Restriction Site Associated DNA Sequencing based Single Nucleotide Polymorphism Discovery in Selected Tef (Eragrostis tef) and Wild Eragrostis Species

አህፅሮት ጤፍ በኢትዮጵያ የተገኘ እና በኢትዮጵያዊያን አርሶአደሮች ለብዙ አመታት ሲመረት የኖረ ዘርፈ ብዙ ጥቅሞች ያሉት የሰብል አይነት ነዉ፡፡ ጤፍ የሚመረተውና ጥቅም ላይ የሚውለው በአብዛኛው በኢትዮጵያ በመሆኑ በአለም አቀፍ ደረጃ ጤፍ ላይ የሚደረጉ ምርምሮች ውሱን ናቸው፡፡  በተለይ የጤፍን ምርምር ለማሳለጥ የተዘጋጁ የሞለኪውላር ሳይንስ ግብአቶች ውሱን በመሆናቸው በሌሎች ሰብሎች ላይ ጥቅም ላይ የዋሉ የዲ ኤን ኤ ሲኩዌንሲንግ ቴክኖሎጂን መሰረት ያደረጉ የምርምር አቅጣጫዎችን መከተል ወሳኝ ነው፡፡  እዚህ በቀረበው  የምርምር ስራ የዲ ኤን ኤ ሲኩዌንሲንግ ቴክኖሎጂን መሰረት ያደረገን የምርምር ስልት በመከተል ከአገሪቱ የተለያዩ አካባቢዎች የተሰበሰቡ አርባ ሁለት የጤፍ ዝርያዎች፡ አንድ ሚውታንት ላይን እና ሁለት ለጤፍ ቅርብ የሆኑ ዋይልድ ሬላቲቭስ ላይ ጥናት ተደርጎ የጤፍን ምርምር በዘመናዊ መልኩ የሚያግዙ ግብአቶች ተገኝተዋል፡፡ Abstract Genome-wide knowledge about the nature and extent of genetic diversity present in tef (Eragrostis tef), the most consumed food grain in Ethiopia is limited. Adopting next generation sequencing (NGS) protocols to enhance its genomics and breeding is essential. Here, we applied the Restriction Site Associated DNA (RAD) sequencing protocol and surveyed the genomes of 43 tef landraces, one mutant line and two wild Eragrostis species. After mapping sequencing reads to the de novo assembled unitag and the tef reference genome, a total of 9,024 and 58,735 high quality single nucleotide polymorphisms (SNPs) were identified, respectively. We identified greater number of SNPs and greater nucleotide diversity in the two wild Eragrostis species than in the tef landraces. The tef landrace populations in this study were poorly differentiated with FST values of 0.015. In the phylogenetic analysis, grouping of the landraces was not consistent with the area of collection, but few localized grouping of the landraces was evident, probably showing the communality of tef seed use across geographical boundaries. The improved tef varieties show reduced genetic diversity compared to the landraces and were all grouped into one cluster reflecting the nature of tef breeding which largely targets common genomic regions. We suggest that future work needs to aim beyond common genomic regions. The work presented here is a valuable addition to the growing molecular resources developed for tef genetic improvement

AtPTR4 and AtPTR6 are differentially expressed, tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectivel

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

Technology generation to dissemination:lessons learned from the tef improvement project

Indigenous crops also known as orphan crops are key contributors to food security, which is becoming increasingly vulnerable with the current trend of population growth and climate change. They have the major advantage that they fit well into the general socio-economic and ecological context of developing world agriculture. However, most indigenous crops did not benefit from the Green Revolution, which dramatically increased the yield of major crops such as wheat and rice. Here, we describe the Tef Improvement Project, which employs both conventional- and molecular-breeding techniques to improve tef\u2014an orphan crop important to the food security in the Horn of Africa, a region of the world with recurring devastating famines. We have established an efficient pipeline to bring improved tef lines from the laboratory to the farmers of Ethiopia. Of critical importance to the long-term success of this project is the cooperation among participants in Ethiopia and Switzerland, including donors, policy makers, research institutions, and farmers. Together, European and African scientists have developed a pipeline using breeding and genomic tools to improve the orphan crop tef and bring new cultivars to the farmers in Ethiopia. We highlight a new variety, Tesfa, developed in this pipeline and possessing a novel and desirable combination of traits. Tesfa\u2019s recent approval for release illustrates the success of the project and marks a milestone as it is the first variety (of many in the pipeline) to be released

