Management and distribution of experimental data from prebreeding projects is important to ensure uptake of germplasm into breeding and research programs. Being able to access and share this data in standard formats is essential. The adoption of a common informatics platform for crops that may have limited resources brings economies of scale, allowing common informatics components to be used across multiple species. The close integration of such a platform with commonly used breeding software, visualization, and analysis tools reduces the barrier for entry to researchers and provides a common framework to facilitate collaborations and data sharing. This work presents significant updates to the Germinate platform and highlights its value in distributing prebreeding data for 14 crops as part of the project ‘Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives’ (hereafter Crop Trust Crop Wild Relatives project) led by the Crop Trust ( https://www.cwrdiversity.org ). The addition of data on these species compliments data already publicly available in Germinate. We present a suite of updated Germinate features using examples from these crop species and their wild relatives. The use of Germinate within the Crop Trust Crop Wild Relatives project demonstrates the usefulness of the system and the benefits a shared informatics platform provides. These data resources provide a foundation on which breeding and research communities can develop additional online resources for their crops, harness new data as it becomes available, and benefit collectively from future developments of the Germinate platform.

Wheat (Triticum sp.) is one of the world’s most important crops, and constantly increasing its productivity is crucial to the livelihoods of millions of people. However, more than a century of intensive breeding and selection processes have eroded genetic diversity in the elite genepool, making new genetic gains difficult. Therefore, the need to introduce novel genetic diversity into modern wheat has become increasingly important. This review provides an overview of the plant genetic resources (PGR) available for wheat. We describe the most important taxonomic and phylogenetic relationships of these PGR to guide their use in wheat breeding. In addition, we present the status of the use of some of these resources in wheat breeding programs. We propose several introgression schemes that allow the transfer of qualitative and quantitative alleles from PGR into elite germplasm. With this in mind, we propose the use of a stage-gate approach to align the pre-breeding with main breeding programs to meet the needs of breeders, farmers, and end-users. Overall, this review provides a clear starting point to guide the introgression of useful alleles over the next decade.

KEY MESSAGE:Diagnostic markers for Rrs1Rh4 have been identified by testing for associations between SNPs within the Rrs1 interval in 150 barley genotypes and their resistance to Rhynchosporium commune isolates recognised by lines containing Rrs1. Rhynchosporium or barley scald, caused by the destructive fungal pathogen Rhynchosporium commune, is one of the most economically important diseases of barley in the world. Barley landraces from Syria and Jordan demonstrated high resistance to rhynchosporium in the field. Genotyping of a wide range of barley cultivars and landraces, including known sources of different Rrs1 genes/alleles, across the Rrs1 interval, followed by association analysis of this genotypic data with resistance phenotypes to R. commune isolates recognised by Rrs1, allowed the identification of diagnostic markers for Rrs1Rh4. These markers are specific to Rrs1Rh4 and do not detect other Rrs1 genes/alleles. The Rrs1Rh4 diagnostic markers represent a resource that can be exploited by breeders for the sustainable deployment of varietal resistance in new cultivars. Thirteen out of the 55 most resistant Syrian and Jordanian landraces were shown to contain markers specific to Rrs1Rh4. One of these lines came from Jordan, with the remaining 12 lines from different locations in Syria. One of the Syrian landraces containing Rrs1Rh4 was also shown to have Rrs2. The remaining landraces that performed well against rhynchosporium in the field are likely to contain other resistance genes and represent an important novel resource yet to be exploited by European breeders.

