Genome-wide epigenetic modifications in sports horses during training as an adaptation phenomenon.

With his bicentennial breeding history based on athletic performance, the Thoroughbred horse can be considered the equine sport breed. Although genomic and transcriptomic tools and knowledge are at the state of the art in equine species, the epigenome and its modifications in response to environmental stimuli, such as training, are less studied. One of the major epigenetic modifications is cytosine methylation at 5' of DNA molecules. This crucial biochemical modification directly mediates biological processes and, to some extent, determines the organisms' phenotypic plasticity. Exercise indeed affects the epigenomic state, both in humans and in horses. In this study, we highlight, with a genome-wide analysis of methylation, how the adaptation to training in the Thoroughbred can modify the methylation pattern throughout the genome. Twenty untrained horses, kept under the same environmental conditions and sprint training regimen, were recruited, collecting peripheral blood at the start of the training and after 30 and 90 days. Extracted leukocyte DNA was analyzed with the methylation content sensitive enzyme ddRAD (MCSeEd) technique for the first time applied to animal cells. Approximately one thousand differently methylated genomic regions (DMRs) and nearby genes were called, revealing that methylation changes can be found in a large part of the genome and, therefore, referable to the physiological adaptation to training. Functional analysis via GO enrichment was also performed. We observed significant differences in methylation patterns throughout the training stages: we hypothesize that the methylation profile of some genes can be affected early by training, while others require a more persistent stimulus.

The Application of High-Resolution Melting Analysis to trnL (UAA) Intron Allowed a Qualitative Identification of Apple Juice Adulterations.

Food authenticity plays a pivotal role in the modern age since an increased consumers awareness has led them to pay more attention to food commodities. For this reason, it is important to have reliable and fast techniques able to detect possible adulterations in food, which affect qualitative and economic value. Therefore, the aim of this study was to detect possible adulterations in apple juice from others fruit species (i.e., pear, peach, and kiwi) combining DNA barcoding approach, using trnL (UAA) intron, with high resolution melting analysis (HRMA). A preliminary phylogenetic analysis, using sequences retrieved by the GenBank, confirmed the discriminatory power of trnL (UAA) intron among the four fruit species examined. Moreover, the sequencing of the trnL (UAA) fragments obtained from apple, pear, peach, and kiwi, demonstrated the suitability of an inner shorter sequence, P6 loop, to differentiate the considered species. The HRMA coupled with trnL (UAA) intron allowed discrimination among the four fruits but provided incomplete results for juices. Whereas the HRMA targeting the P6 loop amplicons confirmed the suitability of the technique to qualitatively distinguish fruit juices composed by the combination of apple/pear and apple/peach. However, the impossibility of discriminating apple/kiwi juices from the pure kiwi sample highlighted limitations, most likely related to the DNA extraction process. This hypothesis was further confirmed by analyzing DNA blends obtained by combining nucleic acids extracted from pure matrixes (i.e., apple and kiwi fruits). In this specific case, the application of HRMA allowed both qualitative and quantitative assessment of the samples.

Genome-Wide Identification and Spatial Expression Analysis of Histone Modification Gene Families in the Rubber Dandelion Taraxacum kok-saghyz.

Taraxacum kok-saghyz (Tks), also known as the Russian dandelion, is a recognized alternative source of natural rubber quite comparable, for quality and use, to the one obtained from the so-called rubber tree, Hevea brasiliensis. In addition to that, Tks roots produce several other compounds, including inulin, whose use in pharmaceutical and dietary products is quite extensive. Histone-modifying genes (HMGs) catalyze a series of post-translational modifications that affect chromatin organization and conformation, which, in turn, regulate many downstream processes, including gene expression. In this study, we present the first analysis of HMGs in Tks. Altogether, we identified 154 putative Tks homologs: 60 HMTs, 34 HDMs, 42 HATs, and 18 HDACs. Interestingly, whilst most of the classes showed similar numbers in other plant species, including M. truncatula and A. thaliana, HATs and HMT-PRMTs were indeed more abundant in Tks. Composition and structure analysis of Tks HMG proteins showed, for some classes, the presence of novel domains, suggesting a divergence from the canonical HMG model. The analysis of publicly available transcriptome datasets, combined with spatial expression of different developmental tissues, allowed us to identify several HMGs with a putative role in metabolite biosynthesis. Overall, our work describes HMG genomic organization and sets the premises for the functional characterization of epigenetic modifications in rubber-producing plants.

