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High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence-absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat. © 2014 The Authors Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 ( EGT1 ) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Disease lesion mimic (DLM) or necrotic mutants display necrotic lesions in the absence of pathogen infections. They can show improved resistance to some pathogens and their molecular dissection can contribute to revealing components of plant defense pathways. Although forward-genetics strategies to find genes causal to mutant phenotypes are available in crops, these strategies require the production of experimental cross populations, mutagenesis, or gene editing and are time- and resource-consuming or may have to deal with regulated plant materials. In this study, we described a collection of 34 DLM mutants in barley (Hordeum vulgare L.) and applied a novel method called complementation by sequencing (CBS), which enables the identification of the gene responsible for a mutant phenotype given the availability of two or more chemically mutagenized individuals showing the same phenotype. Complementation by sequencing relies on the feasibility to obtain all induced mutations present in chemical mutants and on the low probability that different individuals share the same mutated genes. By CBS, we identified a cytochrome P450 CYP71P1 gene as responsible for orange blotch DLM mutants, including the historical barley nec3 locus. By comparative phylogenetic analysis we showed that CYP71P1 gene family emerged early in angiosperm evolution but has been recurrently lost in some lineages including Arabidopsis thaliana (L.) Heynh. Complementation by sequencing is a straightforward cost-effective approach to clone genes controlling phenotypes in a chemically mutagenized collection. The TILLMore (TM) collection will be instrumental for understanding the molecular basis of DLM phenotypes and to contribute knowledge about mechanisms of host-pathogen interaction., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Key Message: Spikelet indeterminacy and supernumerary spikelet phenotypes in barley multiflorus2.b mutant show polygenic inheritance. Genetic analysis of multiflorus2.b revealed major QTLs for spikelet determinacy and supernumerary spikelet phenotypes on 2H and 6H chromosomes. Understanding the genetic basis of yield forming factors in small grain cereals is of extreme importance, especially in the wake of stagnation of further yield gains in these crops. One such yield forming factor in these cereals is the number of grain-bearing florets produced per spikelet. Wild-type barley (Hordeum vulgare L.) spikelets are determinate structures, and the spikelet axis (rachilla) degenerates after producing single floret. In contrast, the rachilla of wheat (Triticum ssp.) spikelets, which are indeterminate, elongates to produce up to 12 florets. In our study, we characterized the barley spikelet determinacy mutant multiflorus2.b (mul2.b) that produced up to three fertile florets on elongated rachillae of lateral spikelets. Apart from the lateral spikelet indeterminacy (LS-IN), we also characterized the supernumerary spikelet phenotype in the central spikelets (CS-SS) of mul2.b. Through our phenotypic and genetic analyses, we identified two major QTLs on chromosomes 2H and 6H, and two minor QTLs on 3H for the LS-IN phenotype. For, the CS-SS phenotype, we identified one major QTL on 6H, and a minor QTL on 5H chromosomes. Notably, the 6H QTLs for CS-SS and LS-IN phenotypes co-located with each other, potentially indicating that a single genetic factor might regulate both phenotypes. Thus, our in-depth phenotyping combined with genetic analyses revealed the quantitative nature of the LS-IN and CS-SS phenotypes in mul2.b, paving the way for cloning the genes underlying these QTLs in the future., (© 2021. The Author(s).)

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 ( enhanced gravitropism 2 ) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain-containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Clonal hematopoiesis predisposes to hematological malignancies. However, clonal hematopoiesis is understudied in classical Hodgkin lymphoma (cHL), a mature B-cell neoplasm exhibiting the most abundant microenvironment. We analyzed clonal hematopoiesis in 40 cHL cases by sequencing microdissected tumor cells and matched normal cells from blood and/or lymph nodes. Five patients had blood and/or tissue clonal hematopoiesis. In three of five patients (all failing first-line therapy), clonal hematopoiesis spread through the tissue microenvironment extensively, and featured mutant DNMT3A R882H , KRAS G60D and DNMT3A R882H + TET2 Q1274 * in 33%, 92% and 60% of non-neoplastic cells, respectively. In the latter case, DNMT3A/TET2- mutant clonal hematopoiesis seeded the neoplastic clone, which was infected by the Epstein-Barr virus and showed almost no other somatic mutations exome-wide. In the former case, DNMT3A -mutant clonal hematopoiesis did not originate the neoplastic clone despite dominating the blood and B-cell lineage (~94% leukocytes; ~96% mature blood B cells), yet led to NPM1 -mutated acute myeloid leukemia 6 years after therapy for cHL. Our results expand to cHL the spectrum of hematologic malignancies associated with clonal hematopoiesis., Competing Interests: Conflict of interest: M.P. Martelli reports personal fees from AbbVie, Amgen, Pfizer, Jazz Pharmaceuticals, Novartis, and Janssen outside the submitted work. The authors declare no potential conflicts of interest.

