The role of omics in improving the orphan crop tef.

Tef or teff [Eragrostis tef (Zucc.) Trotter] is a cereal crop indigenous to the Horn of Africa, where it is a staple food for a large population. The popularity of tef arises from its resilience to environmental stresses and its nutritional value. For many years, tef has been considered an orphan crop, but recent research initiatives from across the globe are helping to unravel its undisclosed potential. Advanced omics tools and techniques have been directed toward the exploration of tef's diversity with the aim of increasing its productivity. In this review, we report on the most recent advances in tef omics that brought the crop into the spotlight of international research.

A role for codon order in translation dynamics.

The genetic code is degenerate. Each amino acid is encoded by up to six synonymous codons; the choice between these codons influences gene expression. Here, we show that in coding sequences, once a particular codon has been used, subsequent occurrences of the same amino acid do not use codons randomly, but favor codons that use the same tRNA. The effect is pronounced in rapidly induced genes, involves both frequent and rare codons and diminishes only slowly as a function of the distance between subsequent synonymous codons. Furthermore, we found that in S. cerevisiae codon correlation accelerates translation relative to the translation of synonymous yet anticorrelated sequences. The data suggest that tRNA diffusion away from the ribosome is slower than translation, and that some tRNA channeling takes place at the ribosome. They also establish that the dynamics of translation leave a significant signature at the level of the genome.

Complex evolution of novel red floral color in Petunia.

Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate upregulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE, restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through rebalancing the expression of three hydroxylating genes. Furthermore, the downregulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition toward the gain of a novel red color.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

Identification and characterization of durum wheat microRNAs in leaf and root tissues.

MicroRNAs are a class of post-transcriptional regulators of plant developmental and physiological processes and responses to environmental stresses. Here, we present the study regarding the annotation and characterization of MIR genes conducted in durum wheat. We characterized the miRNAome of leaf and root tissues at tillering stage under two environmental conditions: irrigated with 100% (control) and 55% of evapotranspiration (early water stress). In total, 90 microRNAs were identified, of which 32 were classified as putative novel and species-specific miRNAs. In addition, seven microRNA homeologous groups were identified in each of the two genomes of the tetraploid durum wheat. Differential expression analysis highlighted a total of 45 microRNAs significantly differentially regulated in the pairwise comparisons leaf versus root. The miRNA families, miR530, miR395, miR393, miR5168, miR396 and miR166, miR171, miR319, and miR167, were the most expressed in leaves in comparison to roots. Putative microRNA targets were predicted for both five and three prime sequences derived from the stem-loop of the MIR gene. Gene ontology analysis showed significant overrepresented gene categories in microRNA targets belonging to transcription factors, phenylpropanoids, oxydases, and lipid binding-protein. This work represents one of the first genome wide characterization of MIR genes in durum wheat, identifying leaf and root tissue-specific microRNAs. This genomic identification of microRNAs together with the analysis of their expression profiles is a well-accepted starting point leading to a better comprehension of the role of MIR genes in the genus Triticum.

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast.

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to >1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p's molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.

Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene.

Genetic improvement of native crops is a new and promising strategy to combat hunger in the developing world. Tef is the major staple food crop for approximately 50 million people in Ethiopia. As an indigenous cereal, it is well adapted to diverse climatic and soil conditions; however, its productivity is extremely low mainly due to susceptibility to lodging. Tef has a tall and weak stem, liable to lodge (or fall over), which is aggravated by wind, rain, or application of nitrogen fertilizer. To circumvent this problem, the first semi-dwarf lodging-tolerant tef line, called kegne, was developed from an ethyl methanesulphonate (EMS)-mutagenized population. The response of kegne to microtubule-depolymerizing and -stabilizing drugs, as well as subsequent gene sequencing and segregation analysis, suggests that a defect in the α-Tubulin gene is functionally and genetically tightly linked to the kegne phenotype. In diploid species such as rice, homozygous mutations in α-Tubulin genes result in extreme dwarfism and weak stems. In the allotetraploid tef, only one homeologue is mutated, and the presence of the second intact α-Tubulin gene copy confers the agriculturally beneficial semi-dwarf and lodging-tolerant phenotype. Introgression of kegne into locally adapted and popular tef cultivars in Ethiopia will increase the lodging tolerance in the tef germplasm and, as a result, will improve the productivity of this valuable crop.

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef).

BACKGROUND: Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. RESULTS: The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. CONCLUSIONS: It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

The origins and progress of genomics research on Tef (Eragrostis tef).

