Genotype and Environment Affect the Grain Quality and Yield of Winter Oats (Avena sativa L.).

The extent to which the quality and yield of plant varieties are influenced by the environment is important for their successful uptake by end users particularly as climatic fluctuations are resulting in environments that are highly variable from one growing season to another. The genotype-by-environment interaction (GEI) of milling quality and yield was studied using four winter oat varieties in multi-locational trials over 4 years in the U.K. Significant differences across the 22 environments were found between physical grain quality and composition as well as grain yield, with the environment having a significant effect on all of the traits measured. Grain yield was closely related to grain number m-2 whereas milling quality traits were related to grain size attributes. Considerable genotype by environment interaction was obtained for all grain quality traits and stability analysis revealed that the variety Mascani was the least sensitive to the environment for all milling quality traits measured whereas the variety Balado was the most sensitive. Examination of environmental conditions at specific within-year stages of crop development indicated that both temperature and rainfall during grain development were correlated with grain yield and ß-glucan content and with the ease of removing the hull (hullability).

Subtelomeric assembly of a multi-gene pathway for antimicrobial defense compounds in cereals.

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat-the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a 'self-poisoning' scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.

A noncanonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat.

Plants produce an array of natural products with important ecological functions. These compounds are often decorated with oligosaccharide groups that influence bioactivity, but the biosynthesis of such sugar chains is not well understood. Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits, as exemplified by avenacins, antimicrobial defense compounds produced by oats. Avenacins have a branched trisaccharide moiety consisting of l-arabinose linked to 2 d-glucose molecules that is critical for antifungal activity. Plant natural product glycosylation is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). We previously characterized the arabinosyltransferase that initiates the avenacin sugar chain; however, the enzymes that add the 2 remaining d-glucose molecules have remained elusive. Here we characterize the enzymes that catalyze these last 2 glucosylation steps. AsUGT91G16 is a classical cytosolic UGT that adds a 1,2-linked d-glucose molecule to l-arabinose. Unexpectedly, the enzyme that adds the final 1,4-linked d-glucose (AsTG1) is not a UGT, but rather a sugar transferase belonging to Glycosyl Hydrolase family 1 (GH1). Unlike classical UGTs, AsTG1 is vacuolar. Analysis of oat mutants reveals that AsTG1 corresponds to Sad3, a previously uncharacterized locus shown by mutation to be required for avenacin biosynthesis. AsTG1 and AsUGT91G16 form part of the avenacin biosynthetic gene cluster. Our demonstration that a vacuolar transglucosidase family member plays a critical role in triterpene biosynthesis highlights the importance of considering other classes of carbohydrate-active enzymes in addition to UGTs as candidates when elucidating pathways for the biosynthesis of glycosylated natural products in plants.

Genomic insights from the first chromosome-scale assemblies of oat (Avena spp.) diploid species.

BACKGROUND: Cultivated hexaploid oat (Common oat; Avena sativa) has held a significant place within the global crop community for centuries; although its cultivation has decreased over the past century, its nutritional benefits have garnered increased interest for human consumption. We report the development of fully annotated, chromosome-scale assemblies for the extant progenitor species of the As- and Cp-subgenomes, Avena atlantica and Avena eriantha respectively. The diploid Avena species serve as important genetic resources for improving common oat's adaptive and food quality characteristics. RESULTS: The A. atlantica and A. eriantha genome assemblies span 3.69 and 3.78 Gb with an N50 of 513 and 535 Mb, respectively. Annotation of the genomes, using sequenced transcriptomes, identified ~ 50,000 gene models in each species-including 2965 resistance gene analogs across both species. Analysis of these assemblies classified much of each genome as repetitive sequence (~ 83%), including species-specific, centromeric-specific, and telomeric-specific repeats. LTR retrotransposons make up most of the classified elements. Genome-wide syntenic comparisons with other members of the Pooideae revealed orthologous relationships, while comparisons with genetic maps from common oat clarified subgenome origins for each of the 21 hexaploid linkage groups. The utility of the diploid genomes was demonstrated by identifying putative candidate genes for flowering time (HD3A) and crown rust resistance (Pc91). We also investigate the phylogenetic relationships among other A- and C-genome Avena species. CONCLUSIONS: The genomes we report here are the first chromosome-scale assemblies for the tribe Poeae, subtribe Aveninae. Our analyses provide important insight into the evolution and complexity of common hexaploid oat, including subgenome origin, homoeologous relationships, and major intra- and intergenomic rearrangements. They also provide the annotation framework needed to accelerate gene discovery and plant breeding.

