Department of Veterinary and Biomedical Sciences: 2005 Annual Report

Veterinary and Biomedical Sciences Personnel VBMS Teaching Program - Courses 2005 VBMS Grants and Contracts Program, 2005 Publications by VBMS Faculty in 2005 Selected Committees, Editorial and Other Appointments. 2005 Articles Regarding the Department in 2005 Departmental Budget Summaries, 2005 Nebraska Agricultural Statistics, 2004/2005

Department of Veterinary and Biomedical Sciences: 2005 Annual Report

Veterinary and Biomedical Sciences Personnel VBMS Teaching Program - Courses 2005 VBMS Grants and Contracts Program, 2005 Publications by VBMS Faculty in 2005 Selected Committees, Editorial and Other Appointments. 2005 Articles Regarding the Department in 2005 Departmental Budget Summaries, 2005 Nebraska Agricultural Statistics, 2004/2005

Genomics-assisted breeding strategies for quantitative resistances to Northern corn leaf blight in maize (Zea mays L.) and Fusarium diseases in maize and in triticale (× Triticosecale Wittm.)

Fusarium head blight (FHB) in triticale (× Triticosecale Wittm.), Gibberella ear rot (GER) and Northern corn leaf blight (NCLB) in maize (Zea mays L.) are devastating crop diseases causing yield losses and/or reducing grain quality worldwide. Resistance breeding is the most efficient and sustainable approach to reduce the damages caused by these diseases. For all three pathosystems, a quantitative inheritance based on many genes with small effects has been described in previous studies. Hence, this thesis aimed to assess the potential of genomics-assisted breeding strategies to reduce FHB, GER and NCLB in applied breeding programs. In particular, the objectives were to: (i) Dissect the genetic architecture underlying quantitative variation for FHB, GER and NCLB through different quantitative trait loci (QTL) and association mapping approaches; (ii) assess the potential of genomics-assisted selection to select superior triticale genotypes harboring FHB resistance; (iii) phenotype and characterize Brazilian resistance donors conferring resistance to GER and NCLB in multi-environment trials in Brazil and in Europe; and (iv) evaluate approaches for the introgression and integration of NCLB and GER resistances from tropical to adapted germplasm. The genome-wide association study (GWAS) conducted for FHB resistance in triticale revealed six QTL that reduced damages by 5 to 8%. The most prominent QTL identified in our study was mapped on chromosome 5B and explained 30% of the genotypic variance. To evaluate the potential of genomic selection (GS), we performed a five-fold cross-validation study. Here, weighted genomic selection increased the prediction accuracy from 0.55 to 0.78 compared to the non-weighted GS model, indicating the high potential of the weighted genomic selection approach. The successful application of GS requires large training sets to develop robust models. However, large training sets based on the target trait deoxynivalenol (DON) are usually not available. Due to the rather moderate correlation between FHB and DON, we recommend a negative selection based on genomic estimated breeding values (GEBVs) for FHB severity in early breeding stages. In the long-run, however, we encourage breeders to build and test GS calibrations for DON content in triticale. The genetic architecture of GER caused by Fusarium graminearum in maize was investigated in Brazilian tropical germplasm in multi-environment trials. We observed high genotype-by-environment interactions which requires trials in many environments for the identification of stable QTL. We identified four QTL that explained between 5 to 22% of the genotypic variance. Most of the resistance alleles identified in our study originated from the Brazilian tropical parents indicating the potential of this exotic germplasm as resistance source. The QTL located on chromosome bin 1.02 was identified both in Brazilian and in European trials, and across all six biparental populations. This QTL is likely stable, an important feature for its successful employment across different genetic backgrounds and environments. This stable QTL is a great