Tef, Eragrostis tef (Zucc.) Trotter, is the most important cereal in Ethiopia. Tef is cultivated by more than five million small-scale farmers annually and constitutes the staple food for more than half of the population of 80 million. The crop is preferred by both farmers and consumers due to its beneficial traits associated with its agronomy and utilization. The genetic and phenotypic diversity of tef in Ethiopia is a national treasure of potentially global importance. In order for this diversity to be effectively conserved and utilized, a better understanding at the genomic level is necessary. In the recent years, tef has become the subject of genomic research in Ethiopia and abroad. Genomic-assisted tef improvement holds tremendous potential for improving productivity, thereby benefiting the smallholder farmers who have cultivated and relied on the crop for thousands of years. It is hoped that such research endeavours will provide solutions to some of the age-old problems of tef's husbandry. In this review, we provide a brief description of the genesis and progress of tef genomic research to date, suggest ways to utilize the genomic tools developed so far, discuss the potential of genomics to enable sustainable conservation and use of tef genetic diversity and suggest opportunities for the future research.

FastML: a web server for probabilistic reconstruction of ancestral sequences.

Ancestral sequence reconstruction is essential to a variety of evolutionary studies. Here, we present the FastML web server, a user-friendly tool for the reconstruction of ancestral sequences. FastML implements various novel features that differentiate it from existing tools: (i) FastML uses an indel-coding method, in which each gap, possibly spanning multiples sites, is coded as binary data. FastML then reconstructs ancestral indel states assuming a continuous time Markov process. FastML provides the most likely ancestral sequences, integrating both indels and characters; (ii) FastML accounts for uncertainty in ancestral states: it provides not only the posterior probabilities for each character and indel at each sequence position, but also a sample of ancestral sequences from this posterior distribution, and a list of the k-most likely ancestral sequences; (iii) FastML implements a large array of evolutionary models, which makes it generic and applicable for nucleotide, protein and codon sequences; and (iv) a graphical representation of the results is provided, including, for example, a graphical logo of the inferred ancestral sequences. The utility of FastML is demonstrated by reconstructing ancestral sequences of the Env protein from various HIV-1 subtypes. FastML is freely available for all academic users and is available online at http://fastml.tau.ac.il/.

Tight genetic linkage of genes causing hybrid necrosis and pollinator isolation between young species.

The mechanisms of reproductive isolation that cause phenotypic diversification and eventually speciation are a major topic of evolutionary research. Hybrid necrosis is a post-zygotic isolation mechanism in which cell death develops in the absence of pathogens. It is often due to the incompatibility between proteins from two parents. Here we describe a unique case of hybrid necrosis due to an incompatibility between loci on chromosomes 2 and 7 between two pollinator-isolated Petunia species. Typical immune responses as well as endoplasmic reticulum stress responses are induced in the necrotic line. The locus on chromosome 2 encodes ChiA1, a bifunctional GH18 chitinase/lysozyme. The enzymatic activity of ChiA1 is dispensable for the development of necrosis. We propose that the extremely high expression of ChiA1 involves a positive feedback loop between the loci on chromosomes 2 and 7. ChiA1 is tightly linked to major genes involved in the adaptation to different pollinators, a form of pre-zygotic isolation. This linkage of pre- and post-zygotic barriers strengthens reproductive isolation and probably contributes to rapid diversification and speciation.

Support patterns from different outgroups provide a strong phylogenetic signal.

It is known that the accuracy of phylogenetic reconstruction decreases when more distant outgroups are used. We quantify this phenomenon with a novel scoring method, the outgroup score pOG. This score expresses if the support for a particular branch of a tree decreases with increasingly distant outgroups. Large-scale simulations confirmed that the outgroup support follows this expectation and that the pOG score captures this pattern. The score often identifies the correct topology even when the primary reconstruction methods fail, particularly in the presence of model violations. In simulations of problematic phylogenetic scenarios such as rate variation among lineages (which can lead to long-branch attraction artifacts) and quartet-based reconstruction, the pOG analysis outperformed the primary reconstruction methods. Because the pOG method does not make any assumptions about the evolutionary model (besides the decreasing support from increasingly distant outgroups), it can detect cases of violations not treated by a specific model or too strong to be fully corrected. When used as an optimization criterion in the construction of a tree of 23 mammals, the outgroup signal confirmed many well-accepted mammalian orders and superorders. It supports Atlantogenata, a clade of Afrotheria and Xenarthra, and suggests an Artiodactyla-Chiroptera clade.

Panicle Angle is an Important Factor in Tef Lodging Tolerance.

