To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool 1 . Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43 , which was crossed into bread wheat from the wild grass Thinopyrum elongatum 2 , 3 . Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.

Fusarium head blight (FHB), a fungus disease of small grain cereal crops, results in reduced yields and dimin ished value of harvested grain due to the presence of deoxynivalenol (DON), a mycotoxin produced by the causal pathogen Fusarium graminearum. DON and other tricothecene mycotoxins pose serious health risks to both humans and livestock, especially swine. Due to these health concerns, barley used for malting, food or feed is routinely assayed for DON levels. Various methods are available for assaying DON levels in grain samples including enzyme-linked immunosorbent assay (ELISA) and gas chromatography-mass spectrometry (GC-MS). ELISA and GC-MS are very accurate; however, assaying grain samples by these techniques are laborious, expensive and destructive. In this study, we explored the feasibility of using hyperspectral imaging (382-1030 nm) to develop a rapid and non-destructive protocol for assaying DON in barley kernels. Samples of 888 and 116 from various genetic lines were selected for calibration and prediction. Full-wavelength locally weighted partial least squares regression (LWPLSR) achieved high accuracy with the coefficient of determination in prediction (R-P(2)) of 0.728 and root mean square error of prediction (RMSEP) of 3.802. Competitive adaptive reweighted sampling (CARS) was used to choose potential feature wavelengths, and these selected variables were further optimized using the iterative selection of successive projections algorithm (ISSPA). The CARS-ISSPA-LWPLSR model developed using 7 feature variables yielded R-P(2) of 0.680 and RMSEP of 4.213 in DON content prediction. Based on the 7 wavelengths selected by CARS-ISSPA, partial least square discriminant analysis (PLSDA) discriminated barley kernels having lower DON (less than1.25 mg/kg) levels from those with higher levels (including 1.25-3 mg/kg, 3-5 mg/kg, and 5-10 mg/kg), with Matthews correlation coefficient in crossvalidation (M-RCV) of as high as 0.931. The results demonstrate that hyperspectral imaging have potential for accelerating non-destructive DON assays of barley samples.

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel.

Genetic resistance is the most economic and environmentally sustainable approach for crop disease protection. Disease resistance (R) genes from wild relatives are a valuable resource for breeding resistant crops. However, introgression of R genes into crops is a lengthy process often associated with co-integration of deleterious linked genes 1, 2 and pathogens can rapidly evolve to overcome R genes when deployed singly 3 . Introducing multiple cloned R genes into crops as a stack would avoid linkage drag and delay emergence of resistance-breaking pathogen races 4 . However, current R gene cloning methods require segregating or mutant progenies 5–10 , which are difficult to generate for many wild relatives due to poor agronomic traits. We exploited natural pan-genome variation in a wild diploid wheat by combining association genetics with R gene enrichment sequencing (AgRenSeq) to clone four stem rust resistance genes in <6 months. RenSeq combined with diversity panels is therefore a major advance in isolating R genes for engineering broad-spectrum resistance in crops.

We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen.Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F-6 recombinant inbred line and an F-2 population, two genes were identified that mapped to the short arm of chromosome 1S(sh), designated as Sr-1644-1Sh, and the long arm of chromosome 5S(sh), designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

Muscle is a highly heterogeneous tissue. Practical approaches to sample selectively small regions of muscle cross sections would help to effectively utilize analytical techniques on muscle studies while taking into account tissue heterogeneity. In this report, semimembranosus muscle tissue cross sections were directly sampled and analyzed by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Prior to CE-LIF analysis, a small region in the muscle cross section was stained with 10-nonyl acridine orange (NAO) which is a mitochondrion-selective fluorescent probe known to form a stable complex with cardiolipin, a phospholipid found only in mitochondria. By micromanipulation, the injection end of the capillary was brought into contact with the tissue exhibiting fluorescently labeled mitochondria. Sampling from a region similar in size to the cross section of a single fiber was carried out by applying 11 kPa of negative pressure for 3 s. When an electric field of -200V/cm was applied, fluorescently labeled mitochondria electromigrated and were individually detected by postcolumn LIF detection. For each sample, the electropherogram displays a migration time window with a collection of narrow peaks. The collection of individual peak measurements is represented as a distribution of individual intensities related to cardiolipin content of mitochondria and a distribution of individual electrophoretic mobilities. Positioning the capillary injection end was sufficiently spatially accurate to deplete mitochondria in the sampled region upon repetitive injections. Treatment of a muscle cross section with a protease (trypsin) prior to mitochondria sampling resulted in a higher number of detected mitochondria, suggesting that one of the effects of this enzyme is a partial digestion of the muscles myofibrils, which eases the release of interfibrillar mitochondria entangled within these fibers. The protease treatment also resulted in changes to the electrophoretic mobility distribution of individual mitochondria, which may imply that partial digestion of proteins bound to the mitochondria contributes to the alteration in the electrophoretic mobility of mitochondria. The ability to sample a region as small as a single muscle fiber cross section and its direct CE-LIF analysis opens exciting possibilities for the direct analysis of muscle biopsies and mapping the mitochondrial electrophoretic properties in highly heterogeneous tissues.