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background: In this study, we aimed to evaluate the effects of tocilizumab in adult patients admitted to hospital with COVID-19 with both hypoxia and systemic inflammation. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. Those trial participants with hypoxia (oxygen saturation <92% on air or requiring oxygen therapy) and evidence of systemic inflammation (C-reactive protein ≥75 mg/L) were eligible for random assignment in a 1:1 ratio to usual standard of care alone versus usual standard of care plus tocilizumab at a dose of 400 mg–800 mg (depending on weight) given intravenously. A second dose could be given 12–24 h later if the patient's condition had not improved. The primary outcome was 28-day mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN (50189673) and ClinicalTrials.gov (NCT04381936). Findings: Between April 23, 2020, and Jan 24, 2021, 4116 adults of 21 550 patients enrolled into the RECOVERY trial were included in the assessment of tocilizumab, including 3385 (82%) patients receiving systemic corticosteroids. Overall, 621 (31%) of the 2022 patients allocated tocilizumab and 729 (35%) of the 2094 patients allocated to usual care died within 28 days (rate ratio 0·85; 95% CI 0·76–0·94; p=0·0028). Consistent results were seen in all prespecified subgroups of patients, including those receiving systemic corticosteroids. Patients allocated to tocilizumab were more likely to be discharged from hospital within 28 days (57% vs 50%; rate ratio 1·22; 1·12–1·33; p<0·0001). Among those not receiving invasive mechanical ventilation at baseline, patients allocated tocilizumab were less likely to reach the composite endpoint of invasive mechanical ventilation or death (35% vs 42%; risk ratio 0·84; 95% CI 0·77–0·92; p<0·0001). Interpretation: In hospitalised COVID-19 patients with hypoxia and systemic inflammation, tocilizumab improved survival and other clinical outcomes. These benefits were seen regardless of the amount of respiratory support and were additional to the benefits of systemic corticosteroids. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Restriction Site Associated DNA Sequencing based Single Nucleotide Polymorphism Discovery in Selected Tef (Eragrostis tef) and Wild Eragrostis Species

