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.

Genetic diversity and population structure of selected tef core germplasm lines based on microsatellite markers.

BACKGROUND: Tef is an indigenous and important food, feed, and cash crop for smallholder Ethiopian farmers. Knowledge of the natural genetic composition of the crop provides the option to further exploit its genetic potential through breeding. However, there are insufficient reports on the genetic variability of Ethiopian tef using a medium-throughput marker system. Hence, the current study was designed to evaluate the genetic variability of released and core germplasm that was collected earlier. METHODS AND RESULTS: Eighty-one tef genotypes collected from eight Ethiopian ecological zones and released varieties were targeted using 14 SSR markers. The study yielded a total of 122 alleles across the entire locus and population. The molecular variance analysis indicated the existence of large genetic differentiation (FIS and FIT = 0.87), with 86% and 13% of the total variation accounted for among genotypes within the population and across all genotypes used for this study, respectively. However, low genetic differentiation among the populations (FST = 0.014, which accounts for 1%) was observed. Multivariate analyses such as clustering and PCoA did not cluster genotypes into distinct groups according to their geographical areas of population. This is presumably due to gene flow among populations. CONCLUSION: In conclusion, our findings show that there is significant genetic diversity within populations, particularly in the Jimma, Tigray, and released varieties, as well as the presence of private alleles and heterozygosity. The study also indicates the existence of genotypic admixture in the studied materials. The identification of private alleles and their differentiation will be helpful in selecting breeding materials and creating breeding plans.

CRISPR/Cas9-mediated tetra-allelic mutation of the 'Green Revolution' SEMIDWARF-1 (SD-1) gene confers lodging resistance in tef (Eragrostis tef).

Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.

Significance and prospects of an orphan crop tef.

MAIN CONCLUSION: Tef is a resilient crop from the Horn of Africa with significant importance in food and nutrition security, and currently gaining global popularity as health and performance food. Tef [Eragrostis tef (Zucc.) Trotter] is the most important cereal of Ethiopia in terms of production, consumption and cash crop value. In Ethiopia, tef is annually grown on about 3 million ha with total grain production of over 5 million tons. As such, it accounts for about 30% of the total cultivated area and one-fifth of the gross grain production of all cereals cultivated in the country. In spite of its supreme economic and agricultural significance in Ethiopia, its productivity is relatively low with national average yield of about 1.7 t/ha. This has primarily been due to the very little scientific improvement done on the crop. Tef has still been an "orphan crop" since it is globally a very much under-researched crop owing to its localized importance. Scientific research on tef in Ethiopia began in the late 1950s. The main objective of this paper is to provide an overview of the significance and major production constraints of tef, and the major achievements made to date in various tef research aspects including breeding, agronomy, crop protection, and agricultural economics and extension. Based on these reviews, the paper eventually concludes with remarks on the way forward by emphasizing on the identification of the major gaps and the improvement efforts required for realizing the ever-needed breakthrough in the productivity and production of the crop. The major focal areas of future efforts include increasing productivity of both grain and biomass, systematic conservation and mining of the genetic resources, tackling the lodging malady, mechanization of the crop's husbandry, understanding the overall physiology of the crop especially with respect to stress tolerance, unraveling the nutritional qualities, and development of recipes and value-added products.

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.

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.

Genetic diversity in tef [Eragrostis tef (Zucc.) Trotter].

Tef [Eragrostis tef (Zucc.) Trotter] is a cereal crop resilient to adverse climatic and soil conditions, and possessing desirable storage properties. Although tef provides high quality food and grows under marginal conditions unsuitable for other cereals, it is considered to be an orphan crop because it has benefited little from genetic improvement. Hence, unlike other cereals such as maize and wheat, the productivity of tef is extremely low. In spite of the low productivity, tef is widely cultivated by over six million small-scale farmers in Ethiopia where it is annually grown on more than three million hectares of land, accounting for over 30% of the total cereal acreage. Tef, a tetraploid with 40 chromosomes (2n = 4x = 40), belongs to the family Poaceae and, together with finger millet (Eleusine coracana Gaerth.), to the subfamily Chloridoideae. It was originated and domesticated in Ethiopia. There are about 350 Eragrostis species of which E. tef is the only species cultivated for human consumption. At the present time, the gene bank in Ethiopia holds over five thousand tef accessions collected from geographical regions diverse in terms of climate and elevation. These germplasm accessions appear to have huge variability with regard to key agronomic and nutritional traits. In order to properly utilize the variability in developing new tef cultivars, various techniques have been implemented to catalog the extent and unravel the patterns of genetic diversity. In this review, we show some recent initiatives investigating the diversity of tef using genomics, transcriptomics and proteomics and discuss the prospect of these efforts in providing molecular resources that can aid modern tef breeding.

