Gefu Wang-Pruski

Potato Expressed Sequence Tag Generation and Analysis using Standard and Unique cDNA Libraries

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5′ end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3′ ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences.

Potato after-cooking darkening

After-cooking darkening (ACD) is one of the most widespread, undesirable characteristics of cultivated potato. With the current expansion of the potato-processing industry around the world, there is a renewed interest in the development of new ways to prevent ACD. After-cooking darkening is caused by the oxidation of the ferri-chlorogenic acid in the boiled or fried potatoes. The severity of the darkening is dependent on the ratio of chlorogenic acid to citric acid concentrations in the potato tubers. Higher ratio normally results in darker tubers. The concentration of the chlorogenic and citric acids is genetically controlled and influenced by environmental conditions. This paper outlines the history of ACD and current status of knowledge of the chemistry of the dark pigment formation and its genetic and environmental determinants. Also discussed are the methods of chemical prevention of ACD presently used by the potato-processing industry and potential strategies for reducing tuber after cooking darkening using molecular approaches.ResumenEl oscurecimiento después de la cocción (ACD siglas en Inglés) es una de las características indeseables mas difundidas en el cultivo de la papa. Con la expansión creciente de la industria de procesamiento de papa en la actualidad, existe un interés renovado en desarrollar formas nuevas para prevenir el ACD. El oscurecimiento después de la cocción es causado por oxidación del ácido ferri-clorogénico en las papas hervidas o fritas. La severidad del oscurecimiento depende de la proporción de ácido clorogénico y la concentración de ácido cítrico en los tubérculos de papa. Una mayor proporción da normalmente como resultado tubérculos más oscuros. La concentración de los ácidos clorogénico y cítrico es controlada genéticamente e influenciada por las condiciones del medio ambiente. Este artículo destaca la historia del ACD y el estado actual del conocimiento de la química involucrada en la formación del pigmento oscuro y sus determinantes genético y medio ambiental. También discute los métodos de prevención química del ACD, actualmente usados por la industria de procesamiento de papa y las estrategias potenciales para reducir el oscurecimiento después de la cocción, utilizando un enfoque molecular.

Proteomics analysis suggests broad functional changes in potato leaves triggered by phosphites and a complex indirect mode of action against Phytophthora infestans

UNLABELLED Phosphite (salts of phosphorous acid; Phi)-based fungicides are increasingly used in controlling oomycete pathogens, such as the late blight agent Phytophthora infestans. In plants, low amounts of Phi induce pathogen resistance through an indirect mode of action. We used iTRAQ-based quantitative proteomics to investigate the effects of phosphite on potato plants before and after infection with P. infestans. Ninety-three (62 up-regulated and 31 down-regulated) differentially regulated proteins, from a total of 1172 reproducibly identified proteins, were identified in the leaf proteome of Phi-treated potato plants. Four days post-inoculation with P. infestans, 16 of the 31 down-regulated proteins remained down-regulated and 42 of the 62 up-regulated proteins remained up-regulated, including 90% of the defense proteins. This group includes pathogenesis-related, stress-responsive, and detoxification-related proteins. Callose deposition and ultrastructural analyses of leaf tissues after infection were used to complement the proteomics approach. This study represents the first comprehensive proteomics analysis of the indirect mode of action of Phi, demonstrating broad effects on plant defense and plant metabolism. The proteomics data and the microscopy study suggest that Phi triggers a hypersensitive response that is responsible for induced resistance of potato leaves against P. infestans. BIOLOGICAL SIGNIFICANCE Phosphie triggers complex functional changes in potato leaves that are responsible for the induced resistance against Phytophthora infestans. This article is part of a Special Issue entitled: Translational Plant Proteomics.

RAPD and pedigree-based genetic diversity estimates in cultivated diploid potato hybrids.

