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Blueberries ( Vaccinium sect. Cyanococcus ) are a dietary source of phenolic acids, including chlorogenic acid (CGA) and related compounds such as acetylated caffeoylquinic acid (ACQA) and caffeoylarbutin (CA). These compounds are known to be potent antioxidants with potential health benefits. While the chemistry of these compounds has been extensively studied, the genetic analysis has lagged behind. Understanding the genetic basis for traits with potential health implications may be of great use in plant breeding. By characterizing genetic variation related to fruit chemistry, breeders can make more efficient use of plant diversity to develop new cultivars with higher concentrations of these potentially beneficial compounds. Using a large interspecific F1 population, developed from a cross between the temperate V. corymbosum var. ceasariense and the subtropical V. darrowii , with 1025 individuals genotyped using genotype-by-sequencing methods, of which 289 were phenotyped for phenolic acid content, with data collected across 2019 and 2020, we have identified loci associated with phenolic acid content. Loci for the compounds clustered on the proximal arm of Vc02, suggesting that a single gene or several closely associated genes are responsible for the biosynthesis of all four tested compounds. Within this region are multiple gene models similar to hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) and UDP glucose:cinnamate glucosyl transferase (UGCT), genes known to be involved in the CGA biosynthesis pathway. Additional loci on Vc07 and Vc12 were associated with caffeoylarbutin content, suggesting a more complicated biosynthesis of that compound.

Germplasm collections are rich sources of genetic variation to improve crops for many valuable traits. Nested association mapping (NAM) populations can overcome the limitations of genome-wide association studies (GWAS) in germplasm collections by reducing the effect of population structure. We exploited the genetic diversity of the USDA-ARS wheat (Triticum aestivum L.) core collection by developing the Spring Wheat Multiparent Introgression Population (SWMIP). To develop this population, twenty-five core parents were crossed and backcrossed to the Minnesota spring wheat cultivar RB07. The NAM population and 26 founder parents were genotyped using genotyping-by-sequencing and phenotyped for heading date, height, test weight, and grain protein content. After quality control, 20,312 markers with physical map positions were generated for 2,038 recombinant inbred lines (RILs). The number of RILs in each family varied between 58 and 96. Three GWAS models were utilized for quantitative trait loci (QTL) detection and accounted for known family stratification, genetic kinship, and both covariates. GWAS was performed on the whole population and also by bootstrap sampling of an equal number of RILs from each family. Greater power of QTL detection was achieved by treating families equally through bootstrapping. In total 16, 15, 12, and 13 marker-trait associations (MTAs) were identified for heading date, height, test weight, and grain protein content, respectively. Some of these MTAs were coincident with major genes known to control the traits, but others were novel and contributed by the wheat core parents. The SWMIP will be a valuable source of genetic variation for spring wheat breeding., (© 2020 The Authors. The Plant Genome published by Wiley Periodicals, Inc. on behalf of Crop Science Society of America.)

The β-ketoacyl-[acyl carrier protein] synthase 1 ( KASI ) gene has been shown in model plant systems to be critical for the conversion of sucrose to oil. A previous study characterized the morphological and seed composition phenotypes associated with a reciprocal chromosomal translocation that disrupted one of the KASI genes in soybean. The principle findings of this work included a wrinkled seed phenotype, an increase in seed sucrose, a decrease in seed oil, and a low frequency of transmission of the translocation. However, it remained unclear which, if any, of these phenotypes were directly caused by the loss of KASI gene function, as opposed to the chromosomal translocation or other associated factors. In this study, CRISPR/Cas9 mutagenesis was used to generate multiple knockout alleles for this gene, and also one in-frame allele. These soybean plants were evaluated for morphology, seed composition traits, and genetic transmission. Our results indicate that the CRISPR/Cas9 mutants exhibited the same phenotypes as the chromosomal translocation mutant, validating that the observed phenotypes are caused by the loss of gene function. Furthermore, the plants harboring homozygous in-frame mutations exhibited similar phenotypes compared to the plants harboring homozygous knockout mutations. This result indicates that the amino acids lost in the in-frame mutant are essential for proper gene function. In-frame edits for this gene may need to target less essential and/or evolutionarily conserved domains in order to generate novel seed composition phenotypes., (Copyright © 2020 Virdi, Spencer, Stec, Xiong, Merry, Muehlbauer and Stupar.)