Tef [Eragrostis tef (Zucc.) Trotter] is an important crop in the Horn of Africa, particularly in Ethiopia, where it is a staple food for over 60 million people. However, the productivity of tef remains extremely low in part due to its susceptibility to lodging. Lodging is the displacement of the plant from the upright position, and it is exacerbated by rain, wind and the application of fertilizer. In order to address the issue of global food security, especially in the Horn of Africa, greater insight into the causes of tef lodging is needed. In this study, we combine modeling and biomechanical measurements to compare the properties relating to lodging tolerance in high yielding, improved tef genotypes, and lower yielding natural landraces. Our results indicate that the angle of the panicle contributes to the likelihood of lodging in tef. Varieties with compact panicles and reduced height had increased lodging resistance compared to the other varieties. By comparing different varieties, we found that overall, the landraces of tef lodged less than improved varieties. We constructed a model of stem bending and found that panicle angle was an important determinant of the amount of lodging. The findings from this study provide key information to those involved in tef improvement, especially those interested in lodging tolerance.

A phylogenomic study of human, dog, and mouse.

In recent years the phylogenetic relationship of mammalian orders has been addressed in a number of molecular studies. These analyses have frequently yielded inconsistent results with respect to some basal ordinal relationships. For example, the relative placement of primates, rodents, and carnivores has differed in various studies. Here, we attempt to resolve this phylogenetic problem by using data from completely sequenced nuclear genomes to base the analyses on the largest possible amount of data. To minimize the risk of reconstruction artifacts, the trees were reconstructed under different criteria-distance, parsimony, and likelihood. For the distance trees, distance metrics that measure independent phenomena (amino acid replacement, synonymous substitution, and gene reordering) were used, as it is highly improbable that all of the trees would be affected the same way by any reconstruction artifact. In contradiction to the currently favored classification, our results based on full-genome analysis of the phylogenetic relationship between human, dog, and mouse yielded overwhelming support for a primate-carnivore clade with the exclusion of rodents.

Identification of miRNAs linked with the drought response of tef [Eragrostis tef (Zucc.) Trotter].

Tef [Eragrostis tef (Zucc.) Trotter], a staple food crop in the Horn of Africa and particularly in Ethiopia, has several beneficial agronomical and nutritional properties, including waterlogging and drought tolerance. In this study, we performed microRNA profiling of tef using the Illumina HiSeq 2500 platform, analyzing both shoots and roots of two tef genotypes, one drought-tolerant (Tsedey) and one drought-susceptible (Alba). We obtained more than 10 million filtered reads for each of the 24 sequenced small cDNA libraries. Reads mapping to known miRNAs were more abundant in the root than shoot tissues. Thirteen and 35 miRNAs were significantly modulated in response to drought, in Alba and Tsedey roots, respectively. One miRNA was upregulated under drought conditions in both genotypes. In shoots, nine miRNAs were modulated in common between the two genotypes and all showed similar trends of expression. One-hundred and forty-seven new miRNA mature sequences were identified in silico, 22 of these were detected in all relevant samples and seven were differentially regulated when comparing drought with normal watering. Putative targets of the miRNA regulated under drought in root and shoot tissues were predicted. Among the targets were transcription factors such as CCAAT-HAP2, MADS and NAC. Verification with qRT-PCR revealed that five of six potential targets showed a pattern of expression that was consistent with the correspondent miRNA amount measured by RNA-Seq. In general, candidate miRNAs involved in the post-transcriptional regulation of the tef response to drought could be included in next-generation breeding programs.

Waterlogging affects plant morphology and the expression of key genes in tef (Eragrostis tef).

Tef [Eragrostis tef (Zucc.) Trotter], an allotetraploid cereal that is a staple food to over 60 million people in the Horn of Africa, has a high nutritional content and is resistant to many biotic and abiotic stresses such as waterlogging and drought. Three tef genotypes, Alba, Tsedey, and Quncho, were subjected to waterlogging conditions and their growth, physiology, and change in transcript expression were measured with the goal of identifying targets for breeding cultivars with improved waterlogging tolerance. Root and shoot growth and dry weight were observed over 22 days. Stomatal conductance and chlorophyll and carotenoid contents were quantified. Microscopy was used to monitor changes in the stem cross sections. Illumina RNA sequencing was used to obtain the expression profiles of tef under flooding and control conditions and was verified using qPCR. Results indicated differences in growth between the three genotypes. Waterlogged Tsedey plants grew higher and had more root biomass than normally watered Tsedey plants. Quncho and Alba genotypes were more susceptible to the excess moisture stress. The effects of these changes were observed on the plant physiology. Among the three tested tef genotypes, Tsedey formed more aerenchyma than Alba and had accelerated growth under waterlogging. Tsedey and Quncho had constitutive aerenchyma. Genes affecting carbohydrate metabolism, cell growth, response to reactive oxygen species, transport, signaling, and stress responses were found to change under excess moisture stress. In general, these results show the presence of substantial anatomical and physiological differences among tef genotypes when waterlogged during the early growth stage.

SynPAM-a distance measure based on synonymous codon substitutions.

