Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations

A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications

Global characterization of the root transcriptome of a wild species of rice, Oryza longistaminata, by deep sequencing

<p>Abstract</p> <p>Background</p> <p><it>Oryza longistaminata</it>, an AA genome type (2 n = 24), originates from Africa and is closely related to Asian cultivated rice (<it>O. sativa L</it>.). It contains various valuable traits with respect to tolerance to biotic and abiotic stress, QTLs with agronomically important traits and high ability to use nitrogen efficiently (NUE). However, only limited genomic or transcriptomic data of <it>O. longistaminata </it>are currently available.</p> <p>Results</p> <p>In this study we present the first comprehensive characterization of the <it>O. longistaminata </it>root transcriptome using 454 pyrosequencing. One sequencing run using a normalized cDNA library from <it>O. longistaminata </it>roots adapted to low N conditions generated 337,830 reads, which assembled into 41,189 contigs and 30,178 singletons. By similarity search against protein databases, putative functions were assigned to over 34,510 uni-ESTs. Comparison with ESTs derived from cultivated rice collections revealed expressed genes across different plant species, however 16.7% of the <it>O. longistaminata </it>ESTs had not been detected as expressed in <it>O. sativa</it>. Additionally, 15.7% had no significant similarity to known sequences. RT-PCR and Southern blot analyses confirmed the expression of selected novel transcripts in <it>O. longistaminata</it>.</p> <p>Conclusion</p> <p>Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates sufficient genomic information for adequate de novo assembly of a large number of transcripts in a wild rice species, <it>O. longistaminata</it>. The generated sequence data are publicly available and will facilitate gene discovery in <it>O. longistaminata </it>and rice functional genomic studies. The large number of abundant of novel ESTs suggests different metabolic activity in <it>O. longistaminata </it>roots in comparison to <it>O. sativa </it>roots.</p

Diversity of Bradyrhizobia in Subsahara Africa: A Rich Resource

Making use of biological nitrogen fixation (BNF) with pulses and green manure legumes can help to alleviate nitrogen deficiencies and increase soil fertility, problems faced particularly in smallholder agriculture in Subsahara Africa (SSA). The isolation of indigenous rhizobia provides a basis for the formulation of rhizobial inoculants. Moreover, their identification and characterization contribute to the general understanding of species distribution and ecology. Here we discuss global species discovery of Bradyrhizobium spp. Although recently the number of validly published Bradyrhizobium species is rapidly increasing, their diversity in SSA is not well-represented. We summarize the recent knowledge on species diversity in the Bradyrhizobium yuanmingense lineage to which most SSA isolates belong, and their biogeographic distribution and adaptations. Most indigenous rhizobia appear to differ from species found on other continents. We stress that an as yet hidden diversity may be a rich resource for inoculant development in future. As some species are exceptionally temperature tolerant, they may be potential biofertilizer candidates for global warming scenarios

Catch crop diversity increases rhizosphere carbon input and soil microbial biomass

Catch crops increase plant species richness in crop rotations, but are most often grown as pure stands. Here, we investigate the impacts of increasing plant diversity in catch crop rotations on rhizosphere C input and microbial utilization. Mustard (Sinapis alba L.) planted as a single cultivar was compared to diversified catch crop mixtures of four (Mix4) or 12 species (Mix12). We traced the C transfer from shoots to roots towards the soil microbial community and the soil respiration in a 13C pulse labelling field experiment. Net CO2-C uptake from the atmosphere increased by two times in mix 4 and more than three times in mix 12. Higher net ecosystem C production was linked to increasing catch crop diversity and increased belowground transfer rates of recently fixed photoassimilates. The higher rhizosphere C input stimulated the growth and activity of the soil microbiome, which was investigated by phospholipid fatty acid (PLFA) analyses. Total microbial biomass increased from 14 to 22 g m−2 as compared to the fallow and was 18 and 8% higher for mix 12 and mix 4 as compared to mustard. In particular, the fungal and actinobacterial communities profited the most from the higher belowground C input and their biomass increased by 3.4 and 1.3 times as compared to the fallow. The residence time of the 13C pulse, traced in the CO2 flux from the soil environment, increased with plant diversity by up to 1.8 times. The results of this study suggest positive impacts of plant diversity on C cycling by higher atmospheric C uptake, higher transport rates towards the rhizosphere, higher microbial incorporation and prolonged residence time in the soil environment. We conclude that diversified catch crop mixtures improve the efficiency of C cycling in cropping systems and provide a promising tool for sustainable soil management