Barley ( Hordeum vulgare ssp. vulgare ) is cultivated from the equator to the Arctic Circle. The wild progenitor species, Hordeum vulgare ssp. spontaneum , occupies a relatively narrow latitudinal range (∼30 - 40° N) primarily at low elevation (< 1,500 m). Adaptation to the range of cultivation has occurred over ∼8,000 years. The genetic basis of adaptation is amenable to study through environmental association. An advantage of environmental association in a well-characterized crop is that many loci that contribute to climatic adaptation and abiotic stress tolerance have already been identified. This provides the opportunity to determine if environmental association approaches effectively identify these loci of large effect. Using published genotyping from 7,864 SNPs in 803 barley landraces, we examined allele frequency differentiation across multiple partitions of the data and mixed model associations relative to bioclimatic variables. Using newly generated resequencing data from a subset of these landraces, we tested for linkage disequilibrium (LD) between SNPs queried in genotyping and SNPs in neighboring loci. Six loci previously reported to contribute to adaptive differences in flowering time and abiotic stress in barley and six loci previously identified in other plant species were identified in our analyses. In many cases, patterns of LD are consistent with the causative variant occurring in the immediate vicinity of the queried SNP. The identification of barley orthologs to well-characterized genes may provide a new understanding of the nature of adaptive variation and could permit a more targeted use of potentially adaptive variants in barley breeding and germplasm improvement., (Copyright © 2019 Lei et al.)

The explosion of RNA-Seq data has enabled the identification of expressed genes without relying on gene models with biases toward open reading frames, allowing the identification of many more long noncoding RNAs (lncRNAs) in eukaryotes. Various tissue enrichment strategies and deep sequencing have also enabled the identification of an extensive list of genes expressed in maize gametophytes, tissues that are intractable to both traditional genetic and gene expression analyses. However, the function of very few genes from the lncRNA and gametophyte sets (or from their intersection) has been tested. Methods for isolating and identifying lncRNAs from gametophyte samples of maize are described here. This method is transferable to any maize gametophyte mutant enabling the development of gene networks involving both protein-coding genes and lncRNAs. Additionally, these methods can be adapted to apply to other grass model systems to test for evolutionary conservation of lncRNA expression patterns.

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. 'Morex' was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent in Asia but co-exist with deoxynivalenol producers in lower frequency in North America and Europe. Previous studies identified a barley UDP-glucosyltransferase, HvUGT13248, that efficiently detoxifies deoxynivalenol, and when expressed in transgenic wheat results in high levels of type II resistance against deoxynivalenol-producing F. graminearum. Here we show that HvUGT13248 is also capable of converting nivalenol into the non-toxic nivalenol-3-O-β-d-glucoside. We describe the enzymatic preparation of a nivalenol-glucoside standard and its use in development of an analytical method to detect the nivalenol-glucoside conjugate. Recombinant Escherichia coli expressing HvUGT13248 glycosylates nivalenol more efficiently than deoxynivalenol. Overexpression in yeast, Arabidopsis thaliana, and wheat leads to increased nivalenol resistance. Increased ability to convert nivalenol to nivalenol-glucoside was observed in transgenic wheat, which also exhibits type II resistance to a nivalenol-producing F. graminearum strain. Our results demonstrate the HvUGT13248 can act to detoxify deoxynivalenol and nivalenol and provide resistance to deoxynivalenol- and nivalenol-producing Fusarium., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Key Message: Three adjacent and distinct sequence rearrangements were identified at a NAP1 locus in a soybean mutant. Genetic dissection and validation revealed the function of this gene in soybean trichome development. A soybean (Glycine max (L.) Merr.) gnarled trichome mutant, exhibiting stunted trichomes compared to wild-type, was identified in a fast neutron mutant population. Genetic mapping using whole genome sequencing-based bulked segregant analysis identified a 26.6 megabase interval on chromosome 20 that co-segregated with the phenotype. Comparative genomic hybridization analysis of the mutant indicated that the chromosome 20 interval included a small structural variant within the coding region of a soybean ortholog (Glyma.20G019300) of Arabidopsis Nck-Associated Protein 1 (NAP1), a regulator of actin nucleation during trichome morphogenesis. Sequence analysis of the candidate allele revealed multiple rearrangements within the coding region, including two deletions (approximately 1-2 kb each), a translocation, and an inversion. Further analyses revealed that the mutant allele perfectly co-segregated with the phenotype, and a wild-type soybean NAP1 transgene functionally complemented an Arabidopsis nap1 mutant. In addition, mapping and exon sequencing of NAP1 in a spontaneous soybean gnarled trichome mutant (T31) identified a frame shift mutation resulting in a truncation of the coding region. These data indicate that the soybean NAP1 gene is essential for proper trichome development and show the utility of the soybean fast neutron population for forward genetic approaches for identifying genes.

