Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe-set, consisting of up to 16 probe-pairs. Signal intensities across probe-pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays

Effects of green seaweed extract on Arabidopsis early development suggest roles for hormone signalling in plant responses to algal fertilisers

The growing population requires sustainable, environmentally-friendly crops. The plant growth enhancing properties of algal extracts have suggested their use as biofertilisers. The mechanism(s) by which algal extracts affect plant growth are unknown. We examined the effects of extracts from the common green seaweed Ulva intestinalis on germination and root development in the model land plant Arabidopsis thaliana. Ulva extract concentrations above 0.1% inhibited Arabidopsis germination and root growth. Ulva extract less than 0.1% stimulated root growth. All concentrations of Ulva extract inhibited lateral root formation.An abscisic-acid-insensitive mutant, abi1, showed altered sensitivity to germination- and root growth-inhibition. Ethylene- and cytokinin-insensitive mutants were partly insensitive to germination-inhibition. This suggests that different mechanisms mediate each effect of Ulva extract on early Arabidopsis development and that multiple hormones contribute to germination inhibition. Elemental analysis showed that Ulva contains high levels of Aluminium ions (Al3+). Ethylene and cytokinin have been suggested to function in Al3+-mediated root growth inhibition: our data suggest that if Ulva Al3+ levels inhibit root growth, this is via a novel mechanism. We suggest algal extracts should be used cautiously as fertilisers, as the inhibitory effects on early development may outweigh any benefits if the concentration of extract is too high

Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa

<p>Abstract</p> <p>Background</p> <p>'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip^®microarrays to profile the response of the banana (<it>Musa </it>spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed <it>Musa </it>transcripts.</p> <p>Results</p> <p>Following cross-hybridisation of <it>Musa </it>gDNA to the Rice GeneChip^®Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the <it>Musa </it>cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 <it>Musa </it>gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive <it>Musa </it>transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments.</p> <p>Conclusion</p> <p>Our results demonstrate that despite the general paucity of nucleotide sequence data in <it>Musa </it>and only distant phylogenetic relations to rice, gDNA probe-based cross-hybridisation to the Rice GeneChip^®is a highly promising strategy to study complex biological responses and illustrates the potential of such strategies for gene discovery in non-model species.</p

Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray

Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process

Evaluating the Use of Daily Care Notes Software for Older People with Dementia

There has been little research to investigate the impact of software to support the care for older people with dementia care. This article reports the evaluation of software adapted to support one key person-centered task for the care of older residents with dementia – recording and sharing daily care notes. The evaluation on the dementia wing of 1 residential home for over 6 months revealed that use of the software on mobile devices carried by the carers increased the number and volume of daily care notes recorded, but only for the types of content that were already being recorded by carers. Carers reported more advantages that resulted from daily care notes once in digital form than from the documenting task, as well as barriers to the use of mobile digital software to record daily care notes

Seasonal total methane depletion in limestone caves

Methane concentration in caves is commonly much lower than the external atmosphere, yet the cave CH4 depletion causal mechanism is contested and dynamic links to external diurnal and seasonal temperature cycles unknown. Here, we report a continuous 3-year record of cave methane and other trace gases in Jenolan Caves, Australia which shows a seasonal cycle of extreme CH4 depletion, from ambient ∼1,775 ppb to near zero during summer and to ∼800 ppb in winter. Methanotrophic bacteria, some newly-discovered, rapidly consume methane on cave surfaces and in external karst soils with lifetimes in the cave of a few hours. Extreme bacterial selection due to the absence of alternate carbon sources for growth in the cave environment has resulted in an extremely high proportion 2-12% of methanotrophs in the total bacteria present. Unexpected seasonal bias in our cave CH4 depletion record is explained by a three-step process involving methanotrophy in aerobic karst soil above the cave, summer transport of soil-gas into the cave through epikarst, followed by further cave CH4 depletion. Disentangling cause and effect of cave gas variations by tracing sources and sinks has identified seasonal speleothem growth bias, with implied palaeo-climate record bias

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background: Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results: We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions: Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service

Infestation by Myzus persicae Increases Susceptibility of Brassica napus cv. “Canard” to Rhizoctonia solani AG 2-1

Activation of plant defense pathways can be influenced by the presence of different species of attacking organisms. Understanding the complicated interactions triggering plant defense mechanisms is of great interest as it may allow the development of more effective and sustainable disease control methods. Myzus persicae and Rhizoctonia solani anastomosis group (AG) 2-1 are two important organisms attacking oilseed rape (OSR), causing disease and reduced yields. At present, is unclear how these two interact with each other and with OSR defenses and therefore the aim of the present study was to gain a better insight into the indirect interaction between aphids and pathogen. In separate experiments, we assessed the effect of AG 2-1 infection on aphid performance, measured as growth rate and population increase and then the effect of aphid infestation on AG 2-1 by quantifying disease and the amount of fungal DNA in plant stems and compost for two OSR varieties, “Canard” and “Temple.” Additionally, we examined the expression of genes related to jasmonic acid (JA) and salicylic acid (SA) defense pathways. There was no significant effect of AG 2-1 infection on M. persicae performance. However, aphid infestation in one of the varieties, “Canard,” resulted in significantly increased disease symptoms caused by AG 2-1, although, the amount of fungal DNA was not significantly different between treatments. This meant that “Canard” plants had become more susceptible to the disease. Expression of LOX3 and MYC2 was elevated under AG 2-1 treatment but downregulated in plants with both aphids and pathogen. Therefore it seems plausible that alterations in the JA signaling due to aphid infestation resulted in the increased susceptibility to AG 2-1

Low-intensity microwave irradiation does not substantially alter gene expression in late larval and adult Caenorhabditis elegans.

Reports that low-intensity microwave radiation induces heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg-1 for 6-well plates) that minimises temperature differentials between sham and exposed conditions (≤0.1 °C). Parallel measurement and simulation studies of SAR distribution within this exposure system are presented. We compared 5 Affymetrix gene-arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against 5 gene-arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run). No genes showed consistent expression changes across all 5 comparisons, and all expression changes appeared modest after normalisation (≤ 40% up- or down-regulated). The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131). We conclude that the pattern of gene expression in L4/adult C. elegans is substantially unaffected by low-intensity microwave radiation; the minor changes observed in this study could well be false positives. As a positive control, we compared RNA samples from N2 worms subjected to a mild heat-shock treatment (30ºC) against controls at 26 ºC (2 gene arrays per condition). As expected, heat-shock genes are strongly up-regulated at 30ºC, particularly an hsp-70 family member (C12C8.1) and hsp-16.2 . Under these heat-shock conditions, we confirmed that an hsp-16.2::GFP transgene was strongly up-regulated, whereas two non-heat-inducible transgenes (daf-16::GFP; cyp-34A9::GFP) showed little change in expression

Reference Intervals: Strengths, Weaknesses, and Challenges

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