The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

The wheat Seven in absentia gene is associated with increases in biomass and yield in hot climates.

Wheat (Triticum aestivum L.) productivity is severely reduced by high temperatures. Breeding of heat-tolerant cultivars can be achieved by identifying genes controlling physiological and agronomical traits when high temperatures occur and using these to select superior genotypes, but no gene underlying genetic variation for heat tolerance has previously been described. We advanced the positional cloning of qYDH.3BL, a quantitative trait locus (QTL) on bread wheat chromosome 3B associated with increased yield in hot and dry climates. The delimited genomic region contained 12 putative genes and a sequence variant in the promoter region of one gene, Seven in absentia, TaSINA. This was associated with the QTL's effects on early vigour, root growth, plant biomass, and yield components in two distinct wheat populations grown under various growth conditions. Near isogenic lines carrying the positive allele at qYDH.3BL underexpressed TaSINA and had increased vigour and water use efficiency early in development, as well as increased biomass, grain number, and grain weight following heat stress. A survey of worldwide distribution indicated that the positive allele became widespread from the 1950s through the CIMMYT wheat breeding programme but, to date, has been selected only in breeding programmes in Mexico and Australia.

Harnessing translational research in wheat for climate resilience.

Despite being the world's most widely grown crop, research investments in wheat (Triticum aestivum and Triticum durum) fall behind those in other staple crops. Current yield gains will not meet 2050 needs, and climate stresses compound this challenge. However, there is good evidence that heat and drought resilience can be boosted through translating promising ideas into novel breeding technologies using powerful new tools in genetics and remote sensing, for example. Such technologies can also be applied to identify climate resilience traits from among the vast and largely untapped reserve of wheat genetic resources in collections worldwide. This review describes multi-pronged research opportunities at the focus of the Heat and Drought Wheat Improvement Consortium (coordinated by CIMMYT), which together create a pipeline to boost heat and drought resilience, specifically: improving crop design targets using big data approaches; developing phenomic tools for field-based screening and research; applying genomic technologies to elucidate the bases of climate resilience traits; and applying these outputs in developing next-generation breeding methods. The global impact of these outputs will be validated through the International Wheat Improvement Network, a global germplasm development and testing system that contributes key productivity traits to approximately half of the global wheat-growing area.

Breeding for drought and heat tolerance in wheat.

KEY MESSAGE: Many approaches have been adopted to enhance the heat and drought tolerance of wheat with mixed success. An assessment of the relative merits of different strategies is presented. Wheat is the most widely grown crop globally and plays a key role in human nutrition. However, it is grown in environments that are prone to heat and drought stress, resulting in severely reduced yield in some seasons. Increased climate variability is expected to have a particularly adverse effect of wheat production. Breeding for stable yield across both good and bad seasons while maintaining high yield under optimal conditions is a high priority for most wheat breeding programs and has been a focus of research activities. Multiple strategies have been explored to enhance the heat and drought tolerance of wheat including extensive genetic analysis and modify the expression of genes involved in stress responses, targeting specific physiological traits and direct selection under a range of stress scenarios. These approaches have been combined with improvements in phenotyping, the development of genetic and genomic resources, and extended screening and analysis techniques. The results have greatly expanded our knowledge and understanding of the factors that influence yield under stress, but not all have delivered the hoped-for progress. Here, we provide an overview of the different strategies and an assessment of the most promising approaches.

Breeding crops for climate resilience.

In enhancing the resilience of our crops to the impacts of climate change, selection objectives need to address increased variability in the production environment. This encompasses the effects of more variable rainfall and temperatures than currently experienced, including extreme weather events, and changes in pest and pathogens distribution with the increased likelihood of major pest and disease outbreaks as well as occurrence of novel pathogens. Farmers manage the inevitable risks associated with cropping by planting varieties that deliver high yields and good quality under optimal conditions but minimise losses when the seasons are bad. Breeders and agronomists work to support farmers in specific target environments, but increased climate variability has meant that they need to broaden the adaptability of varieties grown and increase the yield stability to help minimise climate-induced risks and build resilience.

Expression of Specific Alleles of Zinc-Finger Transcription Factors, HvSAP8 and HvSAP16, and Corresponding SNP Markers, Are Associated with Drought Tolerance in Barley Populations.

