barley. This QTL was not reported before and the thick leaf allele from the wild barley could provide a useful source for improving grain yield through breeding.ConclusionsOur results also provided valuable evidence that source traits and sink traits in barley are tightly connected and suggest further improvement of barley yield potential with enhanced and balanced source and sink relationships by exploiting potentialities of the wild barley resources. Moreover, this study will provide a novel sight on understanding the evolution and development of leaf morphology in barley and improving barley production by rewilding for lost superior traits during plant evolution.

Tracking Changes in the Spring Barley Gene Pool in Poland during 120 Years of Breeding.

This study was undertaken to investigate the diversity and population structure of 83 spring barley (Hordeum vulgare L.) cultivars, which corresponded to 120 years of this crop’s breeding in Poland. The analysis was based on 11,655 DArTseq-derived SNPs evenly distributed across seven barley chromosomes. Five groups were assigned in the studied cultivars according to the period of their breeding. A decrease in observed heterozygosity within the groups was noted along with the progress in breeding, with a simultaneous increase in the inbreeding coefficient value. As a result of breeding, some of the unique allelic variation present in old cultivars was lost, but crosses with foreign materials also provided new alleles to the barley gene pool. It is important to mention that the above changes affected different chromosomes to varying degrees. The internal variability of the cultivars ranged from 0.011 to 0.236. Internal uniformity was lowest among the oldest cultivars, although some highly homogeneous ones were found among them. This is probably an effect of genetic drift or selection during their multiplications and regenerations in the period from breeding to the time of analysis. The population genetic structure of the studied group of cultivars appears to be quite complex. It was shown that their genetic makeup consists of as many as eleven distinct gene pools. The analysis also showed traces of directed selection on chromosomes 3H and 5H. Detailed data analysis confirmed the presence of duplicates for 11 cultivars. The performed research will allow both improvement of the management of barley genetic resources in the gene bank and the reuse of this rich and forgotten variability in breeding programs and research.

Leaf thickness of barley: genetic dissection, candidate genes prediction and its relationship with yield-related traits.

In this first genetic study on assessing leaf thickness directly in cereals, major and environmentally stable QTL were detected in barley and candidate genes underlying a major locus were identified. Leaf thickness (LT) is an important characteristic affecting leaf functions which have been intensively studied. However, as LT has a small dimension in many plant species and technically difficult to measure, previous studies on this characteristic are often based on indirect estimations. In the first study of detecting QTL controlling LT by directly measuring the characteristic in barley, large and stable loci were detected from both field and glasshouse trials conducted in different cropping seasons by assessing a population of 201 recombinant inbred lines. Four loci (locating on chromosome arms 2H, 3H, 5H and 6H, respectively) were consistently detected for flag leaf thickness (FLT) in each of these trials. The one on 6H had the largest effect, with a maximum LOD 9.8 explaining up to 20.9% of phenotypic variance. FLT does not only show strong interactions with flag leaf width and flag leaf area but has also strong correlations with fertile tiller number, spike row types, kernel number per spike and heading date. Though with reduced efficiency, these loci were also detectable from assessing second last leaf of fully grown plants or even from assessing the third leaves of seedlings. Taking advantage of the high-quality genome assemblies for both parents of the mapping population used in this study, three candidate genes underlying the 6H QTL were predicted based on orthologous analysis. These results do not only broaden our understanding on genetic basis of LT and its relationship with other traits in cereal crops but also form the bases for cloning and functional analysis of genes regulating LT in barley.