Plant domestication and crop improvement have resulted in reduced genetic diversity in most of our cultivated crops, thus limiting their potential to adapt to future challenges (Byrne et al., 2018; Keneni, Bekele, Imtiaz, & Dagne, 2012; Tanksley & McCouch, 1997; Swarup et al., 2020). One response to mitigate the impact of climate change on agricultural systems is to develop improved varieties that are genetically tolerant or resistant to the new range of abiotic and biotic challenges. Improvement via crop breeding requires access to novel variants of genes for complex adaptive traits. Crop wild relatives (CWR) and landraces are a potentially valuable source of these alleles (Cossani & Reynolds, 2015; Seiler, QiL, & Marek, 2017). However, these materials are often difficult to work with and require genetic selection to make them agronomic and useful in a breeding program, a process commonly referred to as ‘pre-breeding’. This term was first used by Rick (1984) and refers to a wide range of activities designed to (i) identify beneficial traits in CWR and other plant genetic resources (PGR), and (ii) transfer these traits into breeding lines (Ortiz, 2002; Sharma, Upadhyaya, Varshney, & Gowda, 2013). Pre-breeding forms a bridge between the genebanks that hold and safeguard CWR and landraces, and breeders and farmers who use them. Pre-breeding is a long, laborious process, which includes multiple crossings and trait evaluations, and is risky and costly (Dempewolf et al., 2017. Thus, pre-breeding efforts tend to be avoided whenever possible, especially by advanced breeding programs. However, climate change is forcing breeders to seek useful traits from all available sources, no matter how challenging. Utilizing CWR in pre-breeding can access diversity for specific traits of importance that are not present in the elite crop genotypes that are generally used in breeding programs. As the pace of change of temperature, rainfall, and pest and pathogen attack increases, and extremes become normal, long-term efforts and substantial investment in pre-breeding are required to develop improved varieties that are well adapted to the various challenges ahead of us. One unique, global initiative to facilitate the use of CWR in breeding new, improved crop varieties is the project “Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives” (http://www.cwrdiversity.org/; (Dempewolf et al., 2014, 2017). This is the first systematic global long-term investment supporting the use of CWR across multiple genepools to generate knowledge and pre-breeding materials contributing to adaptation to climate change. The aim is to build capacities in developing countries in order to use this diversity where it is needed most. The project focussed its pre-breeding efforts on 19 crops important for food security. Of key importance has been the establishment of partnerships between CGIAR centers, advanced research organizations and national breeding programs around the world. A total of 19 pre-breeding partnerships are being supported in 50 countries by the project. Partners from more than 100 organizations worked together, including farmer organizations and the private sector. The pre-breeding lines and information produced are being made available via the multilateral system (MLS) of the Plant Treaty. Most projects have already deposited advanced material in genebanks for conservation and future use. Our intention in this special issue of Crop Science is to promote global awareness of the progress made by the pre-breeding component of the project and to highlight newly available pre-breeding materials and related information obtained from this major global initiative. We present a series of 17 research articles on 13 crops reporting successful implementation of CWR utilization in pre-breeding. The contributions cover all activity areas in the pre-breeding continuum, and thus connect genebanks with breeding pipelines. We have also included a review article and a collaborative paper describing an advanced database that provides a common platform for storing, visualizing and sharing pre-breeding data. In the wide range of pre-breeding activities considered, the starting point is to fill diversity gaps in genebank collections by acquiring new germplasm with unique priority traits. In "Filling the gaps in gene banks: Collecting, characterizing and phenotyping wild banana relatives of Papua New Guinea,” Eyland et al. (2021) deliver a proof of the principle that wild banana diversity is not yet fully represented in ex situ genebanks and that wild Musa acuminata ssp. banksii populations (the ancestor of most edible bananas) contain individuals with beneficial traits, useful for drought tolerance breeding programs. The next step in the pre-breeding process is to characterize and evaluate CWR populations and CWR-derived