Detached Leaf Assays for Resistance to Crown Rust Reveal Diversity Within Populations of Avena sterilis.

Crown rust is the most widespread and damaging disease of oat (Avena species). Genetic resistance to the pathogen is the preferred method for crop protection but widespread deployment of limited numbers of major effect genes has promoted the rapid emergence and spread of pathogen races that are able to overcome these genes. Combining genes with even partial resistance may help develop durable cultivars that are less vulnerable to changes in pathogen virulence. Partial resistance is expected to be relatively common in populations of wild species where constant pathogen pressure encourages diversity in host resistance mechanisms, but it may be discarded in conventional screens for major gene resistance. Here, we used a detached leaf assay to detect resistance to the crown rust pathogen, Puccinia coronata Cda. f. sp. avenae, in previously uncharacterized collections of the hexaploid wild oat relative A. sterilis made by the Polish National Centre for Plant Genetic Resources. Many of the accessions were collected in Morocco, the center of diversity for the Avena genus. The detached leaf assessment allowed individual plants to be challenged with multiple pathotypes and their responses compared with 34 known differentials. Broad-spectrum resistance was identified within accession PL 51855, which behaved as a single major locus on crossing to three cultivars. The locus provided resistance to over 50 rust pathotypes, a greater range than seen for any of the known host resistance (Pc) genes. Strong resistance was identified in other accessions, and heterogeneity in response within accessions was common. Several accessions show multiple partial resistance responses that may be of value for developing durable resistance in cultivars. Because the sources of resistance in all but two differential lines were collected outside of Morocco, resistance in all accessions tested here are potentially novel. This study demonstrates that diversity within A. sterilis accessions collected in Morocco could be a very valuable source of resistance to crown rust, and it provides new germplasm for use in resistance breeding programs. Detached leaf assessment provides a valuable first step in the identification of promising candidates in complex gene bank accessions.

Towards take-all control: a C-21ß oxidase required for acylation of triterpene defence compounds in oat.

Oats produce avenacins, antifungal triterpenes that are synthesized in the roots and provide protection against take-all and other soilborne diseases. Avenacins are acylated at the carbon-21 position of the triterpene scaffold, a modification critical for antifungal activity. We have previously characterized several steps in the avenacin pathway, including those required for acylation. However, transfer of the acyl group to the scaffold requires the C-21ß position to be oxidized first, by an as yet uncharacterized enzyme. We mined oat transcriptome data to identify candidate cytochrome P450 enzymes that may catalyse C-21ß oxidation. Candidates were screened for activity by transient expression in Nicotiana benthamiana. We identified a cytochrome P450 enzyme AsCYP72A475 as a triterpene C-21ß hydroxylase, and showed that expression of this enzyme together with early pathway steps yields C-21ß oxidized avenacin intermediates. We further demonstrate that AsCYP72A475 is synonymous with Sad6, a previously uncharacterized locus required for avenacin biosynthesis. sad6 mutants are compromised in avenacin acylation and have enhanced disease susceptibility. The discovery of AsCYP72A475 represents an important advance in the understanding of triterpene biosynthesis and paves the way for engineering the avenacin pathway into wheat and other cereals for control of take-all and other diseases.

Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity.