Validation of Molecular Markers of Barley Net Blotch Resistance Loci on Chromosome 3H for Marker-Assisted Selection

The most widespread and harmful disease of barley is net form of net blotch caused by the ascomycete Pyrenophora teres f. teres Drechsler (Ptt). A cost effective and environmentally sustainable strategy for barley protection against Ptt is to develop barley cultivars possessing genetic resistance. In previous GWA analysis, we identified SNP-markers associated with a resistance locus on chromosome 3H in the interval of 45.82–54.53 cM. These SNPs have been described previously in the literature to be located within the same region of chromosome 3H. The aim of the study was to validate QTL markers controlling resistance to Ptt on chromosome 3H in this region by KASP genotyping in four F2 populations of crosses between the resistant cultivars, Morex, Fox, and Zolo, and the accession, Local k-21578, with the susceptible barley cv. Gesine and in a doubled haploid (DH) population of Canadian Lake Shore (CLS)/Harrington. Eleven of fifteen studied markers showed high efficacy (97.5–100%) for co-segregation with resistance to Ptt in the DH population, CLS/Harrington. Three of these markers located at 54.53 cM and one at 51.27 cM were effective in two F2 populations of crosses of Morex and Fox with susceptible cv. Gesine. These markers are also located close to each other on the physical map (442,203,921–443,119,491 bp). Apparently, in cultivars, CLS, Morex, and Fox, resistance to Ptt is determined by the same locus. Markers JHI-Hv50k-2016-166392 (47.1 cM, 112,536,071 bp), Clone ID 3255462_1 (51.63 cM, 363,531,898 bp), and Clone ID 3255462_2 (51.63 cM, 363,531,871 bp) showed high efficacy in the DH population and in the F2 population, Local k-21578/Gesine. Apparently, at least two loci controlling Ptt resistance exist in the chromosome region of 47.0–54.3 cM: one at 46.0–48.44 cM and another at 51.27–54.8 cM. These regions were found to harbor several genes involved in important plant functions, including disease response and signaling pathways. Allele-specific PCR markers were developed based on the KASP assay data and tested on six resistant, two moderately resistant, and two susceptible barley genotypes. Four markers were found to be effective to differentiate susceptible and resistant barley genotypes. The KASP and allele-specific PCR markers associated with Ptt resistance on chromosome 3H will be useful for pyramiding resistance QTLs in barley marker-assisted selection.

Genome-Wide Association Study Reveals Marker Trait Associations (MTA) for Waterlogging-Triggered Adventitious Roots and Aerenchyma Formation in Barley.

Waterlogging is an environmental stress, which severely affects barley growth and development. Limited availability of oxygen in the root zone negatively affects the metabolism of the whole plant. Adventitious roots (AR) and root cortical aerenchyma (RCA) formation are the most important adaptive traits that contribute to a plant’s ability to survive in waterlogged soil conditions. This study used a genome-wide association (GWAS) approach using 18,132 single nucleotide polymorphisms (SNPs) in a panel of 697 barley genotypes to reveal marker trait associations (MTA) conferring the above adaptive traits. Experiments were conducted over two consecutive years in tanks filled with soil and then validated in field experiments. GWAS analysis was conducted using general linear models (GLM), mixed linear models (MLM), and fixed and random model circulating probability unification models (FarmCPU model), with the FarmCPU showing to be the best suited model. Six and five significant (approximately −log10 (p) ≥ 5.5) MTA were identified for AR and RCA formation under waterlogged conditions, respectively. The highest −log10 (p) MTA for adventitious root and aerenchyma formation were approximately 9 and 8 on chromosome 2H and 4H, respectively. The combination of different MTA showed to be more effective in forming RCA and producing more AR under waterlogging stress. Genes from major facilitator superfamily (MFS) transporter and leucine-rich repeat (LRR) families for AR formation, and ethylene responsive factor (ERF) family genes and potassium transporter family genes for RCA formation were the potential candidate genes involved under waterlogging conditions. Several genotypes, which performed consistently well under different conditions, can be used in breeding programs to develop waterlogging-tolerant varieties.

