TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes.

The discovery of an apoplastic enzyme responsible for esterifying carotenoids that is unexpectedly spatially separated from its plastidic substrate, lutein, aff

Wheat grain proteins responses to mixing and thermal treatment were investigated using Mixolab-dough analysis systems with flour from two cultivars, Ventura-26 (normal amylose content) and Ventura-19 (reduced amylose content). Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis revealed that, stress associated and metabolic proteins largely interacted with dough matrix of Ventura-26 after 26min (56°C); gliadins, avenin-like b proteins, LMW-GSs, and partial globulins showed stronger interactions within the dough matrix of Ventura-26 at 32min/C3 (80°C), thereafter, however, stronger protein interactions were observed within the dough matrix of Ventura-19 at 38min/C4 (85°C) and 43min (80°C). Thirty-seven proteins associated with changes in dough matrix due to reduced amylose content were identified by mass spectrometry and mainly annotated to the chromosome group 1, 4, and 6. The findings provide new entry points for modifying final product attributes.

Triacylglycerol is a major component of vegetable oil in seeds and fruits of many plants, but its production in vegetative tissues is rather limited. It would be intriguing and important to explore any possibility to expand current oil production platforms, for example from the plant vegetative tissues. By expressing a suite of transgenes involved in the triacylglycerol biosynthesis, we have previously observed substantial accumulation of triacylglycerol in tobacco (Nicotiana tabacum) leaf and potato (Solanum tuberosum) tuber. In this study, simultaneous RNA interference (RNAi) downregulation of ADP-glucose pyrophosphorylase (AGPase) and Sugar-dependent1 (SDP1), was able to increase the accumulation of triacylglycerol and other lipids in both wild type potato and the previously generated high oil potato line 69. Particularly, a 16-fold enhancement of triacylglycerol production was observed in the mature transgenic tubers derived from the wild type potato, and a two-fold increase in triacylglycerol was observed in the high oil potato line 69, accounting for about 7% of tuber dry weight, which is the highest triacylglycerol accumulation ever reported in potato. In addition to the alterations of lipid content and fatty acid composition, sugar accumulation, starch content of the RNAi potato lines in both tuber and leaf tissues were also substantially changed, as well as the tuber starch properties. Microscopic analysis further revealed variation of lipid droplet distribution and starch granule morphology in the mature transgenic tubers compared to their parent lines. This study reflects that the carbon partitioning between lipid and starch in both leaves and non-photosynthetic tuber tissues, respectively, are highly orchestrated in potato, and it is promising to convert low-energy starch to storage lipids via genetic manipulation of the carbon metabolism pathways.

In mammals Orexin-A and -B are neuropeptides involved in the hypothalamic regulation of diverse physiological functions including food intake and the sleep-wake cycle. This generalisation was investigated in meat-(broiler) and layer-type juvenile domestic chickens by immunocytochemical localisation of orexin A/B in the hypothalamus, and by measurements of hypothalamic hypocretin mRNA which encodes for orexin A/B after chronic food restriction, and during the sleep-wake cycle. Orexin immunoreactive fibres were observed throughout the hypothalamus with cell bodies in and around the paraventricular nucleus. No differences were observed in the pattern of immunoreactivity using anti- human orexin-A, or -B antisera. The amount of hypothalamic hypocretin mRNA in food -restricted broilers was higher than in broilers fed ad libitum, but the same as in layer- type hens fed ad libitum. Hypothalamic hypocretin mRNA was increased (P<0.01) in 12-week-old broilers fed 25% of their ad libitum intake between 6-12 weeks of age. No difference in hypothalamic hypocretin mRNA was seen in 12-week-old layer- type hens when they were awake (1-2h after lights on) or sleeping (1-2h after lights off). It is concluded that in the chicken, we could not find evidence that hypothalamic orexin plays a role in the sleep-wake cycle and it may be involved in aspects of energy balance.