Glycosylation of small molecules is critical for numerous biological processes in plants, including hormone homeostasis, neutralization of xenobiotics, and synthesis and storage of specialized metabolites. Glycosylation of plant natural products is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits such as disease resistance and flavor and have numerous pharmaceutical applications. Most characterized plant natural product UGTs are glucosyltransferases, and little is known about enzymes that add other sugars. Here we report the discovery and characterization of AsAAT1 (UGT99D1), which is required for biosynthesis of the antifungal saponin avenacin A-1 in oat (Avena strigosa). This enzyme adds l-Ara to the triterpene scaffold at the C-3 position, a modification critical for disease resistance. The only previously reported plant natural product arabinosyltransferase is a flavonoid arabinosyltransferase from Arabidopsis (Arabidopsis thaliana). We show that AsAAT1 has high specificity for UDP-ß-l-arabinopyranose, identify two amino acids required for sugar donor specificity, and through targeted mutagenesis convert AsAAT1 into a glucosyltransferase. We further identify a second arabinosyltransferase potentially implicated in the biosynthesis of saponins that determine bitterness in soybean (Glycine max). Our investigations suggest independent evolution of UDP-Ara sugar donor specificity in arabinosyltransferases in monocots and eudicots.

Multi-Environmental Trials Reveal Genetic Plasticity of Oat Agronomic Traits Associated With Climate Variable Changes.

Although oat cultivation around the Mediterranean basin is steadily increasing, its yield in these regions lags far behind those of Northern Europe. This results mainly from the poor adaptation of current oat cultivars to Mediterranean environments. Local landraces may act as reservoirs of favorable traits that could contribute to increase oat resilience in this region. To aid selection of suitable agro-climate adapted genotypes we integrated genome-wide association approaches with analysis of field assessed phenotypes of genetic variants and of the weight of associated markers across different environmental variables. Association models accounting for oat population structure were applied on either arithmetic means or best linear unbiased prediction (BLUPs) to ensure robust identification of associations with the agronomic traits evaluated. The meta-analysis of the six joint environments (mega-environment) identified several markers associated with several agronomic traits and crown rust severity. Five of these associated markers were located within expressed genes. These associations were only mildly influenced by climatic variables indicating that these markers are good candidates to improve the genetic potential of oat under Mediterranean conditions. The models also highlighted several marker-trait associations, strongly affected by particular climatic variables including high rain pre- or post-heading dates and high temperatures, revealing strong potential for oat adaptation to specific agro-climatic conditions. These results will contribute to increase oat resilience for particular climatic conditions and facilitate breeding for plant adaptation to a wider range of climatic conditions in the current scenario of climate change.

Characteristics of Resistance to Puccinia coronata f. sp. avenae in Avena fatua.

Crown rust, caused by Puccinia coronata f. sp. avenae, is the most widespread and harmful fungal disease of oat. The best defense against the pathogen is use of cultivars with genetic resistance, which is effective, economic, and an environmentally friendly alternative to chemical control. However, the continuous evolution of the pathogen can rapidly overcome major gene resistance, creating an urgent need to identify new sources. Wild oat accessions have already proven to be valuable donors of many resistance genes, but the weed species Avena fatua remains underexploited. Its abundance across multiple environments and the frequent occurrence of herbicide-resistant populations demonstrate its ready ability to adapt to biotic and abiotic stresses; yet, surprisingly, there are no extensive studies which describe crown rust resistance occurrence in gene bank stocks of A. fatua. In this study, 204 accessions of A. fatua maintained in the collections of the United States Department of Agriculture (USDA) and Polish National Centre for Plant Genetic Resources were evaluated at the seedling stage for crown rust reaction using host-pathogen tests with five highly diverse and virulent races of P. coronata. Of tested genotypes, 85% showed a heterogeneous infection pattern, while 61% were susceptible or moderately susceptible to all races. Of the 79 resistant A. fatua accessions, seedling resistance to at least two P. coronata isolates was recognized within 19 accessions, with 13 displaying a homogeneously resistant phenotype to one or two races. Accessions showing multiple single seedling resistance to three or four isolates were observed. Based on the seedling reaction to isolates used in the study, 18 infection profiles (IP) were determined. Using UPGMA clustering, resistant accessions were divided into six main clusters encompassing samples with similar IPs. Twelve of 18 patterns allowed us to postulate the likely presence of novel crown rust resistance genes, whose origin was predominantly from Kenya or Egypt. Future work will clarify the genetic basis of the resistances observed here, as well as confirm their potential utility in breeding resistant oat cultivars.