Measuring evolutionary distances between DNA or protein sequences forms the basis of many applications in computational biology and evolutionary studies. Of particular interest are distances based on synonymous substitutions, since these substitutions are considered to be under very little selection pressure and therefore assumed to accumulate in an almost clock-like manner. SynPAM, the method presented here, allows the estimation of distances between coding DNA sequences based on synonymous codon substitutions. The problem of estimating an accurate distance from the observed substitution pattern is solved by maximum-likelihood with empirical codon substitution matrices employed for the underlying Markov model. Comparisons with established measures of synonymous distance indicate that SynPAM has less variance and yields useful results over a longer time range.

A Proteomic Approach to Investigate the Drought Response in the Orphan Crop Eragrostis tef.

The orphan crop, Eragrostis tef, was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants tolerated water loss to 50% relative water content (RWC) before adverse effects in leaf tissues were observed. Proteomic analysis using isobaric tag for relative and absolute quantification (iTRAQ) mass spectrometry and appropriate database searching enabled the detection of 5727 proteins, of which 211 proteins, including a number of spliced variants, were found to be differentially regulated with the imposed stress conditions. Validation of the iTRAQ dataset was done with selected stress-related proteins, fructose-bisphosphate aldolase (FBA) and the protective antioxidant proteins, monodehydroascorbate reductase (MDHAR) and peroxidase (POX). Western blot analyses confirmed protein presence and showed increased protein abundance levels during water deficit while enzymatic activity for FBA, MDHAR and POX increased at selected RWC points. Gene ontology (GO)-term enrichment and analysis revealed terms involved in biotic and abiotic stress response, signaling, transport, cellular homeostasis and pentose metabolic processes, to be enriched in tef upregulated proteins, while terms linked to reactive oxygen species (ROS)-producing processes under water-deficit, such as photosynthesis and associated light harvesting reactions, manganese transport and homeostasis, the synthesis of sugars and cell wall catabolism and modification, to be enriched in tef downregulated proteins.

Gibberellin Deficiency Confers Both Lodging and Drought Tolerance in Small Cereals.

Tef [Eragrostis tef (Zucc.) Trotter] and finger millet [Eleusine coracana Gaertn] are staple cereal crops in Africa and Asia with several desirable agronomic and nutritional properties. Tef is becoming a life-style crop as it is gluten-free while finger millet has a low glycemic index which makes it an ideal food for diabetic patients. However, both tef and finger millet have extremely low grain yields mainly due to moisture scarcity and susceptibility of the plants to lodging. In this study, the effects of gibberellic acid (GA) inhibitors particularly paclobutrazol (PBZ) on diverse physiological and yield-related parameters were investigated and compared to GA mutants in rice (Oryza sativa L.). The application of PBZ to tef and finger millet significantly reduced the plant height and increased lodging tolerance. Remarkably, PBZ also enhanced the tolerance of both tef and finger millet to moisture deficit. Under moisture scarcity, tef plants treated with PBZ did not exhibit drought-related symptoms and their stomatal conductance was unaltered, leading to higher shoot biomass and grain yield. Semi-dwarf rice mutants altered in GA biosynthesis, were also shown to have improved tolerance to dehydration. The combination of traits (drought tolerance, lodging tolerance and increased yield) that we found in plants with altered GA pathway is of importance to breeders who would otherwise rely on extensive crossing to introgress each trait individually. The key role played by PBZ in the tolerance to both lodging and drought calls for further studies using mutants in the GA biosynthesis pathway in order to obtain candidate lines which can be incorporated into crop-breeding programs to create lodging tolerant and climate-smart crops.

Empirical codon substitution matrix.

BACKGROUND: Codon substitution probabilities are used in many types of molecular evolution studies such as determining Ka/Ks ratios, creating ancestral DNA sequences or aligning coding DNA. Until the recent dramatic increase in genomic data enabled construction of empirical matrices, researchers relied on parameterized models of codon evolution. Here we present the first empirical codon substitution matrix entirely built from alignments of coding sequences from vertebrate DNA and thus provide an alternative to parameterized models of codon evolution. RESULTS: A set of 17,502 alignments of orthologous sequences from five vertebrate genomes yielded 8.3 million aligned codons from which the number of substitutions between codons were counted. From this data, both a probability matrix and a matrix of similarity scores were computed. They are 64 x 64 matrices describing the substitutions between all codons. Substitutions from sense codons to stop codons are not considered, resulting in block diagonal matrices consisting of 61 x 61 entries for the sense codons and 3 x 3 entries for the stop codons. CONCLUSION: The amount of genomic data currently available allowed for the construction of an empirical codon substitution matrix. However, more sequence data is still needed to construct matrices from different subsets of DNA, specific to kingdoms, evolutionary distance or different amount of synonymous change. Codon mutation matrices have advantages for alignments up to medium evolutionary distances and for usages that require DNA such as ancestral reconstruction of DNA sequences and the calculation of Ka/Ks ratios.