head blight (FHB) is a disease caused predominantly by the fungal pathogen that affects wheat and other small-grain cereals and can lead to severe yield loss and reduction in grain quality. Trichothecene mycotoxins, such as deoxynivalenol (DON), accumulate during infection and increase pathogen virulence and decrease grain quality. The locus on wheat chromosome 3BS confers Type II resistance to FHB and resistance to the spread of infection on the spike and has been associated with resistance to DON accumulation. To gain a better genetic understanding of the functional role of and resistance or susceptibility to FHB, we examined DON and ergosterol accumulation, FHB resistance, and the whole-genome transcriptomic response using RNA-seq in a near-isogenic line (NIL) pair carrying the resistant and susceptible alleles for during infection and DON treatment. Our results provide a gene expression atlas for the resistant and susceptible wheat- interaction. The DON concentration and transcriptomic results show that the rachis is a key location for conferring Type II resistance. In addition, the wheat transcriptome analysis revealed a set of -responsive genes that may play a role in resistance and a set of DON-responsive genes that may play a role in trichothecene resistance. Transcriptomic results from the pathogen show that the genome responds differently to the host level of resistance. The results of this study extend our understanding of host and pathogen responses in the wheat- interaction., (Copyright © 2016 Crop Science Society of America.)

Barley was introduced to North America ∼400 yr ago but adaptation to modern production environments is more recent. Comparisons of allele frequencies among growth habits and spike (inflorescence) types in North America indicate that significant genetic differentiation has accumulated in a relatively short evolutionary time span. Allele frequency differentiation is greatest among barley with two-row vs. six-row spikes, followed by spring vs. winter growth habit. Large changes in allele frequency among breeding programs suggest a major contribution of genetic drift and linked selection on genetic variation. Despite this, comparisons of 3613 modern North American cultivated barley breeding lines that differ for spike-type and growth habit permit the discovery of 142 single nucleotide polymorphism (SNP) outliers putatively linked to targets of selection. For example, SNPs within the Cbf4, Ppd-H1, and Vrn-H1 loci, which have previously been associated with agronomically adaptive phenotypes, are identified as outliers. Analysis of extended haplotype sharing identifies genomic regions shared within and among breeding populations, suggestive of a number of genomic regions subject to recent selection. Finally, we are able to identify recent bouts of gene flow between breeding populations that could point to the sharing of agronomically adaptive variation. These results are supported by pedigrees and breeders' understanding of germplasm sharing., (Copyright © 2016 Poets et al.)