Expression of a fungal ferulic acid esterase in suspension cultures of tall fescue (Festuca arundinacea) decreases cell wall feruloylation and increases rates of cell wall digestion.

In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase (faeA) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells.

A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement.

Hexaploid oat ( L., 2 = 6 = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice ( L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.

High-density marker profiling confirms ancestral genomes of Avena species and identifies D-genome chromosomes of hexaploid oat.

KEY MESSAGE: Genome analysis of 27 oat species identifies ancestral groups, delineates the D genome, and identifies ancestral origin of 21 mapped chromosomes in hexaploid oat. We investigated genomic relationships among 27 species of the genus Avena using high-density genetic markers revealed by genotyping-by-sequencing (GBS). Two methods of GBS analysis were used: one based on tag-level haplotypes that were previously mapped in cultivated hexaploid oat (A. sativa), and one intended to sample and enumerate tag-level haplotypes originating from all species under investigation. Qualitatively, both methods gave similar predictions regarding the clustering of species and shared ancestral genomes. Furthermore, results were consistent with previous phylogenies of the genus obtained with conventional approaches, supporting the robustness of whole genome GBS analysis. Evidence is presented to justify the final and definitive classification of the tetraploids A. insularis, A. maroccana (=A. magna), and A. murphyi as containing D-plus-C genomes, and not A-plus-C genomes, as is most often specified in past literature. Through electronic painting of the 21 chromosome representations in the hexaploid oat consensus map, we show how the relative frequency of matches between mapped hexaploid-derived haplotypes and AC (DC)-genome tetraploids vs. A- and C-genome diploids can accurately reveal the genome origin of all hexaploid chromosomes, including the approximate positions of inter-genome translocations. Evidence is provided that supports the continued classification of a diverged B genome in AB tetraploids, and it is confirmed that no extant A-genome diploids, including A. canariensis, are similar enough to the D genome of tetraploid and hexaploid oat to warrant consideration as a D-genome diploid.

Functional co-expression of a fungal ferulic acid esterase and a ß-1,4 endoxylanase in Festuca arundinacea (tall fescue) modifies post-harvest cell wall deconstruction.

MAIN CONCLUSION: Improved post-harvest cell wall deconstruction of tall fescue leaves has been demonstrated by in-planta co-expression of a constitutively expressed ferulic acid esterase together with a senescence-induced ß-1,4 endoxylanase. Tall fescue plants (Festuca arundinacea) constitutively expressing vacuole- or apoplast-targeted ferulic acid esterase from Aspergillus niger were retransformed with a senescence-induced and apoplast-targeted ß-1,4 endo-xylanase from Trichoderma reesei. Enzyme activities in co-expressing plants stabilized after repeated vegetative propagation, with xylanase activity in senescent leaves increasing and ferulic acid esterase activity decreasing after tillering. Plants co-expressing both enzymes in the apoplast, with the lowest levels of ferulate monomers and dimers and the lowest levels of cell wall arabinoxylans, released ten times more cell wall hydroxycinnamic acids and five times more arabinoxylan from the cell wall on autodigestion compared to expression of ferulic acid esterase or xylanase alone. These plants also showed a 31 % increase in cellulase-mediated release of reducing sugars, a 5 % point increase in in vitro dry matter digestibility and a 23 % increase in acetyl bromide-soluble lignin. However, plant growth was adversely affected by expressing FAE in the apoplast, giving plants with narrower shorted leaves, and a 71 % decrease in biomass.