The 125th anniversary of the first postulation of the soil origin of endophytic bacteria – a tribute to M.L.V. Galippe

In both managed and natural ecosystems, a wide range of various non-nodulating bacteria can thrive as endophytes in the plant interior, and some can be beneficial to their hosts (Hallmann and Berg 2007; Reinhold-Hurek and Hurek 2011). Colonizationmechanisms, the ecology and functioning of these endophytic bacteria as well as their interactions with plants have been investigated (Hardoim et al. 2008; Compant et al. 2010). Although the source of colonization can also be the spermosphere, anthosphere, caulosphere, and the phyllosphere,most endophytic bacteria are derived from the soil environment (Hallmann and Berg 2007; Compant et al. 2010)

Environmental Factors Affecting the Expression of pilAB as Well as the Proteome and Transcriptome of the Grass Endophyte Azoarcus sp. Strain BH72

gene encoding the structural protein of type IV pili that are essential for plant colonization appears to be regulated in a population density-dependent manner.. The expression of one of them was shown to be induced in plant roots. sp. to analyze mechanisms and molecules involved in the population-dependent gene expression in this endophyte in future

Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants

Burkholderia phytofirmans PsJN is a naturally occurring plant-associated bacterial endophyte that effectively colonizes a wide range of plants and stimulates their growth and vitality. Here we analyze whole genomes, of PsJN and of eight other endophytic bacteria. This study illustrates that a wide spectrum of endophytic life styles exists. Although we postulate the existence of typical endophytic traits, no unique gene cluster could be exclusively linked to the endophytic lifestyle. Furthermore, our study revealed a high genetic diversity among bacterial endophytes as reflected in their genotypic and phenotypic features. B. phytofirmans PsJN is in many aspects outstanding among the selected endophytes. It has the biggest genome consisting of two chromosomes and one plasmid, well-equipped with genes for the degradation of complex organic compounds and detoxification, e.g., 24 glutathione-S-transferase (GST) genes. Furthermore, strain PsJN has a high number of cell surface signaling and secretion systems and harbors the 3-OH-PAME quorum-sensing system that coordinates the switch of free-living to the symbiotic lifestyle in the plant-pathogen R. solanacearum. The ability of B. phytofirmans PsJN to successfully colonize such a wide variety of plant species might be based on its large genome harboring a broad range of physiological functions

Catch crop mixtures have higher potential for nutrient carry-over than pure stands under changing environments

Winter catch crops are grown to scavenge nutrients over a period of unfavorable growth conditions and to conserve nutrients for subsequent release to the following main crop. Since environmental conditions have a strong impact on the growth and nutrient capture in roots and shoots of individual catch crop species, we anticipated that mixtures will be more durable and efficient in nutrient capture due to compensatory effects among component species. We tested this hypothesis and determined the nitrogen and phosphorus accumulation in the shoots and roots of four catch crop species grown in pure vs. mixed stands at two sites for two or three years. Element concentrations were determined in the root and shoot biomass of each species and used to calculate the nutrient pool fixed in the root or shoot biomass. A qPCR-based technique was applied to quantify the root biomass of individual species based on species-specific DNA sequences. Despite considerable variation across environments, the overall plant biomass of white mustard (Sinapis alba), lacy phacelia (Phacelia tanacetifolia) and bristle oat (Avena strigosa) was similar and higher than that of Egyptian clover (Trifolium alexandrinum). While pure stands varied 6- to 24-fold in shoot biomass depending on environmental conditions, the variation was only ~3-fold for catch crop mixtures, with less pronounced variation in the root biomass. In general, the root biomass was comparable to the shoot biomass in each species. Roots contributed 26–46% of the nitrogen and 36–48% of the phosphorus to the total accumulation of these nutrients in the catch crop biomass, thus emphasizing the importance of plant roots as belowground nutrient pool for potential carry-over of nutrients to the subsequent crop. Although the mixture was mostly dominated by two of the four species, namely mustard and phacelia, it captured similar or even larger amounts of nutrients than the best-performing pure stand under any growth condition. This was the case for shoot- and for root-bound nutrients. Our results indicate that catch crop mixtures have higher durability than pure cultures to environmental variations. The amount of nitrogen captured by the mixture meets the average postharvest nitrogen that is left over by a wide range of cash crops, thus emphasizing that catch crop mixtures represent an efficient nutrient management tool in crop rotations. © 202