Barley ( Hordeum vulgare L.) is one of the major grain crops worldwide and considered as a model plant for temperate cereals. One of the barley row-type groups, named intermedium- barley, was used in our previous study where we reported that other genetic loci rather than vrs1 and Int-c could play a role in lateral spikelet development and even in setting grains. To continue this work, we used phenotypic and genotypic data of 254 intermedium -spike barley accessions aimed at dissecting the genetic basis of development and grain traits of lateral and central spikelet using genome wide association (GWAS) analysis. After genotypic data filtering, 8,653 single-nucleotide polymorphism (SNPs) were used for GWAS analysis. A total of 169 significant associations were identified and we focused only on the subset of associations that exceeded the p < 10 -4 threshold. Thirty-three highly significant marker-trait-associations (MTAs), represented in 28 different SNPs on all seven chromosomes for the central and/or lateral spikelet traits; such as kernel length, width, area, weight, unfilled spikelet and 1000-kernel weight, were detected. Highly significant associated markers were anchored physically using barley genome sequencing to identify candidate genes to either contain the SNPs or the closest gene to the SNP position. The results showed that 12 MTAs were specific for lateral spikelet traits, nine MTAs were specific for central spikelet traits and seven MTAs for both central and lateral traits. All together, the GWAS and candidate gene results support our hypothesis that lateral spikelet development could be regulated by loci different from those regulating central spikelet development.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

We compared the performance of two commonly used genotyping platforms, genotyping-by-sequencing (GBS) and single nucleotide polymorphism-arrays (SNP), to investigate the extent and pattern of genetic variation within a collection of 1,000 diverse barley genotypes selected from the German Federal ex situ GenBank hosted at IPK Gatersleben. Each platform revealed equivalent numbers of robust bi-allelic SNPs (39,733 and 37,930 SNPs for the 50K SNP-array and GBS datasets respectively). A small overlap of 464 SNPs was common to both platforms, indicating that the methodologies we used selectively access informative polymorphism in different portions of the barley genome. Approximately half of the GBS dataset was comprised of SNPs with minor allele frequencies (MAFs) below 1%, illustrating the power of GBS to detect rare alleles in diverse germplasm collections. While desired for certain applications, the highly robust calling of alleles at the same SNPs across multiple populations is an advantage of the SNP-array, allowing direct comparisons of data from related or unrelated studies. Overall MAFs and diversity statistics (π) were higher for the SNP-array data, potentially reflecting the conscious removal of markers with a low MAF in the ascertainment population. A comparison of similarity matrices revealed a positive correlation between both approaches, supporting the validity of using either for entire GenBank characterization. To explore the potential of each dataset for focused genetic analyses we explored the outcomes of their use in genome-wide association scans for row type, growth habit and non-adhering hull, and discriminant analysis of principal components for the drivers of sub-population differentiation. Interpretation of the results from both types of analysis yielded broadly similar conclusions indicating that choice of platform used for such analyses should be determined by the research question being asked, group preferences and their capabilities to extract and interpret the different types of output data easily and quickly. Access to the requisite infrastructure for running, processing, analyzing, querying, storing, and displaying either datatype is an additional consideration. Our investigations reveal that for barley the cost per genotyping assay is less for SNP-arrays than GBS, which translates to a cost per informative datapoint being significantly lower for the SNP-array.

Genebanks hold comprehensive collections of cultivars, landraces and crop wild relatives of all major food crops, but their detailed characterization has so far been limited to sparse core sets. The analysis of genome-wide genotyping-by-sequencing data for almost all barley accessions of the German ex situ genebank provides insights into the global population structure of domesticated barley and points out redundancies and coverage gaps in one of the world's major genebanks. Our large sample size and dense marker data afford great power for genome-wide association scans. We detect known and novel loci underlying morphological traits differentiating barley genepools, find evidence for convergent selection for barbless awns in barley and rice and show that a major-effect resistance locus conferring resistance to bymovirus infection has been favored by traditional farmers. This study outlines future directions for genomics-assisted genebank management and the utilization of germplasm collections for linking natural variation to human selection during crop evolution.

Multiparental cross designs for mapping quantitative trait loci (QTL) provide an efficient alternative to biparental populations because of their broader genetic basis and potentially higher mapping resolution. We describe the development and deployment of a recombinant inbred line (RIL) population in durum wheat (Triticum turgidum ssp. durum) obtained by crossing four elite cultivars. A linkage map spanning 2664 cM and including 7594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs. QTL analysis was carried out by both interval mapping on founder haplotype probabilities and SNP bi-allelic tests for heading date and maturity date, plant height and grain yield from four field experiments. Sixteen QTL were identified across environments and detection methods, including two yield QTL on chromosomes 2BL and 7AS, with the former mapped independently from the photoperiod response gene Ppd-B1, while the latter overlapped with the vernalization locus VRN-A3. Additionally, 21 QTL with environment-specific effects were found. Our results indicated a prevalence of environment-specific QTL with relatively small effect on the control of grain yield. For all traits, functionally different QTL alleles in terms of direction and size of genetic effect were distributed among parents. We showed that QTL results based on founder haplotypes closely matched functional alleles at known heading date loci. Despite the four founders, only 2.1 different functional haplotypes were estimated per QTL, on average. This durum wheat population provides a mapping resource for detailed genetic dissection of agronomic traits in an elite background typical of breeding programmes., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor (wild emmer, T. turgidum ssp. dicoccoides). The consensus map harboured 30 144 markers (including 26 626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631 cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array including about 90,000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence-absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat., (© 2014 The Authors Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)