Genome-wide association study for crown rust (Puccinia coronata f. sp. avenae) and powdery mildew (Blumeria graminis f. sp. avenae) resistance in an oat (Avena sativa) collection of commercial varieties and landraces.

Diseases caused by crown rust (Puccinia coronata f. sp. avenae) and powdery mildew (Blumeria graminis f. sp. avenae) are among the most important constraints for the oat crop. Breeding for resistance is one of the most effective, economical, and environmentally friendly means to control these diseases. The purpose of this work was to identify elite alleles for rust and powdery mildew resistance in oat by association mapping to aid selection of resistant plants. To this aim, 177 oat accessions including white and red oat cultivars and landraces were evaluated for disease resistance and further genotyped with 31 simple sequence repeat and 15,000 Diversity Arrays Technology (DArT) markers to reveal association with disease resistance traits. After data curation, 1712 polymorphic markers were considered for association analysis. Principal component analysis and a Bayesian clustering approach were applied to infer population structure. Five different general and mixed linear models accounting for population structure and/or kinship corrections and two different statistical tests were carried out to reduce false positive. Five markers, two of them highly significant in all models tested were associated with rust resistance. No strong association between any marker and powdery mildew resistance at the seedling stage was identified. However, one DArT sequence, oPt-5014, was strongly associated with powdery mildew resistance in adult plants. Overall, the markers showing the strongest association in this study provide ideal candidates for further studies and future inclusion in strategies of marker-assisted selection.

Expression of a Trichoderma reesei ß-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses.

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H(2)O(2) production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H(2)O(2).

Evolution and taxonomic split of the model grass Brachypodium distachyon.

BACKGROUND AND AIMS: Brachypodium distachyon is being widely investigated across the world as a model plant for temperate cereals. This annual plant has three cytotypes (2n = 10, 20, 30) that are still regarded as part of a single species. Here, a multidisciplinary study has been conducted on a representative sampling of the three cytotypes to investigate their evolutionary relationships and origins, and to elucidate if they represent separate species. METHODS: Statistical analyses of 15 selected phenotypic traits were conducted in individuals from 36 lines or populations. Cytogenetic analyses were performed through flow cytometry, fluorescence in situ hybridization (FISH) with genomic (GISH) and multiple DNA sequences as probes, and comparative chromosome painting (CCP). Phylogenetic analyses were based on two plastid (ndhF, trnLF) and five nuclear (ITS, ETS, CAL, DGAT, GI) genes from different Brachypodium lineages, whose divergence times and evolutionary rates were estimated. KEY RESULTS: The phenotypic analyses detected significant differences between the three cytotypes and demonstrated stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirmed that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents the allotetraploid derived from them. Phylogenetic analysis demonstrated that the 2n = 20 and 2n = 10 cytotypes emerged from two independent lineages that were, respectively, the maternal and paternal genome donors of the 2n = 30 cytotype. The 2n = 20 lineage was older and mutated significantly faster than the 2n = 10 lineage and all the core perennial Brachypodium species. CONCLUSIONS: The substantial phenotypic, cytogenetic and molecular differences detected among the three B. distachyon sensu lato cytotypes are indicative of major speciation processes within this complex that allow their taxonomic separation into three distinct species. We have kept the name B. distachyon for the 2n = 10 cytotype and have described two novel species as B. stacei and B. hybridum for, respectively, the 2n = 20 and 2n = 30 cytotypes.

Compact genomes and complex evolution in the genus Brachypodium.

The temperate annual grass Brachypodium distachyon is a diploid species with a chromosome base number of 5. It is strikingly different from other Eurasian species of the genus, which are perennial and often polyploid, with the diploids typically having base numbers of 8 or 9. Previously, phylogenies indicated that B. distachyon split from the other species early in the evolution of the genus, while its genome sequence revealed that extensive synteny on a chromosomal scale had been maintained with rice, a tropical grass with a base number of 12. Here we show evidence that B. distachyon may have a homoploid origin, involving ancestral interspecific hybridisation, although it does not appear to be a component of any of the perennial Eurasian allopolyploids. Using a cytogenetic approach, we show that dysploidy in Brachypodium has not followed a simple progression.