Legumes of the Sardinia Island: Knowledge on Symbiotic and Endophytic Bacteria and Interactive Software Tool for Plant Species Determination.

A meta-analysis was carried out on published literature covering the topic of interactive plant microbiology for botanical species of legumes occurring within the boundary of the Italian island Sardinia, lying between the Tyrrhenian and the western Mediterranean seas. Reports were screened for the description of three types of bacterial occurrences; namely, (a) the nitrogen-fixing symbionts dwelling in root nodules; (b) other bacteria co-hosted in nodules but having the ancillary nature of endophytes; (c) other endophytes isolated from different non-nodular portions of the legume plants. For 105 plant species or subspecies, over a total of 290 valid taxonomical descriptions of bacteria belonging to either one or more of these three categories were found, yielding 85 taxa of symbionts, 142 taxa of endophytes in nodules, and 33 in other plant parts. The most frequent cases were within the Medicago, Trifolium, Lotus, Phaseolus, and Vicia genera, the majority of symbionts belonged to the Rhizobium, Mesorhizobium, Bradyrhizobium, and Sinorhizobium taxa. Both nodular and extra-nodular endophytes were highly represented by Gammaproteobacteria (Pseudomonas, Enterobacter, Pantoea) and Firmicutes (Bacillus, Paenibacillus), along with a surprisingly high diversity of the Actinobacteria genus Micromonospora. The most plant-promiscuous bacteria were Sinorhizobium meliloti as symbiont and Bacillus megaterium as endophyte. In addition to the microbial analyses we introduce a practical user-friendly software tool for plant taxonomy determination working in a Microsoft Excel spreadsheet that we have purposely elaborated for the classification of legume species of Sardinia. Its principle is based on subtractive keys that progressively filter off the plants that do not comply with the observed features, eventually leaving only the name of the specimen under examination.

Functional Characterization of MtrGSTF7, a Glutathione S-Transferase Essential for Anthocyanin Accumulation in Medicago truncatula.

Flavonoids are essential compounds widespread in plants and exert many functions such as defence, definition of organ colour and protection against stresses. In Medicago truncatula, flavonoid biosynthesis and accumulation is finely regulated in terms of tissue specificity and induction by external factors, such as cold and other stresses. Among flavonoids, anthocyanin precursors are synthesised in the cytoplasm, transported to the tonoplast, then imported into the vacuole for further modifications and storage. In the present work, we functionally characterised MtrGSTF7, a phi-class glutathione S-transferase involved in anthocyanin transport to the tonoplast. The mtrgstf7 mutant completely lost the ability to accumulate anthocyanins in leaves both under control and anthocyanin inductive conditions. On the contrary, this mutant showed an increase in the levels of soluble proanthocyanidins (Pas) in their seeds with respect to the wild type. By complementation and expression data analysis, we showed that, differently from A. thaliana and similarly to V. vinifera, transport of anthocyanin and proanthocyanidins is likely carried out by different GSTs belonging to the phi-class. Such functional diversification likely results from the plant need to finely tune the accumulation of diverse classes of flavonoids according to the target organs and developmental stages.

Genome-Wide Identification of Histone Modification Gene Families in the Model Legume Medicago truncatula and Their Expression Analysis in Nodules.

Histone methylation and acetylation are key processes in the epigenetic regulation of plant growth, development, and responses to environmental stimuli. The genes encoding for the enzymes that are responsible for these chromatin post-translational modifications, referred to as histone modification genes (HMGs), have been poorly investigated in Leguminosae species, despite their importance for establishment and activity of nitrogen-fixing nodules. In silico analysis of Medicago truncatula HMGs identified 81 histone methyltransferases, 46 histone demethylases, 64 histone acetyltransferases, and 15 histone deacetylases. MtHMGs were analyzed for their structure and domain composition, and some combinations that were not yet reported in other plant species were identified. Genes have been retrieved from M. truncatula A17 and R108 genotypes as well as M. sativa CADL and Zhongmu No.1; the gene number and distribution were compared with Arabidopsis thaliana. Furthermore, by analyzing the expression data that were obtained at various developmental stages and in different zones of nitrogen-fixing nodules, we identified MtHMG loci that could be involved in nodule development and function. This work sets a reference for HMG genomic organization in legumes which will be useful for functional investigation that is aimed at elucidating HMGs involvement in nodule development and symbiotic nitrogen fixation.

Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum.