Genome-wide knowledge about the nature and extent of genetic diversity present in tef (Eragrostis tef), the most consumed food grain in Ethiopia is limited. Adopting next generation sequencing (NGS) protocols to enhance its genomics and breeding is essential. Here, we applied the Restriction Site Associated DNA (RAD) sequencing protocol and surveyed the genomes of 43 tef landraces, one mutant line and two wild Eragrostis species. After mapping sequencing reads to the de novo assembled unitag and the tef reference genome, a total of 9,024 and 58,735 high quality single nucleotide polymorphisms (SNPs) were identified, respectively. We identified greater number of SNPs and greater nucleotide diversity in the two wild Eragrostis species than in the tef landraces. The tef landrace populations in this study were poorly differentiated with FST values of 0.015. In the phylogenetic analysis, grouping of the landraces was not consistent with the area of collection, but few localized grouping of the landraces was evident, probably showing the communality of tef seed use across geographical boundaries. The improved tef varieties show reduced genetic diversity compared to the landraces and were all grouped into one cluster reflecting the nature of tef breeding which largely targets common genomic regions. We suggest that future work needs to aim beyond common genomic regions. The work presented here is a valuable addition to the growing molecular resources developed for tef genetic improvement.ጤፍ በኢትዮጵያ የተገኘ እና በኢትዮጵያዊያን አርሶአደሮች ለብዙ አመታት ሲመረት የኖረ ዘርፈ ብዙ ጥቅሞች ያሉት የሰብል አይነት ነዉ፡፡ ጤፍ የሚመረተውና ጥቅም ላይ የሚውለው በአብዛኛው በኢትዮጵያ በመሆኑ በአለም አቀፍ ደረጃ ጤፍ ላይ የሚደረጉ ምርምሮች ውሱን ናቸው፡፡ በተለይ የጤፍን ምርምር ለማሳለጥ የተዘጋጁ የሞለኪውላር ሳይንስ ግብአቶች ውሱን በመሆናቸው በሌሎች ሰብሎች ላይ ጥቅም ላይ የዋሉ የዲ ኤን ኤ ሲኩዌንሲንግ ቴክኖሎጂን መሰረት ያደረጉ የምርምር አቅጣጫዎችን መከተል ወሳኝ ነው፡፡ እዚህ በቀረበው የምርምር ስራ የዲ ኤን ኤ ሲኩዌንሲንግ ቴክኖሎጂን መሰረት ያደረገን የምርምር ስልት በመከተል ከአገሪቱ የተለያዩ አካባቢዎች የተሰበሰቡ አርባ ሁለት የጤፍ ዝርያዎች፡ አንድ ሚውታንት ላይን እና ሁለት ለጤፍ ቅርብ የሆኑ ዋይልድ ሬላቲቭስ ላይ ጥናት ተደርጎ የጤፍን ምርምር በዘመናዊ መልኩ የሚያግዙ ግብአቶች ተገኝተዋል፡

Genotyping by Sequencing Reasserts the Close Relationship between Tef and Its Putative Wild Eragrostis Progenitors

The genus Eragrostis consists of 350 species, including tef (Eragrostis tef (Zucc.) Trotter), the only cultivated species in this genus. Very little is known about the genetic potential of these species for tef improvement and genomics research. Here, we investigated a germplasm panel consisting of 40 Eragrostis species and 42 tef lines with single nucleotide polymorphism (SNP) data generated using the genotyping by sequencing (GBS) protocol. Thousands of SNPs were identified genome-wide from the germplasm panel. High-quality SNPs were used to assess sequence similarity and/or divergence, genetic diversity, population structure, and phylogenetic relationships. Mapping individual reads to the tef reference genome revealed that of the 40 wild Eragrostis species included in this study, E. pilosa, E. aethiopica, E. obtusa, E. ferruginea, E. lugens, and E. lehmanniana had 92% of their sequences represented in the tef reference genome. In the maximum likelihood phylogenetic analysis, these wild species clearly showed grouping in the clade consisting of the entire tef germplasm. Population structure analysis showed two major clusters consistent with the germplasm class information and the inferred phylogenetic relationships. The wild Eragrostis species were more diverse than the tef cultivars and could therefore potentially be used to enrich the tef gene pool. The SNP dataset and the results documented here are taxonomically the most inclusive to date and could be a useful informational tool for the design of genomics-informed tef breeding and research

Identification of Drought Tolerant Mutant Lines of Tef [Eragrostis tef (Zucc.) Trotter]

Introduction: Tef [Eragrostis tef (Zucc.) Trotter] is the major food crop in Ethiopia where it is annually cultivated on about 30% of the total area allocated to cereals. The productivity of tef is, however, significantly reduced by drought which can occur at either the early or the late developmental stage of the crop. Methodology: A total of 10,000 EMS (ethyl methane sulfonate) mutagenized M2 tef populations were screened first for early and second for terminal drought tolerance. The performance of candidate lines was investigated using diverse agronomical and physiological parameters. Results and discussion: Several promising lines were obtained for both early and terminal drought tolerance. Among these, genotypes with outstanding performance were found for two drought tolerant tef lines targeting early drought tolerance (dtt2 and dtt13) and three terminal drought tolerant lines targeting terminal drought tolerance (tdt9, tdt15 and tdt19). Conclusions: Candidate lines for both early and terminal drought tolerance will play vital roles in deve- loping drought tolerance in tef improvement

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