candidate for validation and fine mapping, and subsequent introgression in European germplasm but possible negative linkage drag should be tackled. NCLB is another economically important disease in maize and the most devastating leaf disease in maize grown in Europe. Virulent races have already overcome the majority of known qualitative resistances. Therefore, a constant monitoring of S. turcica races is necessary to assist breeders on the choice of effective resistances in each target environment. We investigated the genetic architecture of NCLB in Brazilian tropical germplasm and identified 17 QTL distributed along the ten chromosomes of maize explaining 4 to 31% of the trait genotypic variance each. Most of the alleles reducing the infections originated from Brazilian germplasm and reduced NCLB between 0.3 to 2.5 scores in the 1-9 severity scale, showing the potential of Brazilian germplasm to reduce not only GER but also NCLB severity in maize. These QTL were identified across a wide range of environments comprising different S. turcica race compositions indicating race non-specific resistance and most likely stability. Indeed, QTL 7.03 and 9.03/9.04 were identified both in Brazil and in Europe being promising candidates for trait introgression. These major and stable QTL identified for GER and NCLB can be introgressed into elite germplasm by marker-assisted selection. Subsequently, an integration step is necessary to account for possible negative linkage drag. A rapid genomics-assisted breeding approach for the introgression and integration of exotic into adapted germplasm has been proposed in this thesis. Jointly, our results demonstrate the high potential of genomics-assisted breeding strategies to efficiently increase the quantitative resistance levels of NCLB in maize and Fusarium diseases in maize and in triticale. We identified favorable QTL to increase resistance levels in both crops. In addition, we successfully characterized Brazilian germplasm for GER and NCLB resistances. After validation and fine mapping, the introgression and integration of the QTL identified in this study might contribute to the release of resistant cultivars, an important pillar to cope with global food security.Ährenfusariosen (Fusarium head blight, FHB) in Triticale (× Triticosecale Wittm.), sowie Kolbenfusariosen (Gibberella ear rot, GER) und Turcicum-Blattdürre (Northern corn leaf blight, NCLB) im Mais (Zea mays L.), sind weltweit verheerende Schaderreger, die zu Ertragseinbußen und verminderter Erntegutqualität führen können. Die Resistenzzüchtung ist die effizienteste und nachhaltigste Methode, um die auftretenden Schadwirkungen dieser Pflanzenkrankheiten zu minimieren. Für alle drei Pathosysteme wurden bereits quantitative Resistenzen in der Literatur beschrieben. Die Zielsetzung der vorliegenden Arbeit war daher, das Potenzial genomisch unterstützter Zuchtstrategien zur Reduktion von FHB, GER und NCLB zu untersuchen. Insbesondere wurden dabei die nachfolgenden Ziele verfolgt: (i) die genetische Architektur von FHB, GER und NCLB mit verschiedenen QTL- und Assoziationskartierungsansätzen zu untersuchen; (ii) das Potential genomisch unterstützter Zuchtstrategien zur Selektion von Triticale Genotypen mit verbesserter FHB Resistenz zu bewerten; (iii) die Charaktierisierung und Phänotypisierung brasilianischer Resistenzdonoren für GER und NCLB in mehreren Umwelten in Brasilien und Europa; und (iv) Ansätze für die Einkreuzung und Intergration von tropischen NCLB und GER Resistenzquellen in europäisches Zuchtmaterial zu bewerten. Die genomweite Assoziationskartierung (GWAS) für FHB Resistenz in Triticale identifizierte sechs QTL, die eine Reduktion im Befallswert zwischen 5 und 8% erklärten. Der vielversprechendste QTL wurde auf Chromosome 5B identifizert und erklärte 30% der genotypischen Varianz. Um das Potenzial der genomischen Selektion (GS) zu evaluieren, wurde eine fünffache Kreuzvalidierung durchgeführt. Hier zeigte eine gewichtete genomische Selektion (wGS) einen Anstieg in der Vorhersagegenauigkeit von 0.55 auf 0.78, verglichen zum ungewichteten GS Modell. Dies unterstreicht das Potenzial