Characterization of Rsg2.a3: A new greenbug resistance allele at the Rsg2 locus from wild barley (Hordeum vulgare ssp. spontaneum)

Greenbug (Schizaphis graminum Rondani) is a destructive insect pest that not only damages plants, but also serves as a vector for many viruses. Host plant resistance is the preferred strategy for managing greenbug. Two greenbug resistance genes, Rsg1 and Rsg2, have been reported in barley. To breed cultivars with effective resistance against various greenbug biotypes, additional resistance genes are urgently needed to sustain barley production. Wild barley accession WBDC053 (PI 681777) was previously found to be resistant to several greenbug biotypes. In this study, a recombinant inbred line (RIL) population derived from Weskan × WBDC053 was evaluated for response to two greenbug biotypes (E and TX1) and genotyped using genotyping by sequencing (GBS). A set of 3347 high quality GBS-derived single nucleotide polymorphisms (SNPs) were then used to map the greenbug resistance gene in this wild barley accession. Linkage analysis placed the greenbug resistance gene in a 2.35 Mb interval (0–2,354,645 bp) in the terminal region of the short arm of chromosome 2H. This interval harbors 15 genes with leucine-rich-repeat (LRR) protein domains. An allelism test indicated that the greenbug resistance gene in WBDC053, designated Rsg2.a3, is likely allelic or closely linked to Rsg2. GBS-SNPs 2H_1318811 and 2H_1839499 co-segregating with Rsg2.a3 in the RIL population were converted to Kompetitive allele specific PCR (KASP) markers KASP-Rsg2.a3-1 and KASP-Rsg2.a3-2, respectively. The two KASP markers can be used to select Rsg2.a3 and have the potential to tag Rsg2 in barley improvement programs.

Physical geography, isolation by distance and environmental variables shape genomic variation of wild barley (Hordeum vulgare L. ssp. spontaneum) in the Southern Levant

Determining the extent of genetic variation that reflects local adaptation in crop-wild relatives is of interest for the purpose of identifying useful genetic diversity for plant breeding. We investigated the association of genomic variation with geographical and environmental factors in wild barley (Hordeum vulgare L. ssp. spontaneum) populations of the Southern Levant using genotyping by sequencing (GBS) of 244 accessions in the Barley 1K+ collection. The inference of population structure resulted in four genetic clusters that corresponded to eco-geographical habitats and a significant association between lower gene flow rates and geographical barriers, e.g. the Judaean Mountains and the Sea of Galilee. Redundancy analysis (RDA) revealed that spatial autocorrelation explained 45% and environmental variables explained 15% of total genomic variation. Only 4.5% of genomic variation was solely attributed to environmental variation if the component confounded with spatial autocorrelation was excluded. A synthetic environmental variable combining latitude, solar radiation, and accumulated precipitation explained the highest proportion of genomic variation (3.9%). When conditioned on population structure, soil water capacity was the most important environmental variable explaining 1.18% of genomic variation. Genome scans with outlier analysis and genome-environment association studies were conducted to identify adaptation signatures. RDA and outlier methods jointly detected selection signatures in the pericentromeric regions, which have reduced recombination, of the chromosomes 3H, 4H, and 5H. However, selection signatures mostly disappeared after correction for population structure. In conclusion, adaptation to the highly diverse environments of the Southern Levant over short geographical ranges had a limited effect on the genomic diversity of wild barley. This highlighted the importance of nonselective forces in genetic differentiation.

Two dominant genes in barley (Hordeum vulgare L.) complementarily encode perfect resistance to Japanese soil-borne wheat mosaic virus.

Japanese soil-borne wheat mosaic virus (Furovirus) is a damaging pathogen of wheat and barley. This virus can survive in the soil for several decades, so the deployment of resistant cultivars represents the only practical control measure. Here, a genetic analysis has identified two regions of the barley genome-one on chromosome 2H and the other on chromosome 3H-as harboring gene(s) encoding resistance to this virus. The joint presence of both loci, termed Jmv1 and Jmv2, made the plants essentially immune, with resistance being dominant over susceptibility at each locus. Phylogenetic analysis showed that the virus is not closely related to the type Furovirus species Soil-borne wheat mosaic virus. There was a difference between the RNA1- and RNA2-based phylogenies of the virus species in Furovirus implying the independent segregation of the virus subgenomes.