The shoot apical meristem contains a pool of undifferentiated stem cells and controls initiation of all aerial plant organs. In maize (Zea mays), leaves are formed throughout vegetative development; on transition to floral development, the shoot meristem forms the tassel. Due to the regulated balance between stem cell maintenance and organogenesis, the structure and morphology of the shoot meristem are constrained during vegetative development. Previous work identified loci controlling meristem architecture in a recombinant inbred line population. The study presented here expanded on this by investigating shoot apical meristem morphology across a diverse set of maize inbred lines. Crosses of these lines to common parents showed varying phenotypic expression in the F1, with some form of heterosis occasionally observed. An investigation of meristematic growth throughout vegetative development in diverse lines linked the timing of reproductive transition to flowering time. Phenotypic correlations of meristem morphology with adult plant traits showed an association between the meristem and flowering time, leaf shape, and yield traits, revealing links between the control and architecture of undifferentiated and differentiated plant organs. Finally, quantitative trait loci mapping was utilized to map the genetic architecture of these meristem traits in two divergent populations. Control of meristem architecture was mainly population-specific, with 15 total unique loci mapped across the two populations with only one locus identified in both populations., (Copyright © 2015 Thompson et al.)

Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.

The shoot apical meristem contains a pool of undifferentiated stem cells and generates all above-ground organs of the plant. During vegetative growth, cells differentiate from the meristem to initiate leaves while the pool of meristematic cells is preserved; this balance is determined in part by genetic regulatory mechanisms. To assess vegetative meristem growth and genetic control in Zea mays, we investigated its morphology at multiple time points and identified three stages of growth. We measured meristem height, width, plastochron internode length, and associated traits from 86 individuals of the intermated B73 × Mo17 recombinant inbred line population. For meristem height-related traits, the parents exhibited markedly different phenotypes, with B73 being very tall, Mo17 short, and the population distributed between. In the outer cell layer, differences appeared to be related to number of cells rather than cell size. In contrast, B73 and Mo17 were similar in meristem width traits and plastochron internode length, with transgressive segregation in the population. Multiple loci (6-9 for each trait) were mapped, indicating meristem architecture is controlled by many regions; none of these coincided with previously described mutants impacting meristem development. Major loci for height and width explaining 16% and 19% of the variation were identified on chromosomes 5 and 8, respectively. Significant loci for related traits frequently coincided, whereas those for unrelated traits did not overlap. With the use of three near-isogenic lines, a locus explaining 16% of the parental variation in meristem height was validated. Published expression data were leveraged to identify candidate genes in significant regions., (Copyright © 2014 Thompson et al.)

Genetic differentiation in natural populations is driven by geographic distance and by ecological or physical features within and between natural habitats that reduce migration. The primary population structure in wild barley differentiates populations east and west of the Zagros Mountains. Genetic differentiation between eastern and western populations is uneven across the genome and is greatest on linkage groups 2H and 5H. Genetic markers in these two regions demonstrate the largest difference in frequency between the primary populations and have the highest informativeness for assignment to each population. Previous cytological and genetic studies suggest there are chromosomal structural rearrangements (inversions or translocations) in these genomic regions. Environmental association analyses identified an association with both temperature and precipitation variables on 2H and with precipitation variables on 5H., (Copyright © 2014 Fang et al.)

Background: Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown., Results: By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors., Conclusions: We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping.

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Fusarium head blight (FHB) is a threat to barley (Hordeum vulgare L.) production in many parts of the world. A number of barley accessions with partial resistance have been reported and used in mapping experiments to identify quantitative trait loci (QTL) associated with FHB resistance. Here, we present a set of barley germplasm that exhibits FHB resistance identified through screening a global collection of 23,255 wild (Hordeum vulgare ssp. spontaneum) and cultivated (Hordeum vulgare ssp. vulgare) accessions. Seventy-eight accessions were classified as resistant or moderately resistant. The collection of FHB resistant accessions consists of 5, 27, 46 of winter, wild and spring barley, respectively. The population structure and genetic relationships of the germplasm were investigated with 1,727 Diversity Array Technology (DArT) markers. Multiple clustering analyses suggest the presence of four subpopulations. Within cultivated barley, substructure is largely centered on spike morphology and growth habit. Analysis of molecular variance indicated highly significant genetic variance among clusters and within clusters, suggesting that the FHB resistant sources have broad genetic diversity. The haplotype diversity was characterized with DArT markers associated with the four FHB QTLs on chromosome 2H bin8, 10 and 13 and 6H bin7. In general, the wild barley accessions had distinct haplotypes from those of cultivated barley. The haplotype of the resistant source Chevron was the most prevalent in all four QTL regions, followed by those of the resistant sources Fredrickson and CIho4196. These resistant QTL haplotypes were rare in the susceptible cultivars and accessions grown in the upper Midwest USA. Some two- and six-rowed accessions were identified with high FHB resistance, but contained distinct haplotypes at FHB QTLs from known resistance sources. These germplasm warrant further genetic studies and possible incorporation into barley breeding programs.