der wGS Methode. Eine erfolgreiche Implementierung von GS benötigt eine ausreichend große Trainingspopulationen, um robuste Modelle zu kalibrieren. Hierfür ist die Datengrundlage für das Zielmerkmale Deoxynivalenol (DON) üblicherweise jedoch nicht ausreichend. Aufgrund der eher moderaten Korrelation zwischen FHB und DON empfehlen wir zunächst eine Negativselektion basierend auf genomischen Zuchtwerten für FHB Anfälligkeit in frühen Generationen. Langfristig möchten wir jedoch Züchtungsunternehmen motivieren GS Kalibrationen für DON Gehalt in Triticale zu entwickeln und testen. Die genetische Architektur von GER, hervorgerufen durch Fusarium graminearum im Mais, wurde in brasilianischem Zuchtmaterial in mehreren Umwelten untersucht. Wir beobachteten eine große Genotyp-Umwelt Interkation, was Versuche in vielen Umwelten zur Identifikation von stabilen QTL voraussetzt. Wir identifizierten vier QTL die zwischen 5 und 22% der genetischen Varianz erklärten. Die meisten der identifizierten Resistenzallele hatten ihren Ursprung in den tropischen brasialinschen Eltern, was das Potenzial dieses Materials als Resistenzquelle unterstreicht. Der QTL auf dem Chromosom Bin 1.02 wurde in allen sechs Bi-parentalen Populationen sowohl in Brasilien als auch in Europa dentifiziert. Dieser QTL tendiert zu einer hohen Stabilität, was eine sehr wichtige Eigenschaft für die erfolgreiche Anwendung in verschiedenen genetischen Hintergünden und Umwelten ist. Dieser stabile QTL ist daher ein Kandidat für weitere Validierungen, für eine mögliche Feinkartierung und die anschließende Einkreuzung in europäisches Zuchtmaterial. Mögliche negative Einflüsse durch Linkage Drag müssen jedoch berücksichtigt werden. NCLB ist eine weitere wichtige Krankheit mit hoher ökonomischer Bedeutung im Mais. In Europa ist es die wichtigste Blattkrankeit im Mais. Virulente Rassen haben viele der identifizierten qualitativen Resistenzen bereits überwunden. Daher ist ein kontinuierliches Monitoring der S. turcica Rassen notwendig, um Züchter in der Wahl von effektiven Resistenzen für die jeweilige Zielumwelt zu unterstützen. Daher haben wir die genetische Architektur von NCLB in tropischen brasilianischem Zuchtmaterial untersucht und identifizierten insgesamt 17 QTL, verteilt auf allen 10 Chromosomen, die zwischen 4 und 30% der genotypischen Varianz erklärten. Die meisten dieser QTL mit einem reduzierenden Effekt hatten braislianschen Ursprung und reduzierten NCLB zwischen 0.3 und 2.5 Boniturnoten auf einer Skala von 1-9. Dies unterstreicht das hohe Potenzial des brasilianischen Zuchtmaterials zur Reduktion nicht nur von GER aber auch von NCLB im Mais. Die gefundenen QTL wurden in vielen Umwelten mit verschiedenen S. turcica Rassen identifiziert, was eine rassenunabhängige und stabile Resistenzwirkung nahelegt. Die QTL 7.03 und 9.03/9.04 wurden sowohl in Brasilien als auch in Europa identifiziert und sind daher vielversprechende Kandidaten für Einkreuzungen. Die identifizierten stabilen QTL, mit großen Effekten für GER und NCLB, können mit Hilfe Marker gestützter Selektion (MAS) in Elitematerial eingekreuzt werden. Ein anschließender Integrationsschritt ist jedoch nötig, um negativen Effekten durch Linkage Drag zu begegnen. Aus diesem Grund wurde ein schnelles genomisch unterstütztes Zuchtschema zur Einkreuzung und Integration von exotischen Resistenzquellen in den Elitehintergrund in dieser Arbeit vorgestellt. Zusammenfassend zeigen unsere Ergebnisse das hohe Potenzial von genomisch unterstützten Zuchtstrategien zur effizienten Steigerung der quantitativen Resistenz von NCLB im Mais und Fusariumerkrankungen im Mais und in Triticale. Wir identifizierten vielversprechende QTL, um das resistenzniveau in beiden Kulturarten zu erhöhen. Wir waren erfolgreich in der Charakterisierung des brasilianischen Zuchtmaterials für GER und NCLB Resistenz. Nach einer weiteren Validierung und Feinkartierung kann die Einkreuzung und Integration der identifizierten QTL einen signifikanten Beitrag zur Züchtung von Sorten mit verbesserten Resistenzeigenschaften leisten. Dies ist ein wichtiger Baustein, um die globale ernährungssicherheit zu gewährleisten