lines. A set of papers focuses on improving drought, heat or salinity stress-related traits and includes Humphries et al. (2021), “Characterization and pre-breeding of diverse alfalfa wild relatives originating from drought-stressed environments,” who report that newly developed alfalfa hybrids between Medicago sativa x M. arborea and M. sativa x M. truncatula from the tertiary genepool offer new diversity useful to improve growth habit, seed size, fall dormancy and forage yield. Simon et al. (2021), in ”Wild carrot diversity for new sources of abiotic stress tolerance to strengthen vegetable breeding in Bangladesh and Pakistan,” describe new sources of heat and drought tolerance in two wild Daucus species (also see the podcast about their paper: https://fieldlabearth.libsyn.com/carrot-stress-tolerance-and-wild-relative-breeding-with-dr-simon-philipp). Aberkane et al. (2021), in “Evaluation of durum wheat lines derived from interspecific crosses under drought and heat stress,” conclude that gene introgression from wild diploid and wild tetraploid wheat-derived materials pays off and can increase wheat resilience. Drought tolerant genotypes with high values of relative chlorophyll content and more green leaves at maturity have been identified by Ochieng et al. (2021), in ”Novel sources of drought tolerance from landraces and wild sorghum relatives“. Kouassi et al. (2021), used an original approach to estimate drought tolerance scores for vegetative growth and yield related traits in ”Genetic parameters of drought tolerance for agromorphological traits in eggplant, wild relatives, and interspecific hybrids.” Finally, Nhanala and Yencho (2021), in "Assessment of the potential of wild Ipomoea spp. for the improvement of drought tolerance in cultivated sweetpotato (Ipomoea batatas) (L.) Lam,” conclude that cultivated and wild sweetpotatoes respond differently to drought. A second set of papers in this pre-breeding category aims at improving biotic stress-related traits and includes Rehman et al. (2021), “Traits discovery in Hordeum vulgare subsp. spontaneum accessions and in lines derived from interspecific crosses with wild Hordeum species for enhancing barley breeding efforts,” who show that high levels of resistance to the net form of net blotch, scald, leaf rust and powdery mildew were identified in CWR-derived barley lines, including some that were resistant to all four diseases. In ”Novel sources of resistance to blast disease in finger millet,” Dida et al. (2021) show that wild finger millet was generally more resistant to the blast disease caused by Magnaporthe grisea compared to cultivated finger millet. In "Wild Lathyrus species as a great source of resistance for introgression into cultivated grass pea (Lathyrus sativus L.) against broomrape weeds (Orobanche crenata Forsk. and Orobanche foetida Poir.),” Abdallah et al. (2021) identify complete resistance against both weeds in wild L. articulatus. Three papers describe a combination of multiple traits. The usefulness of deploying CWR in durum wheat breeding for several abiotic and biotic traits was assessed by El Haddad et al. (2021), in “Crop wild relatives in durum wheat breeding: Drift or thrift?”, who conclude that CWR-derived lines are a good source of resistance against Septoria leaf blotch. Additionally, under both drought and heat stress conditions, CWR-derived lines revealed a clear superiority for grain yield and yield component traits over the best check varieties. In two papers on pearl millet, the world's hardiest warm-season cereal crop, Sharma et al. (2021a) show that CWR can improve multiple traits even further. Promising introgression lines (ILs) derived from wild pearl millet with improved resistance against five highly virulent blast pathotypes, and also high dry fodder yield and seed set under heat stress conditions, are described by Sharma et al. (2021a), in ”Utilization of wild Pennisetum species for improving biotic and abiotic stress tolerance in pearl millet (Pennisetum glaucum L.).” In an excellent overview entitled "Harnessing wild relatives of pearl millet for germplasm enhancement: challenges and opportunities,” Sharma et al. (2021b) reviewed the availability, importance, and strategies for utilization of CWR for crop improvement, from a breeder's perspective. They proposed a stage-gate process for the efficient management of pre-breeding programs using CWR. Developing nutrient-rich crop cultivars is an important strategy to combat malnutrition due to protein and mineral deficiencies. Sharma et al. (2021c), in "Characterization of cultivated and annual wild Cicer species for seed protein and mineral contents, and identification of promising accessions for chickpea (Cicer arietinum L.) improvement,” identify individuals from different wild Cicer species that can increase concentrations