Genome-wide association studies (GWAS) provide an opportunity to examine the genetic architecture of quantitatively inherited traits in breeding populations. The objectives of this study were to use GWAS to identify chromosome regions governing traits of importance in six-rowed winter barley (Hordeum vulgare L.) germplasm and to identify single-nucleotide polymorphisms (SNPs) markers that can be implemented in a marker-assisted breeding program. Advanced hulled and hulless lines (329 total) were screened using 3,072 SNPs as a part of the US. Barley Coordinated Agricultural Project (CAP). Phenotypic data collected over 4 years for agronomic and food quality traits and resistance to leaf rust (caused by Puccinia hordei G. Otth), powdery mildew [caused by Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal], net blotch (caused by Pyrenophora teres), and spot blotch [caused by Cochliobolus sativus (Ito and Kuribayashi) Drechsler ex Dastur] were analyzed with SNP genotypic data in a GWAS to determine marker-trait associations. Significant SNPs associated with previously described quantitative trait loci (QTL) or genes were identified for heading date on chromosome 3H, test weight on 2H, yield on 7H, grain protein on 5H, polyphenol oxidase activity on 2H and resistance to leaf rust on 2H and 3H, powdery mildew on 1H, 2H and 4H, net blotch on 5H, and spot blotch on 7H. Novel QTL also were identified for agronomic, quality, and disease resistance traits. These SNP-trait associations provide the opportunity to directly select for QTL contributing to multiple traits in breeding programs.

Vegetative axillary meristem (AXM) activity results in the production of branches. In barley (Hordeum vulgare L.), vegetative AXM develop in the crown and give rise to modified branches, referred to as tillers. Mutations in the barley low-tillering mutant uniculm2 block vegetative AXM development and prevent tiller development. The objectives of this work were to examine gene expression in wild-type and cul2 mutant plants, fine map the CUL2 gene, and to examine synteny in the CUL2 region in barley with rice. RNA profiling experiments using two near-isogenic line pairs carrying either the cul2 mutant allele or wild-type CUL2 allele in different genetic backgrounds detected 28 unique gene transcripts exhibiting similar patterns of differential accumulation in both genetic backgrounds, indicating that we have identified key genes impacted by the CUL2 gene. Twenty-four genes had higher abundance in uniculm2 mutant tissues, and nearly half of the annotated genes likely function in stress-response or signal transduction pathways. Genetic mapping identified five co-segregating markers in 1,088 F2 individuals. These markers spanned the centromere region on chromosome 6H, and coincided with a 50-cM region on rice chromosome 2, indicating that it may be difficult to positionally clone CUL2. Taken together, the results revealed stress response and signal transduction pathways that are associated with the CUL2 gene, isolating CUL2 via positional cloning approaches that may be difficult, and the remnants of barley-rice synteny in the CUL2 region.