In this study, RAPD and pedigree data were used to investigate the genetic relationships in a group of 45 diploid hybrid potato clones used in the breeding and genetics program of the Agriculture and Agri-Food Canada Potato Research Centre in Fredericton, New Brunswick, and used for the potato after-cooking darkness program at the Nova Scotia Agricultural College. These hybrids were derived from crossing primitive cultivated South American diploid species such as Solanum phureja or Solanum stenotomum and wild diploid species such as Solanum chacoense and other wild Argentine species with haploids of Solanum tuberosum. These hybrids have subsequently undergone up to 30 years of breeding and selection, for adaptation to local growing and storage conditions, processing traits and pest resistances. The objectives of this study were to estimate the level of genetic similarity (GS) among these sets of clones and to investigate the correlation between RAPD-based GS and f, based on pedigree information. Genetic similarity coefficients varied from 0.29 to 0.90 with a mean of 0.65 when based on the RAPD data, whereas the coefficient of parentage varied from zero to 0.75 with a mean of 0.11. The degree of relationship between the similarity matrices based on RAPD and pedigree was measured by comparing the similarity matrices with the normalized Mantel test. A low positive correlation (R = 0.104, p = 0.999) between the two matrices was observed. Cluster analysis using GS divided the clones into many subgroups that did not correspond well with the grouping based on pedigree. The level of genetic variation present in this set of potato clones is very high. Rigorous selection pressure aimed at different breeding purposes may result in the genetic differentiation of the clones from the same origin.

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Genetic Transformation in Potato: Approaches and Strategies

Improvements in methodology and transformation vectors have helped to increase the transformation efficiency and stable expression of transgenes in potato. Genetic transformation in potato has many benefits and advantages over conventional breeding methods, and potato was one of the first crops to be transformed. Potato transgenic research carried out so far has mainly focused on producing lines resistant to pathogens and modification of potato physiology. This review examines the achievements and current trends in potato transformation technology as well as the biological and practical requirements for the transformation systems. Problems with the application of genetically modified crops for agricultural practices and public concerns are also discussed.ResumenLas mejoras en la metodología y los vectores de transformación han ayudado a incrementar la eficiencia de transformación y lograr la expresión estable de los transgenes en papa. La transformación genética tiene muchas ventajas sobre los métodos convencionales de mejoramiento y la papa fue uno de los primeros cultivos en ser transformados. La investigación transgénica de la papa llevada a cabo hasta ahora se basa en la producción de líneas resistentes a los patógenos y la modificación de la fisiologóa de la planta. Esta revisión examina los logros y tendencias actuales de la tecnología de transformación y los requerimientos biológicos y prácticos de los sistemas de transformación. También se discuten los problemas asociados al uso agrícola de los cultivos genéticamente modificados y las preocupaciones del público.

Relative quantitative proteomic analysis reveals wound response proteins correlated with after-cooking darkening.

Many common potato tuber defects are difficult to elicidate because of the degree of genetic complexity involved, making systems biology approaches necessary. Interaction between chlorogenic acid and iron is responsible for the darkening of potato tuber tissues upon heating – termed after‐cooking darkening (ACD). To explore mechanisms of darkening severity in tuber tissues, we have employed relative quantitative proteomics to discover differentially expressed proteins involved in ACD. Tuber tissue samples were collected from a family of diploid clones which possess a highly segregated degree of the darkening. Exploiting this segregation, as well as the observation that darkening is more prevalent in the stem end of the tuber than the apical end, three sample groups were formed: (i) stem ends of three high‐ACD clones, (ii) stem ends of three low‐ACD clones, and (iii) apical ends of three low‐ACD clones. Protein samples were digested and differentially labeled using isotopic reductive methylation, allowing for an orthogonal two‐way comparison of protein profiles of the sample groups using 2‐D‐LC‐MS/MS. Using a cutoff fold change of 2 between the high‐ and the low‐ACD sample groups, 30 proteins showed a correlation with tissue darkening. Overall, we observed changes in relative protein abundance that showed an enhanced wound‐response program in high‐ACD tissues. Among these proteins, five proteins were further validated at the transcript level using qRT‐PCR. These proteins may be incorporated into design strategies to create potato cultivars with low levels of ACD.

Finding the perfect potato: using functional genomics to improve disease resistance and tuber quality traits

One of the best known plant disease outbreaks of all time was the Irish potato famine of 1845 to 1847 when the late blight pathogen, Phytophthora infestens, devastated the potato crop and caused more than one million deaths in Ireland. Potatoes are still plagued by disease but luckily most countries do not depend on potato as much as they did in Ireland in the 1800s. Producing disease-free potatoes, however, has come at a huge cost. To combat major diseases, such as late blight and common scab, 64 × 106 kg of pesticides are sprayed on potato fields each year. The costs associated with these measures are not only financial; environmental costs, although less easy to quantify, are significant and include negative impacts on natural ecosystems and the contamination of groundwater, lakes, and rivers. Our search for the “perfect” potato, i.e., one that has good processing qualities and disease resistance, has gained new momentum with the emergence of genomic technologies. Through functional genomics we will gain a better understanding of the genes responsible for tuber quality traits and those responsible for disease resistance. With a collection of desirable genes in mind, we can again use genomics as a diagnostic tool to search for these genes in the wide variety of potatoes around the world and to follow their transfer by classical breeding. This paper describes a research program currently underway in Canada that uses functional genomics to improve the potato.