of protein, Ca, Cu, Fe, Mg, Mn, and Zn in the seeds. These resources will facilitate the biofortification of chickpea. The transfer of beneficial alleles and traits from CWR to breeding lines, and ultimately to new cultivars, is the next step in the pre-breeding process. The development of new germplasm resources with novel allelic diversity in a useful background also aids in the identification of genes that contribute to important adaptive traits. Mace et al. (2021), in “A global resource for exploring and exploiting genetic variation in sorghum crop wild relatives,” describe the development of a backcross-nested association mapping (BCNAM) population for sorghum using 9 CWR accessions and two recurrent parents. The BCNAM population of 1,224 lines was genotyped using DArT markers and genome-wide association scans (GWAS) confirmed this resource as a source of new alleles for breeding for future climates. Induced mutations and chromosomal changes can provide breeders with additional tools in the development of new cultivars that are better suited to future climate scenarios. Innes, Denton, Dundas, Peck, and Humphries (2021), demonstrated this in ”The effect of ploidy number on vigor, productivity and potential adaptation to climate change in annual Medicago species.” This paper reported wild or induced tetraploid Medicago lines with the potential to increase early biomass, vigor and leaf size. A key step in pre-breeding is ensuring that the new material appeals to users. In order to incorporate farmers' preferences, on-farm participatory evaluation studies are highly recommended. We took two such studies on board for this special issue of Crop Science: Tin et al. (2021a), in “Phenotypic response of farmer-selected CWR-derived rice lines to salt stress in the Mekong Delta,” describe the development of CWR-derived rice lines and their evaluation in the Mekong Delta. Subsequently, 50 stable BC3F5 lines were selected by farmers and screened in hydroponics to assess their phenotypic response to salt stress. In the second study by Tin et al. (2021b), in ”Participatory selection of CWR-derived salt-tolerant rice lines adapted to the coastal zone of the Mekong Delta,” show that four lines derived from wild Oryza rufipogon and O. nivara are well adapted, with high grain yield (> 6.5 tons/ha), early maturity, and short plant height. In order to provide pre-breeding data relevant to different stakeholders, Raubach et al. (2021) present a relevant and truly collaborative effort to manage, visualize and share data from pre-breeding projects in their paper "From bits to bites: Advancement of the Germinate platform to support prebreeding informatics for crop wild relatives.” The authors describe the status of 12 Germinate databases that are currently publicly accessible. Our intention in creating this special issue of Crop Science is to promote global awareness of the newly available CWR-derived pre-breeding materials and related data sets, in order to support the adaptation of agriculture to climate change. When we reflect on these 19 articles, we conclude that impressive progress has been made by this project in the introgression of beneficial traits from CWR for several crops. Promising CWR-derived lines have been identified and presented here that fully justify the expense and effort. Continuation and further support for this work is needed to achieve practical outcomes, and move the work done so far out of pre-breeding, to breeding, to new varieties contributing to livelihoods and nutrition in farmers’ fields. We would like to thank Elizabeth Gebhardt and Abby Morrison for their excellent support in preparing and publishing this special issue. We greatly acknowledge the following experts for their suggestions for improvement during the peer review process of all 19 articles: Ahmed Amri, Hatice Filiz Boyaci, Paula Bramel, Françoise Carreel, Luigi Cattivelli, Asnake Fikre, Michael Frei, Elcio Guimarães, Jake Howie, Michael Kantar, Colin Khoury, Shiv Kumar, Ljiljana Kuzmanovic, Jan Low, Tian-li Ma, Thiago Mendes, Gary Muehlbauer, Ramakrishnan Nair, Hakan Özkan, Aditya Pratap, Jaime Prohens, Mohamed Rakha, Muhammet Şakiroğlu, Deborah Samac, Stanley Samonte, Radu Sestras, Shivali Sharma, Philipp Simon, Brian Steffenson, Alessandro Tondelli, David Turner, Ramaiah Venuprasad, Andrea Visioni, Eric von Wettberg, Stephen Weise and Om Yadav. Most of this work was undertaken as part of the initiative “Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives” which is supported by the Government of Norway. The pre-breeding projects are coordinated by the Global Crop Diversity Trust and implemented in partnership with national and international genebanks and plant breeding institutes around the world. For further information, please visit the project website: http://www.cwrdiversity.org/.

SummaryBroadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.

Lodging continues to be a major cause of yield loss in important crop species such as Brassica napus. Understanding the genetic regulation of lodging resistance is therefore of key interest to breeders worldwide. Current strategies aimed at minimising lodging risk involve the incorporation of dwarfing genes or the application of plant growth regulators. However, despite these efforts, lodging continues to be a persistent problem and it is therefore of high interest that novel, complimentary strategies for lodging control are implemented. One approach would be to focus on understanding the genetic properties underlying stem mechanical strength. With this in mind, we screened a training genetic diversity panel of B. napus accession for variation in stem mechanical strength and related traits. Using Associative Transcriptomics, we identified molecular markers for a suite of valuable traits. Using an independent test genetic diversity panel, we show that the methods employed are robust for identification of predictive markers. Furthermore, based on conserved synteny with Arabidopsis thaliana, we are able to provide a biological context to the marker associations detected and provide evidence for a role in pectin methylesterification in contributing to stem mechanical strength in Brassicaceae.

The precocious germination of cereal grains before harvest, also known as pre-harvest sprouting, is an important source of yield and quality loss in cereal production. Pre-harvest sprouting is a complex grain defect and is becoming an increasing challenge due to changing climate patterns. Resistance to sprouting is multi-genic, although a significant proportion of the sprouting variation in modern wheat cultivars is controlled by a few major quantitative trait loci, including Phs-A1 in chromosome arm 4AL. Despite its importance, little is known about the physiological basis and the gene(s) underlying this important locus. In this study, we characterized Phs-A1 and show that it confers resistance to sprouting damage by affecting the rate of dormancy loss during dry seed after-ripening. We show Phs-A1 to be effective even when seeds develop at low temperature (13 °C). Comparative analysis of syntenic Phs-A1 intervals in wheat and Brachypodium uncovered ten orthologous genes, including the Plasma Membrane 19 genes (PM19-A1 and PM19-A2) previously proposed as the main candidates for this locus. However, high-resolution fine-mapping in two bi-parental UK mapping populations delimited Phs-A1 to an interval 0.3 cM distal to the PM19 genes. This study suggests the possibility that more than one causal gene underlies this major pre-harvest sprouting locus. The information and resources reported in this study will help test this hypothesis across a wider set of germplasm and will be of importance for breeding more sprouting resilient wheat varieties.

Background: The current approach to reducing the tendency for wheat grown under high fertilizer conditions to collapse (lodge) under the weight of its grain is based on reducing stem height via the introduction of Rht genes. However, these reduce the yield of straw (itself an important commodity) and introduce other undesirable characteristics. Identification of alternative height-control loci is therefore of key interest. In addition, the improvement of stem mechanical strength provides a further way through which lodging can be reduced.Results: To investigate the prospects for genetic alternatives to Rht, we assessed variation for plant height and stem strength properties in a training genetic diversity panel of 100 wheat accessions fixed for Rht. Using mRNAseq data derived from RNA purified from leaves, functional genotypes were developed for the panel comprising 42,066 Single Nucleotide Polymorphism (SNP) markers and 94,060 Gene Expression Markers (GEMs). In the first application in wheat of the recently-developed method of Associative Transcriptomics, we identified associations between trait variation and both SNPs and GEMs. Analysis of marker-trait associations revealed candidates for the causative genes underlying the trait variation, implicating xylan acetylation and the COP9 signalosome as contributing to stem strength and auxin in the control of the observed variation for plant height. Predictive capabilities of key markers for stem strength were validated using a test genetic diversity panel of 30 further wheat accessions.Conclusions: This work illustrates the power of Associative Transcriptomics for the exploration of complex traits of high agronomic importance in wheat. The careful selection of genotypes included in the analysis, allowed for high resolution mapping of novel trait-controlling loci in this staple crop. The use of Gene Expression markers coupled with the more traditional sequence-based markers, provides the power required to understand the biological context of the marker-trait associations observed. This not only adds to the wealth of knowledge that we strive to accumulate regarding gene function and plant adaptation, but also provides breeders with the information required to make more informed decisions regarding the potential consequences of incorporating the use of particular markers into future breeding programmes.