Two genes, HvSAP8 and HvSAP16, encoding Zinc-finger proteins, were identified earlier as active in barley plants. Based on bioinformatics and sequencing analysis, six SNPs were found in the promoter regions of HvSAP8 and one in HvSAP16, among parents of two barley segregating populations, Granal × Baisheshek and Natali × Auksiniai-2. ASQ and Amplifluor markers were developed for HvSAP8 and HvSAP16, one SNP in each gene, and in each of two populations, showing simple Mendelian segregation. Plants of F6 selected breeding lines and parents were evaluated in a soil-based drought screen, revealing differential expression of HvSAP8 and HvSAP16 corresponding with the stress. After almost doubling expression during the early stages of stress, HvSAP8 returned to pre-stress level or was strongly down-regulated in plants with Granal or Baisheshek genotypes, respectively. For HvSAP16 under drought conditions, a high expression level was followed by either a return to original levels or strong down-regulation in plants with Natali or Auksiniai-2 genotypes, respectively. Grain yield in the same breeding lines and parents grown under moderate drought was strongly associated with their HvSAP8 and HvSAP16 genotypes. Additionally, Granal and Natali genotypes with specific alleles at HvSAP8 and HvSAP16 were associated with improved performance under drought via higher 1000 grain weight and more shoots per plant, respectively.

Development of an Australian Bread Wheat Nested Association Mapping Population, a New Genetic Diversity Resource for Breeding under Dry and Hot Climates.

Genetic diversity, knowledge of the genetic architecture of the traits of interest and efficient means of transferring the desired genetic diversity into the relevant genetic background are prerequisites for plant breeding. Exotic germplasm is a rich source of genetic diversity; however, they harbor undesirable traits that limit their suitability for modern agriculture. Nested association mapping (NAM) populations are valuable genetic resources that enable incorporation of genetic diversity, dissection of complex traits and providing germplasm to breeding programs. We developed the OzNAM by crossing and backcrossing 73 diverse exotic parents to two Australian elite varieties Gladius and Scout. The NAM parents were genotyped using the iSelect wheat 90K Infinium SNP array, and the progeny were genotyped using a custom targeted genotyping-by-sequencing assay based on molecular inversion probes designed to target 12,179 SNPs chosen from the iSelect wheat 90K Infinium SNP array of the parents. In total, 3535 BC1F4:6 RILs from 125 families with 21 to 76 lines per family were genotyped and we found 4964 polymorphic and multi-allelic haplotype markers that spanned the whole genome. A subset of 530 lines from 28 families were evaluated in multi-environment trials over three years. To demonstrate the utility of the population in QTL mapping, we chose to map QTL for maturity and plant height using the RTM-GWAS approach and we identified novel and known QTL for maturity and plant height.

Identification, gene expression and genetic polymorphism of zinc finger A20/AN1 stress-associated genes, HvSAP, in salt stressed barley from Kazakhstan.

BACKGROUND: A family of genes designated as the Zinc finger A20/AN1 Transcription factors encoding stress-associated proteins (SAP) are well described in Arabidopsis and rice, and include 14 AtSAP and 18 OsSAP genes that are associated with variable tolerances to multiple abiotic stresses. The SAP gene family displays a great diversity in its structure and across different plant species. The aim of this study was to identify all HvSAP genes in barley (Hordeum vulgare L.), to analyse the expression of selected genes in response to salinity in barley leaves and develop SNP marker for HvSAP12 to evaluate the association between genotypes of barley plants and their grain yield in field trials. RESULTS: In our study, 17 HvSAP genes were identified in barley, which were strongly homologous to rice genes. Five genes, HvSAP5, HvSAP6, HvSAP11, HvSAP12 and HvSAP15, were found to be highly expressed in leaves of barley plants in response to salt stress in hydroponics compared to controls, using both semi-quantitative RT-PCR and qPCR analyses. The Amplifluor-like SNP marker KATU-B30 was developed and used for HvSAP12 genotyping. A strong association (R2 = 0.85) was found between KATU-B30 and grain yield production per plant of 50 F3 breeding lines originating from the cross Granal × Baisheshek in field trials with drought and low to moderate salinity in Northern and Central Kazakhstan. CONCLUSIONS: A group of HvSAP genes, and HvSAP12 in particular, play an important role in the tolerance of barley plants to salinity and drought, and is associated with higher grain yield in field trials. Marker-assisted selection with SNP marker KATU-B30 can be applied in barley breeding to improve grain yield production under conditions of abiotic stress.