Effect of soil type and nutrient management on potato after-cooking darkening

After-cooking darkening (ACD) is a common undesirable trait of potato tubers, caused by the oxidation of the chlorogenic acid-iron compound after cooking, which includes boiling, frying and dehydration. The degree of the darkening is known to be cultivar-dependent and to vary with climatic conditions. However, there is limited information on the effect of different management practices and long-term storage on ACD. In this study, we used three independent experiments to investigate the effects of soil type, different management practices and storage on the susceptibility of ACD. Experiment 1 compared two soil types, two planting dates, and two rates of nitrogen (N), phosphorus (P) and potassium (K) fertilization in two years for ‘Russet Burbank’ potato. Experiment 2 compared seven rates of N fertilization in two years for Russet Burbank and ‘Shepody’ potato. Experiment 3 compared five rates of K fertilization in two years on Russet Burbank potato. In general, the degree of darkening was reduced when climatic conditions and management practices resulted in conditions favorable for crop growth. The degree of ACD generally increased under drier climatic conditions, in soil that was more susceptible to drought, and with delayed planting; however, these effects were not measured in all cases. Similarly, the degree of ACD increased under N deficient conditions. However, N fertilization did not reduce darkening in all trials, and the benefit of N fertilization was not apparent as storage duration was increased. There was a small benefit to K fertilization in Experiment 3; however, K fertilization had no effect on ACD in Experiment 1. There was no effect of P fertilization on ACD. Although significant, the magnitude of the effects of soil type and management practices on ACD was small in comparison to the effects of cultivar and storage duration on ACD.ResumenEl oscurecimiento después de la cocción (ACD) es una característica común e indeseable de los tubérculos de papa y es causado por la oxidación del compuesto ácido clorogénico y el hierro después de cocidos, lo cual incluye hervido, fritura y deshidratado. El grado de oscurecimiento se sabe que depende del cultivar y que varía con las condiciones climáticas. Sin embargo, existe poca información del efecto de diferentes prácticas de mane jo y de almacenamiento por períodos prolongados sobre el ACD. En este estudio, hemos utilizado tres experimentos independientes para investigar los efectos del tipo de suelo, diferentes prácticas de manejo y almacenamiento sobre ACD. El experimento 1 comparó dos tipos de suelo, dos fechas de siembra y dos dosis de fertilización de nitrógeno (N), fósforo (P) y potasio (K) en dos años para papa ‘Russet Burbank’. El experimento 2 comparó siete índices de fertilización con N en dos años para papa Russet Burbank y ‘Shepody’. El experimento 3 comparó cinco índices de fertilización de K en dos años sobre papa Russet Burbank. En general, el grado de oscurecimiento se redujo cuando las condiciones climáticas y prácticas de manejo fueron favorables para el desarrollo del cultivo. El grado de ACD generalmente se incrementó bajo condiciones de clima más seco en suelos susceptibles a la sequía y en la siembra tardía. Similarmente, el grado de ACD se incrementó bajo condiciones de deficiencia de N. Sin embargo, la fertilización con N no redujo el oscurecimiento en todas las pruebas y el beneficio de fertilizar con N no fue evidente a medida que la duración de almacenamiento se incrementó. Hubo un pequeño beneficio con la fertilización con K en el experimento 3; sin embargo, la fertilización con K no tuvo efecto sobre el ACD en el experimento 1. No hubo efecto de la fertilización con N sobre el ACD. Aunque significativa, la magnitud de los efectos del tipo de suelo y prácticas de manejo sobre ACD, fueron pequeños en comparación con los efectos del cultivar y la duración del almacenamiento.

Transformation and Plant Regeneration from Leaf Explants of Solanum tuberosum L. cv. ‘Shepody’

As part of a large-scale genomics project focused on understanding and improving the Shepody potato, we have increased the regeneration and transformation rates for this cultivar. Using combinations of auxins and trans-zeatin, leaf and stem explants were evaluated for callus induction and shoot formation. Several plant growth regulator combinations resulted in higher plant regeneration rates over a previous method. Using the best combination of auxin and cytokinin in combination with Agrobacterium-mediated transformation, we regenerated independent putative transformants from 59.5% of the total explants plated. We ran PCR on a sample of the plants to confirm transformation and 47.1% were nptII positive; giving a confirmed transformation rate of 28.0%.