DNA methylation mediates organisms' adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

Structural characterization and duplication modes of pseudogenes in plants.

We identified and characterized the pseudogene complements of five plant species: four dicots (Arabidopsis thaliana, Vitis vinifera, Populus trichocarpa and Phaseolus vulgaris) and one monocot (Oryza sativa). Retroposition was considered of modest importance for pseudogene formation in all investigated species except V. vinifera, which showed an unusually high number of retro-pseudogenes in non coding genic regions. By using a pipeline for the classification of sequence duplicates in plant genomes, we compared the relative importance of whole genome, tandem, proximal, transposed and dispersed duplication modes in the pseudo and functional gene complements. Pseudogenes showed higher tendencies than functional genes to genomic dispersion. Dispersed pseudogenes were prevalently fragmented and showed high sequence divergence at flanking regions. On the contrary, those deriving from whole genome duplication were proportionally less than expected based on observations on functional loci and showed higher levels of flanking sequence conservation than dispersed pseudogenes. Pseudogenes deriving from tandem and proximal duplications were in excess compared to functional loci, probably reflecting the high evolutionary rate associated with these duplication modes in plant genomes. These data are compatible with high rates of sequence turnover at neutral sites and double strand break repairs mediated duplication mechanisms.

MCSeEd (Methylation Context Sensitive Enzyme ddRAD): A New Method to Analyze DNA Methylation.

Methylation context sensitive enzyme ddRAD (MCSeEd) is a NGS-based method for genome-wide investigations of DNA methylation at different contexts requiring only low to moderate sequencing depth. It is particularly useful for identifying methylation changes in experimental systems challenged by biotic or abiotic stresses or at different developmental stages.

Methylation content sensitive enzyme ddRAD (MCSeEd): a reference-free, whole genome profiling system to address cytosine/adenine methylation changes.

Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/ ) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.

Karyological Analysis and DNA Barcoding of Pompia Citron: A First Step toward the Identification of Its Relatives.

Pompia is a citrus fruit endemic of Sardinia, Italy, with an essential oil profile showing outstanding anti-inflammatory and anti-microbic properties. Despite its remarkable pharmaceutical potential, little taxonomic and genetic information is available for this species. We applied flow cytometry and classical cytogenetic techniques to assess the DNA content and to reconstruct the karyotype of several Pompia accessions. Molecular data from plastid DNA barcoding and nuclear DNA sequencing were used to study the genetic distance between Pompia and other citrus species. Flow cytometric estimates of DNA content and somatic chromosome counts suggest that Pompia is a regular diploid Citrus species. DNA polymorphisms of nuclear and chloroplast markers allowed us to investigate the genetic relationships between Pompia accessions and other Citrus species. Based on DNA polymorphism data we propose that Pompia is a very recent interspecific hybrid generated by a cross between C. aurantium (as seed bearer) and C. medica (as pollen donor). Our findings pave the way for further and more specific investigations of local Pompia germplasm resources that may help the preservation and valorisation of this valuable citrus fruit tree.

Nodule-associated microbiome diversity in wild populations of Sulla coronaria reveals clues on the relative importance of culturable rhizobial symbionts and co-infecting endophytes.

The culturable bacteria from root nodules of Sulla coronaria growing in spontaneous conditions in Sardinia were characterized. This plant's peculiarity is to represent a legume still found in both wild and cropped statuses. We tested whether culturable bacteria would differ from those commonly isolated from its field-cropped varieties, to date exclusively represented by Rhizobium sullae. 63 isolates from 60 surface-sterilized nodules were analyzed by ARDRA and 16S rDNA sequencing. The official nitrogen-fixing symbiont Rhizobium sullae was found only in 25 nodules out of 60. The remaining nodules did not yield culturable rhizobia but a number of different endophytic genera including Pseudomonas sp. (17 nodules), Microbacterium sp. (15 nodules), Pantoea agglomerans (5 nodules). The situation appears therefore a hybrid between what is commonly observed in other Mediterranean legumes occurring only in wild status (featuring non-culturable rhizobia and arrays of culturable endophytes within nodules), as opposed to cropped legumes (endowed with fully culturable rhizobia and minimal endophytic occurrence). These findings, within a species bridging the ecology between native and cropped conditions, suggest insights on the relative importance of endophytic co-occupancy vs. true N-fixing symbiont culturability within nodules. The latter trait thus appears to accompany the domestication path of plants with a main trade-off of renouncing to interactions with a diversity of endophytic co-invaders; the relationships with those being critical in the non-cropped status. In fact, endophytes are known to promote plant growth in harsh conditions, which can be particularly stressful in the Mediterranean due to drought, highly calcareous soils, and pathogens outbreaks.

