Increased efficacy for in-house validation of real-time PCR GMO detection methods

To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSDr and RSDR) were calculated. The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods

Pollen-inferred regional vegetation patterns and demographic change in Southern Anatolia through the Holocene

Southern Anatolia is a highly significant area within the Mediterranean, particularly in terms of understanding how agriculture moved into Europe from neighbouring regions. This study uses pollen, palaeoclimate and archaeological evidence to investigate the relationships between demography and vegetation change, and to explore how the development of agriculture varied spatially. Data from 21 fossil pollen records have been transformed into forested, parkland and open vegetation types using cluster analysis. Patterns of change have been explored using non-metric multidimensional scaling (nMDS) and through analysis of indicator groups, such as an Anthropogenic Pollen Index, and Simpson’s Diversity. Settlement data, which indicate population densities, and summed radiocarbon dates for archaeological sites have been used as a proxy for demographic change. The pollen and archaeological records confirm that farming can be detected earlier in Anatolia in comparison with many other parts of the Mediterranean. Dynamics of change in grazing indicators and the OJCV (Olea, Juglans, Castanea and Vitis) index for cultivated trees appear to match cycles of population expansion and decline. Vegetation and land use change is also influenced by other factors, such as climate change. Investigating the early impacts of anthropogenic activities (e.g. woodcutting, animal herding, the use of fire and agriculture) is key to understanding how societies have modified the environment since the mid–late Holocene, despite the capacity of ecological systems to absorb recurrent disturbances. The results of this study suggest that shifting human population dynamics played an important role in shaping land cover in central and southern Anatolia

Pollen-inferred regional vegetation patterns and demographic change in Southern Anatolia through the Holocene

Southern Anatolia is a highly significant area within the Mediterranean, particularly in terms of understanding how agriculture moved into Europe from neighbouring regions. This study uses pollen, palaeoclimate and archaeological evidence to investigate the relationships between demography and vegetation change, and to explore how the development of agriculture varied spatially. Data from 21 fossil pollen records have been transformed into forested, parkland and open vegetation types using cluster analysis. Patterns of change have been explored using non-metric multidimensional scaling (nMDS) and through analysis of indicator groups, such as an Anthropogenic Pollen Index, and Simpson’s Diversity. Settlement data, which indicate population densities, and summed radiocarbon dates for archaeological sites have been used as a proxy for demographic change. The pollen and archaeological records confirm that farming can be detected earlier in Anatolia in comparison with many other parts of the Mediterranean. Dynamics of change in grazing indicators and the OJCV (Olea, Juglans, Castanea and Vitis) index for cultivated trees appear to match cycles of population expansion and decline. Vegetation and land use change is also influenced by other factors, such as climate change. Investigating the early impacts of anthropogenic activities (e.g. woodcutting, animal herding, the use of fire and agriculture) is key to understanding how societies have modified the environment since the mid–late Holocene, despite the capacity of ecological systems to absorb recurrent disturbances. The results of this study suggest that shifting human population dynamics played an important role in shaping land cover in central and southern Anatolia

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement

Allelic diversity of S‑RNase alleles in diploid potato species

S-ribonucleases (S-RNases) control the pistil specificity of the self-incompatibility (SI) response in the genus Solanum and several other members of the Solanaceae. The nucleotide sequences of S-RNases corresponding to a large number of S-alleles or S-haplotypes have been characterised. However, surprisingly few S-RNase sequences are available for potato species. The identification of new S-alleles in diploid potato species is desirable as these stocks are important sources of traits such as biotic and abiotic resistance. S-RNase sequences are reported here from three distinct diploid types of potato: cultivated Solanum tuberosum Group Phureja, S. tuberosum Group Stenotomum, and the wild species Solanum okadae. Partial S-RNase sequences were obtained from pistil RNA by RT-PCR or 3’RACE (Rapid Amplification of cDNA Ends) using a degenerate primer. Full length sequences were obtained for two alleles by 5’RACE. Database searches with these sequences, identified sixteen S-RNases in total, all of which are novel. The sequence analysis revealed all the expected features of functional S-RNases. Phylogenetic analysis with selected published S-RNase and S-like-RNase sequences from the Solanaceae revealed extensive trans-generic evolution of the S-RNases and a clear distinction from S-like-RNases. Pollination tests were used to confirm the self-incompatibility status and cross-compatibility relationships of the S. okadae accessions. All the S. okadae accessions were found to be self-incompatible as expected with crosses amongst them exhibiting both cross-compatibility and semi-compatibility consistent with the S-genotypes determined from the S-RNase sequence data. The progeny analysis of four semi-compatible crosses examined by allele-specific PCR provided further confirmation that these are functional S-RNases

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

<p>Abstract</p> <p>Background</p> <p>The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (<it>Panicum virgatum </it>L.) ubiquitin genes (<it>PvUbi1 </it>and <it>PvUbi2</it>) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters) fused to the <it>uidA </it>coding region (<it>GUS</it>) and tested for transient and stable expression in a variety of plant species and tissues.</p> <p>Results</p> <p><it>PvUbi1 </it>consists of 607 bp containing <it>cis</it>-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. <it>PvUbi2 </it>consists of 692 bp containing <it>cis</it>-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. <it>PvUbi1 </it>and <it>PvUbi2 </it>were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV <it>35S, 2x35S, ZmUbi1</it>, and <it>OsAct1 </it>promoters. GUS staining following stable transformation in rice demonstrated that the <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drove expression in all examined tissues. When stably transformed into tobacco (<it>Nicotiana tabacum</it>), the <it>PvUbi2+3 </it>and <it>PvUbi2+9 </it>promoter fusion variants showed expression in vascular and reproductive tissues.</p> <p>Conclusions</p> <p>The <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.</p