High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15–45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.     

Analysis of household's energy consumption,forest degradation and plantation requirements in Eastern Tigray,Northern Ethiopia

This study examines the link between household's energy consumption, forest degradation and plantation requirements in Eastern Tigray. Data on household's fuelwood, cow‐dung, kerosene and electricity consumption in the study sites were collected from 557 urban and 114 rural households. Deforestation rate was estimated following FAO and Woody Biomass Inventory Strategy Planning Project (WBISPP's) conversion factors. The study showed that household's fuelwood consumption in urban areas (mean = 2.5 kg/day) was about 52% lower as compared to the rural villages (mean = 3.8 kg/day). The estimated area abandoned annually in the vicinity of urban areas as the result of urban fuelwood consumption varies from 217 ha around Maichew town to 12047 ha around Mekelle City. The estimated area abandoned in rural areas annually as the result of tree cutting for fuelwood also varies from 6.5 ha around Sinkata to 127 ha around Dergajen village. It is therefore recommended that an estimated annual tree plantation of area ranging from 286 ha around Maichew town to 21,684 ha around Mekelle city is required. Moreover, an estimated annual tree plantation of area ranging from 16 ha around Sinkata village to 229 ha around the Dergajen village is required taking the existing population into account.     

Identification and quantification of differentially expressed proteins in E-cadherin deficient SCC9 cells and SCC9 transfectants expressing E-cadherin by dimethyl isotope labeling, LC-MALDI MS and MS/MS

A strategy based on isotope labeling of peptides and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS) has been employed to accurately quantify and confidently identify differentially expressed proteins between an E-cadherin-deficient human carcinoma cell line (SCC9) and its transfectants expressing E-cadherin (SCC9-E). Proteins extracted from each cell line were tryptically digested and the resultant peptides were labeled individually with either d(0)- or d(2)-formaldehyde. The labeled peptides were combined and the peptide mixture was separated and fractionated by a strong cation exchange (SCX) column. Peptides from each SCX fraction were further separated by a microbore reversed-phase (RP) LC column. The effluents were then directly spotted onto a MALDI target using a heated droplet LC-MALDI interface. After mixing with a MALDI matrix, individual sample spots were analyzed by MALDI quadrupole time-of-flight MS, using an initial MS scan to quantify the dimethyl labeled peptide pairs. MS/MS analysis was then carried out on the peptide pairs having relative peak intensity changes of greater than 2-fold. The MS/MS spectra were subjected to database searching for protein identification. The search results were further confirmed by comparing the MS/MS spectra of the peptide pairs. Using this strategy, we detected and compared relative peak intensity changes of 5480 peptide pairs. Among them, 320 peptide pairs showed changes of greater than 2-fold. MS/MS analysis of these changing pairs led to the identification of 49 differentially expressed proteins between the parental SCC9 cells and SCC9-E transfectants. These proteins were determined to be involved in different pathways regulating cytoskeletal organization, cell adhesion, epithelial polarity, and cell proliferation. The changes in protein expression were consistent with increased cell-cell and cell-matrix adhesion and decreased proliferation in SCC9-E cells, in line with E-cadherin tumor suppressor activity. Finally, the accuracy of the MS quantification and subcellular localization for 6 differentially expressed proteins were validated by immunoblotting and immunofluorescence assays.