Targeting expression of a fungal ferulic acid esterase to the apoplast, endoplasmic reticulum or golgi can disrupt feruloylation of the growing cell wall and increase the biodegradability of tall fescue (Festuca arundinacea).

In the cell walls of grasses, ferulic acid is esterified to arabinoxylans and undergoes oxidative reactions to form ferulates dimers, trimers and oligomers. Feruloylation of arabinoxylan is considered important not only because it leads to cross-linked xylans but also because ferulates may act as a nucleating site for the formation of lignin and hence link arabinoxylans to lignin by forming a lignin-ferulate-arabinoxylan complex. Such cross-linking is among the main factors inhibiting the release of fermentable carbohydrates from grasses either for ruminant nutrition or for biofuel production. We have found that significant reductions in the levels of monomeric and dimeric phenolics can be achieved in the growing cell walls during plant development in leaves of Festuca arundinacea by constitutive intracellular targeted expression of Aspergillus niger ferulic acid esterase (FAEA). We propose that this occurred by directly disrupting ester bonds linking phenolics to cell wall polysaccharides by apoplast targeting or by preventing excessive feruloylation of cell wall carbohydrates prior to their incorporation into the cell wall, by targeting to the Golgi membrane system. Plants with lower cell wall ferulate levels, which showed increased digestibility and increased rates of cellulase-mediated release of fermentable sugars, were identified. Targeting FAE to the Golgi was found to be more effective than targeting to the ER, which supports the current theories of the Golgi as the site of feruloylation of arabinoxylans. It is concluded that targeting FAEA expression to the Golgi or apoplast is likely to be an effective strategy for improving wall digestibility in grass species used for fodder or cellulosic ethanol production.

Fragments of the key flowering gene GIGANTEA are associated with helitron-type sequences in the Pooideae grass Lolium perenne.

BACKGROUND: Helitrons are a class of transposable elements which have been identified in a number of species of plants, animals and fungi. They are unique in their proposed rolling-circle mode of replication, have a highly variable copy-number and have been implicated in the restructuring of coding sequences both by their insertion into existing genes and by their incorporation of transcriptionally competent gene fragments. Helitron discovery depends on identifying associated DNA signature sequences and comprehensive evaluation of helitron contribution to a particular genome requires detailed computational analysis of whole genome sequence. Therefore, the role which helitrons have played in modelling non-model plant genomes is largely unknown. RESULTS: Cloning of the flowering gene GIGANTEA (GI) from a BAC library of the Pooideae grass Lolium perenne (perennial ryegrass) identified the target gene and several GI pseudogene fragments spanning the first five exons. Analysis of genomic sequence 5' and 3' of one these GI fragments revealed motifs consistent with helitron-type transposon insertion, specifically a putative 5'-A (downward arrow) T-3' insertion site containing 5'-TC and CTAG-3' borders with a sub-terminal 16 bp hairpin. Screening of a BAC library of the closely related grass species Festuca pratensis (meadow fescue) indicated similar helitron-associated GI fragments present in this genome, as well as non-helitron associated GI fragments derived from the same region of GI. In order to investigate the possible extent of ancestral helitron-activity in L. perenne, a methylation-filtered GeneThresher genomic library developed from this species was screened for potential helitron 3' hairpin sequences associated with a 3'-CTRR motif. This identified 7 potential helitron hairpin-types present between at least 9 and 51 times within the L. perenne methylation-filtered library. CONCLUSION: This represents evidence for a possible ancestral role for helitrons in modelling the genomes of Lolium and related species.

New DArT markers for oat provide enhanced map coverage and global germplasm characterization.

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.