Over the past two decades many quantitative trait loci (QTL) have been detected; however, very few have been incorporated into breeding programs. The recent development of genome-wide association studies (GWAS) in plants provides the opportunity to detect QTL in germplasm collections such as unstructured populations from breeding programs. The overall goal of the barley Coordinated Agricultural Project was to conduct GWAS with the intent to couple QTL detection and breeding. The basic idea is that breeding programs generate a vast amount of phenotypic data and combined with cheap genotyping it should be possible to use GWAS to detect QTL that would be immediately accessible and used by breeding programs. There are several constraints to using breeding program-derived phenotype data for conducting GWAS namely: limited population size and unbalanced data sets. We chose the highly heritable trait heading date to study these two variables. We examined 766 spring barley breeding lines (panel #1) grown in balanced trials and a subset of 384 spring barley breeding lines (panel #2) grown in balanced and unbalanced trials. In panel #1, we detected three major QTL for heading date that have been detected in previous bi-parental mapping studies. Simulation studies showed that population sizes greater than 384 individuals are required to consistently detect QTL. We also showed that unbalanced data sets from panel #2 can be used to detect the three major QTL. However, unbalanced data sets resulted in an increase in the false-positive rate. Interestingly, one-step analysis performed better than two-step analysis in reducing the false-positive rate. The results of this work show that it is possible to use phenotypic data from breeding programs to detect QTL, but that careful consideration of population size and experimental design are required.

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, reduces grain yield and quality in barley. Resistance to FHB is partial and quantitatively inherited. Previously, major FHB resistant QTL were detected on barley chromosome 2H Bin 8 and 2H Bin 10, and another QTL for reduced deoxynivalenol (DON) accumulation was identified on chromosome 3H Bin 6. To develop an understanding of the molecular responses controlled by these loci, we examined DON and fungal biomass levels and the transcriptome differences in near-isogenic line (NIL) pairs carrying contrasting resistant and susceptible alleles at these QTL during F. graminearum infection. No overlap was found among the differentially accumulated transcripts of the three NIL pairs, indicating that the response to infection controlled by the resistance alleles at each QTL may be distinct. Transcripts showing differential accumulation between resistant and susceptible NILs were compared to results from previous wheat/barley-F. graminearum studies and integrated into a wheat/barley-F. graminearum interaction model.

Barley (Hordeum vulgare L.) carrying recessive mutations at the Low number of tillers1 (Lnt1) gene does not develop secondary tillers and only develops one to four tillers by maturity. Double mutant analysis determined that the lnt1 mutant was epistatic to five of the six low and high tillering mutants tested. Double mutants of lnt1 and the low tillering mutant intermedium-b (int-b) resulted in a uniculm plant, indicating a synergistic interaction and that Lnt and Int-b function in separate tillering pathways. RNA profiling identified 70 transcripts with either increased or decreased abundance in the lnt1 mutant compared to wild-type. One gene with reduced transcript levels in the lnt1 mutant was the BELL-like homeodomain transcription factor JuBel2. The JuBel2 allele in the lnt1.a mutant contained a frameshift mutation that eliminated most of the predicted polypeptide, indicating that the Lnt1 gene encodes JuBel2. Previous studies with the low-tillering mutant absent lower laterals (als) showed that the tillering phenotypes and genetic interactions of als and lnt1 with other tillering mutants were very similar. However, the transcriptomes were very different; many transcripts annotated as stress and defense response exhibited increased abundance in the als mutant. This difference suggests a functional separation between Als and Lnt1 in the genetic control of tillering.

Background: High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource., Results: Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM., Conclusion: The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material.

Barley (Hordeum vulgare L.) carrying the recessive mutation absent lower laterals (als) exhibits few tillers and irregular inflorescence development. To gain an increased understanding of the genetic control of tillering in barley, we conducted morphological, genetic, and transcriptome analysis of the als mutant. Axillary buds for primary tillers, but not for secondary tillers, developed in als plants. Double mutant combinations of als with one low-tillering and four high-tillering mutants resulted in a tillering phenotype similar to als, indicating that als was epistatic to these tillering genes. However, double mutant combinations of als with another low-tillering mutant, intermedium-b, reduced tiller numbers, indicating there were at least two genetic pathways regulating tillering in barley. Next, we used simple sequence repeat markers to map the Als gene on the long arm of barley chromosome 3H, Bin 11. Finally, the Affymetrix Barley1 GeneChip was used to identify differentially accumulated transcripts in als compared to wild-type. Forty percent of the transcripts with twofold or greater accumulation in als tissues corresponded to stress and defense response genes. This finding suggested that a tillering pathway may modulate the stress response.