Variation and inter-relationships of quantitative traits in tef (Eragrostis tef (Zucc.) Trotter) germplasm from western and southern Ethiopia.

Three thousand tef (Eragrostis tef (Zucc.) Trotter) lines representing 60 germplasm populations from western and southern Ethiopia were sown on pellic Vertisols at Debre Zeit Agricultural Research Center during the 1999/2000 main season. The objectives were to assess the variation with respect to regions and altitude zones of origin and to study the inter-relationships of 17 pheno-morphic and agronomic traits. The populations showed significant (p < or = 0.05) regional variation in 10 (59%) of the quantitative traits, but clinal variation among altitude zones was significant (p < or = 0.05) only for six (35%) of the traits. On the other hand, the populations revealed consistent variation (p < or = 0.05) within both regions and altitude zones in all the traits evaluated. Likewise, the variation among lines within populations of both regions and altitude zones was significant (p < or = 0.05) in most of the traits. The number of characters showing substantial correlation depicted regional and clinal variation mainly depending on the number of populations. Based on the mean of the populations, grain yield panicle and shoot phytomass plant showed negative correlation with harvest index, and positive correlation with most of the remaining traits. Individual plant grain yield was positively correlated with all the other traits except harvest index, days to maturity, grain filling period and number of primary panicle branches. Overall, the tef germplasm populations showed substantial phenotypic variation which can be utilized in the genetic improvement of the crop.

Inter simple sequence repeat (ISSR) analysis of genetic diversity in tef [Eragrostis tef (Zucc.) Trotter].

The DNA polymorphism among 92 selected tef genotypes belonging to eight origin groups was assessed using eight inter simple sequence repeat (ISSR) primers. The objectives were to examine the possibility of using ISSR markers for unravelling genetic diversity in tef, and to assess the extent and pattern of genetic diversity in the test germplasm with respect to origin groups. The eight primers were able to separate or distinguish all of the 92 tef genotypes based on a total of 110 polymorphic bands among the test lines. The Jaccard similarity coefficient among the test genotypes ranged from 0.26 to 0.86, and at about 60 % similarity level the clustering of this matrix using the unweighted pair-group method based on arithmetic average (UPGMA) resulted in the formation of six major clusters of 2 to 37 lines with further eight lines remaining ungrouped. The standardized Nei genetic distance among the eight groups of origin ranged between 0.03 and 0.32. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of three clusters of the eight groups of origin with bootstrap values ranging from 56 to 97. The overall mean Shannon Weaver diversity index of the test lines was 0.73, indicating better resolution of genetic diversity in tef with ISSR markers than with phenotypic (morphological) traits used in previous studies. This can be attributed mainly to the larger number of loci generated for evaluation with ISSR analysis as compared to the few number of phenotypic traits amenable for assessment and which are further greatly affected by environment and genotype x environment interaction. Analysis of variance of mean Shannon Weaver diversity indices revealed substantial (P < or = 0.05) variation in the level of diversity among the eight groups of origin. In conclusion, our results indicate that ISSR can be useful as DNA-based molecular markers for studying genetic diversity and phylogenetic relationships, DNA fingerprinting for the identification of varieties or cultivars, and also for genome mapping in tef.

Multivariate analysis of diversity of tef (Eragrostis tef (Zucc.) Trotter) germplasm from western and southern Ethiopia.

Sixty tef germplasm populations consisting of 3,000 panicle-derived lines from six western and southern regions of Ethiopia were evaluated for 17 pheno-morphic and agronomic traits on pellic Vertisols at Debre Zeit Agricultural Research Center during the 1999 main season. The objectives were to study the extent and pattern of variation of the germplasm with respect to regions and altitude zones, to classify the populations into relatively homogenous groups and to identify the major traits contributing to the overall diversity of the populations. At 75 % similarity level, the 60 populations aggregated into nine complexes of two to 10, with 12 populations remaining un-grouped. Five principal components (PC) extracted about 81 % of the gross variance among the populations. About 40 % of the variance accounted for by the first PC alone resulted largely from variations in diameters of the two basal culm internodes, grain yield and number of spikelets/panicle, shoot phytomass and grain yield/plant, and number of culm internodes. The entire regional as well as the clinal (altitude zone) variation was explained by five and two PCs, respectively. The discriminant analyses depicted about 77 % correct grouping of the 47 populations into nine clusters and about 62 % and 68 % correct origin-based classification of the germplasm in terms of altitude zones and regions, respectively. In general, the study demonstrated the existence of regional and clinal (altitude zone) variation patterns in the tef germplasm populations. The broad trait variation in the germplasm implies ample opportunities for genetic improvement of tef through selection and hybridization.