Salt-induced expression of intracellular vesicle trafficking genes, CaRab-GTP, and their association with Na+ accumulation in leaves of chickpea (Cicer arietinum L.).

BACKGROUND: Chickpea is an important legume and is moderately tolerant to salinity stress during the growing season. However, the level and mechanisms for salinity tolerance can vary among accessions and cultivars. A large family of CaRab-GTP genes, previously identified in chickpea, is homologous to intracellular vesicle trafficking superfamily genes that play essential roles in response to salinity stress in plants. RESULTS: To determine which of the gene family members are involved in the chickpea salt response, plants from six selected chickpea accessions (Genesis 836, Hattrick, ICC12726, Rupali, Slasher and Yubileiny) were exposed to salinity stress and expression profiles resolved for the major CaRab-GTP gene clades after 5, 9 and 15 days of salt exposure. Gene clade expression profiles (using degenerate primers targeting all members of each clade) were tested for their relationship to salinity tolerance measures, namely plant biomass and Na+ accumulation. Transcripts representing 11 out of the 13 CaRab clades could be detected by RT-PCR, but only six (CaRabA2, -B, -C, -D, -E and -H) could be quantified using qRT-PCR due to low expression levels or poor amplification efficiency of the degenerate primers for clades containing several gene members. Expression profiles of three gene clades, CaRabB, -D and -E, were very similar across all six chickpea accessions, showing a strongly coordinated network. Salt-induced enhancement of CaRabA2 expression at 15 days showed a very strong positive correlation (R2 = 0.905) with Na+ accumulation in leaves. However, salinity tolerance estimated as relative plant biomass production compared to controls, did not correlate with Na+ accumulation in leaves, nor with expression profiles of any of the investigated CaRab-GTP genes. CONCLUSION: A coordinated network of CaRab-GTP genes, which are likely involved in intracellular trafficking, are important for the salinity stress response of chickpea plants.

QTL analysis and fine mapping of a QTL for yield-related traits in wheat grown in dry and hot environments.

Genetic control of grain yield and phenology was examined in the Excalibur/Kukri doubled haploid mapping population grown in 32 field experiments across the climatic zones of southern Australia, India and north-western Mexico where the wheat crop experiences drought and heat stress. A total of 128 QTL were identified for four traits: grain yield, thousand grain weight (TGW), days to heading and grain filling duration. These QTL included 24 QTL for yield and 27 for TGW, showing significant interactions with the environment (Q * E). We also identified 14 QTL with a significant, small main effects on yield across environments. The study focussed on a region of chromosome 1B where two main effect QTL were found for yield and TGW without the confounding effect of phenology. Excalibur was the source of favourable alleles: QYld.aww-1B.2 with a peak at 149.5-150.1 cM and QTgw.aww-1B at 168.5-171.4 cM. We developed near isogenic lines (NIL) for the interval including QYld.aww-1B.2 and QTgw.aww-1B and evaluated them under semi-controlled conditions. Significant differences in four pairs of NIL were observed for grain yield but not for TGW, confirming a positive effect of the Excalibur allele for QYld.aww-1B.2. The interval containing QYld.aww-1B.2 was narrowed down to 2.9 cM which corresponded to a 2.2 Mbp genomic region on the chromosome 1B genomic reference sequence of cv. Chinese Spring and contained 39 predicted genes.

Molecular basis of adaptation to high soil boron in wheat landraces and elite cultivars.

Environmental constraints severely restrict crop yields in most production environments, and expanding the use of variation will underpin future progress in breeding. In semi-arid environments boron toxicity constrains productivity, and genetic improvement is the only effective strategy for addressing the problem. Wheat breeders have sought and used available genetic diversity from landraces to maintain yield in these environments; however, the identity of the genes at the major tolerance loci was unknown. Here we describe the identification of near-identical, root-specific boron transporter genes underlying the two major-effect quantitative trait loci for boron tolerance in wheat, Bo1 and Bo4 (ref. 2). We show that tolerance to a high concentration of boron is associated with multiple genomic changes including tetraploid introgression, dispersed gene duplication, and variation in gene structure and transcript level. An allelic series was identified from a panel of bread and durum wheat cultivars and landraces originating from diverse agronomic zones. Our results demonstrate that, during selection, breeders have matched functionally different boron tolerance alleles to specific environments. The characterization of boron tolerance in wheat illustrates the power of the new wheat genomic resources to define key adaptive processes that have underpinned crop improvement.