The number of viable shoots influences the overall architecture and productivity of wheat (Triticum aestivum L.). The development of lateral branches, or tillers, largely determines the resultant canopy. Tillers develop from the outgrowth of axillary buds, which form in leaf axils at the crown of the plant. Tiller number can be reduced if axillary buds are not formed or if the outgrowth of these buds is restricted. The teosinte branched1 (tb1) gene in maize, and homologs in rice and Arabidopsis, genetically regulate vegetative branching. In maize, increased expression of the tb1 gene restricts the outgrowth of axillary buds into lateral branches. In this study, the maize tb1 gene was introduced through transformation into the wheat cultivar "Bobwhite" to determine the effect of tb1 overexpression on wheat shoot architecture. Examination of multiple generations of plants reveals that tb1 overexpression in wheat results in reduced tiller and spike number. In addition, the number of spikelets on the spike and leaf number were significantly greater in tb1-expressing plants, and the height of these plants was also reduced. These data reveal that the function of the tb1 gene and genetic regulation of lateral branching via the tb1 mode of action is conserved between wheat, rice, maize and Arabidopsis. Thus, the tb1 gene can be used to alter plant architecture in agriculturally important crops like wheat.

Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes alpha-1-purothionin, thaumatin-like protein 1 (tlp-1), and beta-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the alpha-1-purothionin, tlp-1, and beta-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A beta-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.

Transposable elements are ubiquitous genomic parasites with an ancient history of coexistence with their hosts. A few cases have emerged recently where these genetic elements have been recruited for normal function in the host organism. We have identified an expressed hobo/Ac/Tam (hAT) family transposase-like gene in cereal grasses which appears to represent such a case. This gene, which we have called gary, is found in one or two copies in barley, two diverged copies in rice and two very similar copies in hexaploid wheat. No gary homologues are found in Arabidopsis. In all three cereal species, an apparently complete 2.5 kb transposase-like open reading frame is present and nucleotide substitution data show evidence for positive selection, yet the predicted gary protein is probably not an active transposase, as judged by the absence of key amino acids required for transposase function. Gary is expressed in wheat and barley spikes and gary cDNA sequences are also found in rice, oat, rye, maize, sorghum and sugarcane. The short inverted terminal repeats, flanked by an eight-nucleotide host sequence duplication, which are characteristic of a hAT transposon are absent. Genetic mapping in barley shows that gary is located on the distal end of the long arm of chromosome 2H. Wheat homologues of gary map to the same approximate location on the wheat group 2 chromosomes by physical bin-mapping and the more closely related of the two rice garys maps to the syntenic location near the bottom of rice chromosome 4. These data suggest that gary has resided in a single genomic location for at least 60 Myr and has lost the ability to transpose, yet expresses a transposase-related protein that is being conserved under host selection. We propose that the gary transposase-like gene has been recruited by the cereal grasses for an unknown function.

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a major disease problem in wheat (Triticum aestivum). Genetic engineering holds significant potential to enhance FHB resistance in wheat. Due to the requirement of screening for FHB resistance on flowers at anthesis, the number of screens carried out in a year is limited. Our objective was to evaluate the feasibility of using the rapid-maturing dwarf wheat cultivar Apogee as an alternative genotype for transgenic FHB resistance research. Our transformation efficiency (number of transgenic plants/number of embryos) for Apogee was 1.33%. Apogee was also found to exhibit high FHB susceptibility and reached anthesis within 4 weeks. Interestingly, microsatellite marker haplotype analysis of the chromosome 3BS FHB resistant quantitative trait locus (QTL) region indicated that this region maybe deleted in Apogee. Our results indicate that Apogee is particularly well suited for accelerating transgenic FHB resistance research and transgenic wheat research in general.