Municipal solid wastes as a resource for environmental recovery: Impact of water treatment residuals and compost on the microbial and biochemical features of As and trace metal-polluted soils.

In this study we evaluated the microbiological and biochemical impact of iron-based water treatment residuals (Fe-WTRs) and municipal solid waste compost (MSWC), alone and combined, on three different soils co-contaminated with arsenic (As) and trace-metals (TM), i.e. Pb, Cu and Zn. Overall, all the amendments considered significantly increased the abundance of culturable heterotrophic bacteria, with MSWC showing the greatest impact across all soils (up to a 24% increase). In most of treated soils this was accompanied by a significant reduction of both the (culturable) fungal/bacterial ratio, and the proportion of culturable As(V)- and As(III)-resistant bacteria with respect to total bacterial population. The catabolic potential and versatility of the resident microbial communities (assessed by community level physiological profile) was highly soil-dependent and substantial increases of both parameters were observed in the amended soils with the higher total As concentration (from approx. 749 to 22,600 mg kg-1). Moreover, both carbon source utilisation profile and 16S rRNA soil metagenome sequencing indicated a significant impact of MSWC and Fe-WTRs on the structure and diversity of soil microbial communities, with Proteobacteria, Actinobacteria and Firmicutes being the most affected taxa. The assessment of selected soil enzyme activities (dehydrogenase, urease and ß-glucosidase) indicated an increase of metabolic functioning especially in soils treated with MSWC (e.g. dehydrogenase activity increased up to 19.5-fold in the most contaminated soil treated with MSWC). Finally, the microbial and biochemical features of treated (and untreated) contaminated soils (i.e. total bacterial counts, catabolic potential and versatility and soil enzyme activities) were highly correlated with the concentrations of labile As and TM in these latter soils and supported a clear role of the tested amendments (especially MSWC) as As- and TM-immobilising agents.

An analysis of codon bias in six red yeast species.

Red yeasts, primarily species of Rhodotorula, Sporobolomyces, and other genera of Pucciniomycotina, are traditionally considered proficient systems for lipid and terpene production, and only recently have also gained consideration for the production of a wider range of molecules of biotechnological potential. Improvements of transgene delivery protocols and regulated gene expression systems have been proposed, but a dearth of information on compositional and/or structural features of genes has prevented transgene sequence optimization efforts for high expression levels. Here, the codon compositional features of genes in six red yeast species were characterized, and the impact that evolutionary forces may have played in shaping this compositional bias was dissected by using several computational approaches. Results obtained are compatible with the hypothesis that mutational bias, although playing a significant role, cannot alone explain synonymous codon usage bias of genes. Nevertheless, several lines of evidences indicated a role for translational selection in driving the synonymous codons that allow high expression efficiency. These optimal synonymous codons are identified for each of the six species analyzed. Moreover, the presence of intragenic patterns of codon usage, which are thought to facilitate polyribosome formation, was highlighted. The information presented should be taken into consideration for transgene design for optimal expression in red yeast species.

Genetic, Phenotypic, and Commercial Characterization of an Almond Collection from Sardinia.

BACKGROUND: Recent nutritional and medical studies have associated the regular consumption of almonds with a wide range of health benefits. As a consequence, kernel quality has become an important goal for breeding, considering not only the chemical composition conferring a specific organoleptic quality but also physical traits related to industrial processing. METHODS: We characterized an almond collection from Sardinia through analysis of 13 morpho-physiological traits and eight essential oil profiles. The genetic structure of the collection was studied by analyzing the polymorphism of 11 simple sequence repeats (SSR). RESULTS: Both commercial and phenotypic traits showed wide ranges of variation. Most genotypes were early flowering with low yield potential. Several genotypes showed moderate to high yield and very interesting oil compositions of kernels. Based on 11 SSR profiles and Bayesian clustering, the Sardinian cultivars were assigned to groups which were differentiated for several agronomic and commercial traits. CONCLUSIONS: Several cultivars showed a high kernel oil content and high oleic to linoleic content ratio. Based on morphological traits, we propose that some of the analyzed cultivars could be interesting for industrial applications. Finally, we highlight the importance of characterizing early blooming cultivars for sites which are experiencing a rise in mean temperatures due to the effects of global climate changes.

corseq: fast and efficient identification of favoured codons from next generation sequencing reads.