The pseudogenes of barley.

Pseudogenes have a reputation of being 'evolutionary relics' or 'junk DNA'. While they are well characterized in mammals, studies in more complex plant genomes have so far been hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high-quality genome reference assembly available for a Triticeae crop, we conducted a whole-genome assessment of pseudogenes on the barley genome. We identified, characterized and classified 89 440 gene fragments and pseudogenes scattered along the chromosomes, with occasional hotspots and higher densities at the chromosome ends. Full-length pseudogenes (11 015) have preferentially retained their exon-intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints at founding mechanisms. While parent genes related to the defense-response were found to be under-represented in cultivated barley, we detected several defense-related pseudogenes in wild barley accessions. The percentage of transcriptionally active pseudogenes is 7.2%, and these may potentially adopt new regulatory roles.The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non-random duplication and pseudogenization preferences and improve our understanding of the dynamics of gene birth and death in large plant genomes and the mechanisms that lead to evolutionary innovations.

Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins.

KEY MESSAGE: Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.

Correction to: Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins.

Due to an unfortunate turn of events, the panels O to S are missing in Fig. 8 of the original publication. The correct Fig. 8 and its caption is published here and should be treated as definitive.

gRNA validation for wheat genome editing with the CRISPR-Cas9 system.

BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of male-sterile hexaploid wheat lines for use in hybrid seed production.

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male-sterile lines were potentially non-transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.

A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions.

BACKGROUND: The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating. RESULTS: A simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results. CONCLUSIONS: The DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.

Quantifying Wheat Sensitivities to Environmental Constraints to Dissect Genotype × Environment Interactions in the Field.

Yield is subject to strong genotype-by-environment (G × E) interactions in the field, especially under abiotic constraints such as soil water deficit (drought [D]) and high temperature (heat [H]). Since environmental conditions show strong fluctuations during the whole crop cycle, geneticists usually do not consider environmental measures as quantitative variables but rather as factors in multienvironment analyses. Based on 11 experiments in a field platform with contrasting temperature and soil water deficit, we determined the periods of sensitivity to drought and heat constraints in wheat (Triticum aestivum) and determined the average sensitivities for major yield components. G × E interactions were separated into their underlying components, constitutive genotypic effect (G), G × D, G × H, and G × H × D, and were analyzed for two genotypes, highlighting contrasting responses to heat and drought constraints. We then tested the constitutive and responsive behaviors of two strong quantitative trait loci (QTLs) associated previously with yield components. This analysis confirmed the constitutive effect of the chromosome 1B QTL and explained the G × E interaction of the chromosome 3B QTL by a benefit of one allele when temperature rises. In addition to the method itself, which can be applied to other data sets and populations, this study will support the cloning of a major yield QTL on chromosome 3B that is highly dependent on environmental conditions and for which the climatic interaction is now quantified.

Overexpression of the TaSHN1 transcription factor in bread wheat leads to leaf surface modifications, improved drought tolerance, and no yield penalty under controlled growth conditions.

Transcription factors regulate multiple networks, mediating the responses of organisms to stresses, including drought. Here, we investigated the role of the wheat transcription factor TaSHN1 in crop growth and drought tolerance. TaSHN1, isolated from bread wheat, was characterized for molecular interactions and functionality. The overexpression of TaSHN1 in wheat was followed by the evaluation of T2 and T3 transgenic lines for drought tolerance, growth, and yield components. Leaf surface changes were analysed by light microscopy, SEM, TEM, and GC-MS/GC-FID. TaSHN1 behaves as a transcriptional activator in a yeast transactivation assay and binds stress-related DNA cis-elements, determinants of which were revealed using 3D molecular modelling. The overexpression of TaSHN1 in transgenic wheat did not result in a yield penalty under the controlled plant growth conditions of a glasshouse. Transgenic lines had significantly lower stomatal density and leaf water loss and exhibited improved recovery after severe drought, compared with control plants. The comparative analysis of cuticular waxes revealed an increased accumulation of alkanes in leaves of transgenic lines. Our data demonstrate that TaSHN1 may operate as a positive modulator of drought stress tolerance. Positive attributes could be mediated through an enhanced accumulation of alkanes and reduced stomatal density.