D. W. Cullen

Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers

Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g−1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan™ fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan™) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.

A PCR-based method for detection of potato pathogen, Helminthosporium solani, in silver scurf infected tuber tissue and soils.

Silver scurf caused by Helminthosporium solani causes significant economic losses in table stock, seed and processing potatoes. Specific polymerase chain reaction (PCR) primers, Hs1F1/Hs2R1, from H. solani were used for the amplification of a 447-bp product from 20 tissue samples and 54 single spore H. solani isolates, from eastern Canada (27 isolates), western Canada (13 isolates) and North Dakota in USA (14 isolates), but not from other potato fungal pathogens. In addition to PCR analysis, all 54 isolates were studied using conventional detection methods, visual disease symptoms and/or colony morphology and microscopic examination of the morphology of conidiophores and conidia. The PCR assay successfully detected H. solani and the PCR results correlated well with assessments based on conventional techniques. The detection of H. solani by PCR (1 day) is rapid and offers an alternative to the time consuming conventional diagnostic techniques (4-5 weeks). Nested PCR assay was necessary for the detection of H. solani in soils and thus can provide a sensitive technique to study the epidemiology of silver scurf in soils.

Sources of uncertainty in the quantification of genetically modified oilseed rape contamination in seed lots

Testing of seed and grain lots is essential in the enforcement of GM labelling legislation and needs reliable procedures for which associated errors have been identified and minimised. In this paper we consider the testing of oilseed rape seed lots obtained from the harvest of a non-GM crop known to be contaminated by volunteer plants from a GM herbicide tolerant variety. The objective was to identify and quantify the error associated with the testing of these lots from the initial sampling to completion of the real-time PCR assay with which the level of GM contamination was quantified.The results showed that, under the controlled conditions of a single laboratory, the error associated with the real-time PCR assay to be negligible in comparison with sampling error, which was exacerbated by heterogeneity in the distribution of GM seeds, most notably at a small scale, i.e. 25 cm3. Sampling error was reduced by one to two thirds on the application of appropriate homogenisation procedures.

Mapping and expression of genes associated with raspberry fruit ripening and softening

Key messageQTL mapping identifies a range of underlying and unrelated genes with apparent roles in raspberry fruit ripening and softening that show characteristic developing fruit expression profiles.AbstractFruit softening is an important agronomical trait that involves a complex interaction of plant cell processes. We have used both qualitative and quantitative scoring of fruit firmness, length, mass, and resistance to applied force to identify QTL in a raspberry mapping population. QTLs were located primarily on linkage group (LG) 3 with other significant loci on LG 1 and LG 5 and showed mostly additive effects between the two parents. The expression of key genes that underlie these QTLs with roles in cell-wall solubility, water uptake, polyamine synthesis, transcription, and cell respiration was tested across five stages of fruit development, from immature green to red ripe fruit, using real-time RT-qPCR. Gene expression patterns showed variable expression patterns across fruit development with a highly significant positive and negative correlation between genes, supporting precise regulation of expression of different cell processes throughout raspberry fruit development. Variable timing in expression was also found in some genes at different fruit development stages between soft and firm cultivars. Multiple processes have a role to play in fruit softening and this will require development of multiple marker combinations to genes that characterise raspberry fruit softening.

Towards an understanding of the control of ‘crumbly’ fruit in red raspberry

The genetic disorder known as ‘crumbly’ fruit is becoming a serious problem in the European raspberry industry. The study set out to examine the crumbly phenotype in a red raspberry mapping population under two environments (field and polytunnel) across six seasons in an effort to understand variability of the syndrome and to examine whether genetic factors were important and if so, whether QTL associated with the phenotype could be identified. This highlighted that seasonal, environmental (field or polytunnel) and genetic factors all influence the condition. Two QTL that are important for the genetic control of the condition have been located on linkage groups one and three, and an association with ripening time has been identified.

Effect of short-term exposure to high-temperature on total gene expression in the leaves of four raspberry (Rubus idaeus L.) cultivars

Summary The effect of high temperature stress (27ºC or 37ºC for 24 h) on total gene expression profiles in the annual-fruiting raspberry (Rubus idaeus L.) cultivars ‘Autumn Bliss’, ‘Autumn Treasure’, ‘Erika’, and ‘Polka’ were evaluated at the floral initiation stage using a customised Rubus microarray. Significantly affected genes were obtained by pairwise t-tests using ‘volcano plots’ for each cultivar × treatment. A 10ºC elevation in temperature altered levels of expression, in at least one cultivar, of 644 differentially expressed genes in total, with ‘Erika’ and ‘Autumn Treasure’ showing elevated expression of 38 genes compared to ‘Autumn Bliss’ and ‘Polka’. We identified 12 common candidate genes that were modulated differentially in ‘Autumn Bliss’ and ‘Erika’ at 37ºC compared to 27ºC. In addition, two aquaporin genes (PIP1 and TIP2) were down-regulated in ‘Autumn Bliss’, but up-regulated in ‘Autumn Treasure’, ‘Polka’, and ‘Erika’ at 37ºC. Other down-regulated genes from the list of 38 genes included those encoding major latex-like proteins, plasma membrane proteins, cysteine rich proteins, and other stress-related proteins. Validation by real-time quantitative RT-PCR (RT-qPCR) indicated subtle changes in differential gene expression, suggesting a mild response to heat stress. This study used molecular tools to increase our understanding of, and to identify candidate genes involved in, the heat stress response of four annual-fruiting raspberry cultivars.

A Transcript and Metabolite Atlas of Blackcurrant Fruit Development Highlights Hormonal Regulation and Reveals the Role of Key Transcription Factors

Blackcurrant fruit collected at six stages of development were assessed for changes in gene expression using custom whole transcriptome microarrays and for variation in metabolite content using a combination of liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Principal components analysis demonstrated that fruit development could be clearly defined according to their transcript or metabolite profiles. During early developmental stages, metabolite profiles were dominated by amino acids and tannins, whilst transcript profiles were enriched in functions associated with cell division, anatomical structure morphogenesis and cell wall metabolism. During mid fruit development, fatty acids accumulated and transcript profiles were consistent with seed and embryo development. At the later stages, sugars and anthocyanins accumulated consistent with transcript profiles that were associated with secondary metabolism. Transcript data also indicated active signaling during later stages of fruit development. A targeted analysis of signaling networks revealed a dynamic activation and repression of almost 60 different transcripts encoding transcription factors across the course of fruit development, many of which have been demonstrated as pivotal to controlling such processes in other species. Transcripts associated with cytokinin and gibberellin were highly abundant at early fruit development, whilst those associated with ABA and ethylene tended to be more abundant at later stages. The data presented here provides an insight into fruit development in blackcurrant and provides a foundation for further work in the elucidation of the genetic basis of fruit quality.