The ability of roots to extract soil moisture is critical for maintaining yields during drought. However, the extent of genotypic variation for rooting depth and drought tolerance in Northern European wheat (Triticum aestivum L.) germplasm is not known. The objectives of this study were to measure genotypic differences in root activity, test relationships between water use and yield, examine trade-offs between yield potential and investment of biomass in deep roots, and identify genotypes that contrast in deep root activity. A diverse set of 21 wheat genotypes was evaluated under irrigated and managed drought conditions in the field. Root activity was inferred from patterns of water extraction from the soil profile. Genotypes were equally capable of exploiting soil moisture in the upper layers, but there were significant genotypic differences in rates of water uptake after anthesis in deeper soil layers. For example, across the three years of the study, the variety Xi19 showed consistently deeper root activity than the variety Spark; Xi19 also showed greater drought tolerance than Spark. There were positive correlations between water extraction from depth and droughted yields and drought tolerance, but correlations between deep water use and yield potential were not significant or only weakly negative. With appropriate screening tools, selection for genotypes that can better mine deep soil water should improve yield stability in variable rainfall environments.

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)-based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP-based wheat markers have been made available via the use of next-generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial-scale breeding programmes. To identify exome-based co-dominant SNP-based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re-sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95266 putative single nucleotide polymorphisms, of which 10251 were classified as being putatively co-dominant. Validation of a subset of these putative co-dominant markers confirmed that 96% were true polymorphisms and 65% were co-dominant SNP assays. The new co-dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web-based database to facilitate their use on genotyping programmes worldwide.

Many important plant species have polyploidy in their recent ancestry, complicating inferences about the genetic basis of trait variation. Although the principal locus controlling the proportion of polyunsaturated fatty acids (PUFAs) in seeds of Arabidopsis thaliana is known (fatty acid desaturase 2; FAD2), commercial cultivars of a related crop, oilseed rape (Brassica napus), with very low PUFA content have yet to be developed. We showed that a cultivar of oilseed rape with lower than usual PUFA content has non-functional alleles at three of the four orthologous FAD2 loci. To explore the genetic basis further, we developed an ethyl methanesulphonate mutagenised population, JBnaCAB_E, and used it to identify lines that also carried mutations in the remaining functional copy. This confirmed the hypothesised basis of variation, resulting in an allelic series of mutant lines showing a spectrum of PUFA contents of seed oil. Several lines had PUFA content of ~6 % and oleic acid content of ~84 %, achieving a long-standing industry objective: very high oleic, very low PUFA rapeseed without the use of genetic modification technology. The population contains a high rate of mutations and represents an important resource for research in B. napus.

Sequencing a genome and identifying genetic markers lays the groundwork for genome-wide association studies, but can be difficult to achieve for polyploid species. Harper et al . present an approach for performing association studies using genetic maps and markers generated from transcriptome sequencing data alone and apply it to the polyploid crop Brassica napus .

The genomes of cereals such as wheat (Triticum aestivum) and barley (Hordeum vulgare) are large and therefore problematic for the map-based cloning of agronomicaly important traits. However, comparative approaches within the Poaceae permit transfer of molecular knowledge between species, despite their divergence from a common ancestor sixty million years ago. The finding that null variants of the rice gene cytokinin oxidase/dehydrogenase 2 (OsCKX2) result in large yield increases provides an opportunity to explore whether similar gains could be achieved in other Poaceae members. Here, phylogenetic, molecular and comparative analyses of CKX families in the sequenced grass species rice, brachypodium, sorghum, maize and foxtail millet, as well as members identified from the transcriptomes/genomes of wheat and barley, are presented. Phylogenetic analyses define four Poaceae CKX clades. Comparative analyses showed that CKX phylogenetic groupings can largely be explained by a combination of local gene duplication, and the whole-genome duplication event that predates their speciation. Full-length OsCKX2 homologues in barley (HvCKX2.1, HvCKX2.2) and wheat (TaCKX2.3, TaCKX2.4, TaCKX2.5) are characterized, with comparative analysis at the DNA, protein and genetic/physical map levels suggesting that true CKX2 orthologs have been identified. Furthermore, our analysis shows CKX2 genes in barley and wheat have undergone a Triticeae-specific gene-duplication event. Finally, by identifying ten of the eleven CKX genes predicted to be present in barley by comparative analyses, we show that next-generation sequencing approaches can efficiently determine the gene space of large-genome crops. Together, this work provides the foundation for future functional investigation of CKX family members within the Poaceae.

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.