BACKGROUND: Optimization of transgene expression can be achieved by designing coding sequences with the synonymous codon usage of genes which are highly expressed in the host organism. The identification of the so-called "favoured codons" generally requires the access to either the genome or the coding sequences and the availability of expression data. RESULTS: Here we describe corseq, a fast and reliable software for detecting the favoured codons directly from RNAseq data without prior knowledge of genomic sequence or gene annotation. The presented tool allows the inference of codons that are preferentially used in highly expressed genes while estimating the transcripts abundance by a new kmer based approach. corseq is implemented in Python and runs under any operating system. The software requires the Biopython 1.65 library (or later versions) and is available under the 'GNU General Public License version 3' at the project webpage https://sourceforge.net/projects/corseq/files. CONCLUSION: corseq represents a faster and easy-to-use alternative for the detection of favoured codons in non model organisms.

T-DNA Insertional Mutagenesis and Activation Tagging in Medicago truncatula.

The development of plant genetic transformation techniques has greatly enhanced our capacity to investigate and understand gene function. Since T-DNA constructs insert randomly in genomes, in principle, it is possible to construct a population of individuals harboring one or more T-DNA inserted in any region of the genome. Such populations can be screened following two approaches: (1) given a mutant phenotype, one could find the gene subtending the phenotypic alteration (forward approach), or (2) given a gene of interest, one could identify the phenotypic effect of its expression perturbation (reverse approach).Activation tagging is an application of T-DNA mutagenesis aimed at obtaining gain-of-function mutations. This can be achieved by introducing enhancer sequences randomly in the target genome via a T-DNA shuttle and then analyzing the genomic regions flanking the insertion sites in individuals showing phenotypic alterations. In this chapter, we describe the detailed procedure to obtain and screen an activation-tagged population in Medicago truncatula.

Survey of Brevibacillus laterosporus insecticidal protein genes and virulence factors.

The pathogenic action of the bacterium Brevibacillus laterosporus against invertebrates involves a toxin-mediated mechanism. Several studies, conducted with specific strains against diverse targets, suggested the implication of different toxins. Recent genome sequencing and annotation of some insecticidal strains revealed several putative virulence factors highly conserved in this species. After determining the pathogenicity of strain UNISS 18 against different Lepidopteran and Dipteran larvae, in this study we have investigated the actual expression of genes encoding for enzymes (i.e., chitinases, proteases), toxins, and other virulence factors, either in vitro and in vivo at the transcriptional level. Selected genes encode for two chitinases, a collagenase-like protease, a GlcNAc-binding protein, two protective antigen proteins, a bacillolysin, a thermophilic serine proteinase, two spore surface proteins, an insecticidal toxin homologous to Cry75Aa. All target genes were well expressed in pure bacterial cultures with significant differences between bacterial growth phases. Their expression level was generally enhanced in the bacterial population developing in the insect body cavity, compared with pure culture. The expression of certain genes increased substantially over time after insect inoculation. These results support a complex mechanism of action leveraging a variety of available virulence factors, and can also explain the ability of this bacterial species to act against diverse invertebrate targets.

CAR gene cluster and transcript levels of carotenogenic genes in Rhodotorula mucilaginosa.

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

Identification of Pseudogenes in Brachypodium distachyon Chromosomes.

Pseudogenes are gene copies that have lost the capability to encode a functional protein. Based on their structure, pseudogenes are classified in two types. Processed pseudogenes arise by a process of retrotranscription from a spliced mRNA and subsequent integration into the genome. Nonprocessed (or duplicated) pseudogenes are generated by genomic duplication and subsequent mutations that disable their functionality so that they cannot longer encode a functional protein. Differently from processed pseudogenes, duplicated pseudogenes are expected to conserve the exon-intron structure of their functional paralogs.Here, we describe a computational pipeline for identifying pseudogenes of both types in B. distachyon chromosomes. Our pipeline (1) identifies pseudogenes based on tBLASTn searches of B. distachyon proteins against the noncoding genomic complement of the same species, (2) identifies the most homologous pseudogenes functionally paralogous as the pseudogene paternal locus, (3) uses the intron-exon structure of paternal genes to distinguish between pseudogene types.The pipeline is presented in its composing steps and tested on the Brachypodium distachyon Bd1 scaffold.