Evaluation of the relative importance of Ascochyta pisi in the Ascochyta blight complex of pea in Saskatchewan

Ascochyta blight may be caused by the fungal pathogens Peyronellaea pinodes, Ascochyta pisi, Phoma medicaginis var. pinodella, and Phoma koolunga. While P. pinodes has been the most important pathogen in Saskatchewan, A. pisi has become more prevalent in southern and southern-western parts of the province. Conidial germination of A. pisi and P. pinodes on glass slides and infection of plants of pea cultivar CDC Cooper by both pathogens were evaluated under controlled conditions at temperatures ranging from 10 to 30°C and wetness periods of 0 to 12 h to determine whether they had different optimal environmental requirements. For both pathogens, conidial germination and disease severity increased with increasing temperature and leaf wetness period up to the optimum of 20 to 25°C. Overall, P. pinodes had consistently higher germination and disease severity compared to A. pisi. For conidial germination, these differences became obvious starting at 20°C after more than 4 h incubation, and at 25 and 30°C P. pinodes had consistently higher germination after 2 h of incubation. Similarly, disease severity caused by P. pinodes was consistently higher at 20 and 25°C compared to that caused by A. pisi. The role of infected seed in the epidemiology of A. pisi in pea was studied with naturally infected seeds under field conditions in 2012, 2013 and 2014 at two locations where low natural inoculum were expected. Results revealed a significant effect of A. pisi seed infection on emergence of seedlings (P < 0.05). Seed infection levels of 10 and 14.5% resulted in reduced emergence compared to 0.5% seed infection, but the level of seed infection at planting had no impact on A. pisi disease severity, seed infection levels of harvested seed or seed yield. No visible symptoms caused by A. pisi appeared on the aerial parts of the seedlings. Results suggest that disease did not progress from seeds, or contributed to infection of aerial parts of the plants, hence infected seeds cannot be regarded as a source of inoculum in the epidemiology of this pathogen. Assessing seed components of seeds with varying levels of A. pisi infection and seed staining revealed that the pathogen was present in all components of the seed, regardless of the severity of seed staining. Field studies were conducted to assess yield loss caused by A. pisi between 2012 and 2014 at Swift Current and Stewart Valley where a high incidence of A. pisi had been reported in the past, and under irrigation with inoculation at Saskatoon (2014). Two (2012, 2013) or three (2014) fungicides (pyraclostrobin, chlorothalonil, or pyraclostrobin and boscalid) were applied to create plots with low levels of A. pisi infection that were compared with a non-sprayed treatment. The more susceptible pea cultivars Cooper and SW Midas were compared with the more resistant cultivars CDC Bronco and CDC Golden. Disease pressure was low in all three years and no differences in yield between fungicide treated and non-sprayed treatments were observed. Nevertheless, the incidence of A. pisi isolated from harvested seeds of the fungicide treatments was lower than that of the non-sprayed treatment, despite similar A. pisi severity on plants before harvest. The response of the recombinant inbred line (RIL) pea population PR-10 developed from a cross between the susceptible variety Cooper and the partially resistant variety CDC Bronco to A. pisi infection was evaluated in 2012, 2013 and 2014 under field conditions. RILs were inoculated with A. pisi in 2014 at Saskatoon under irrigation. Under low disease pressure in all three years, no difference in A. pisi severity was observed between the parents and among RILs. Yields differed among RILs and were attributed to genetic differences. Considering that the parents did not differ in resistance to A. pisi it was concluded that this population might not be suitable for the study of genetic control of resistance to A. pisi

Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation

5 Summary Viral diseases of Cucurbits are an important limitation in the production of the crop. Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus-2 (WMV-2) are the most important squash (Cucurbita pepo L.) infecting viruses. Mixed infections with these viruses are deleterious for cucurbitaceous plants leading annually to significant yield losses world wide. All varieties of economical importance are susceptible for these viruses and classical breeding did not yield resistance. Therefore, a transgenic approach was chosen to induce resistance against both viruses by post-transcriptional gene silencing (PTGS). Highly conserved regions in the coat protein genes of ZYMV and WMV-2 were chosen to establish an inverted repeat construct. This construct was cloned into binary vector under control of the 35S promoter. Embryogenic callus was induced from different organs of three squash cultivars as target tissues for Agrobacterium transformation. The embryogenic callus was developed within 13-20 weeks incubation on MS medium containing different plant growth regulator combinations of auxin and cytokinin. Induction of embryogenesis in different explants ranged from 5 to 100 % depending on the organ and genotype used. Efficiency of embryo maturation, conversion and germination into entire plants from squash embryogenic callus was found to be callus age depended. Regeneration with young (2 months old) material was efficient, whereas regeneration of material maintained under in vitro conditions for more than 2 years was not possible. Agrobacterium-mediated transformation of squash embryogenic callus was established using transient GUS-gene expression. The highest transformation efficiency was obtained with the supervirulent ATHV strain, bacterial density of 0.85, washing procedure of the embryogenic material prior to Agrobacterium co-culture, application of 1 mM Acetosyringone in induction medium and sub-culturing of embryogenic callus on fresh MS medium 5-9 days prior co-culturing with the Agrobacterium. Selection strategy was optimized using GFP as reporter gene. For the genotype CX3006 300 mg/l Kanamycin showed the highest number of green areas but most efficient selection agent was Paromomycin 150 mg/l. For the genotype Dundoo 200 mg/l Paromomycin was the effective selection agent and showed the highest number of green areas. Selection of transformed calli could be efficient with the used selection agents but regeneration of transgenic plant was not possible because the old material was only one to be used for transformation experiments. It seems that these old materials may have lost their competency when they were maintained for long term in tissue culture. Therefore, the functionality of the inverted repeat construct was evaluated in Nicotiana benthamiana as a model plant. Transgenic lines were analyzed by PCR, Southern blot analysis and segregation analysis of T1 offspring. The transgene-induced PTGS in transgenic lines was confirmed by infiltration of GFP-sensor constructs containing viral derived sequences as silencing target and /or a construct containing the p19 silencing suppressor. In all transgenic lines tested, GFP fluorescence in infiltrated leaves was extinguished three days post-infection with GFP-sensor constructs. In contrast, all transgenic lines showed GFP fluorescence in infiltrated leaves when GFP-sensor constructs were co-infiltrated with a binary vector containing the viral silencing suppressor p19. With this work, tools have been developed to engineer virus resistance in squash. Using the optimized Agrobacterium-mediated transformation procedure together with the efficient RNA-silencing of the inverted inverted repeat construct and freshely induced embryogenic material it is quite possible to establish virus resistance in squash.6 Zusammenfassung Virusbefall verursacht beim Zucchini-Anbau weltweit einen großen wirtschaftlichen Schaden. Früchte von virusinfizierten Pflanzen weisen mosaikähnliche Verfärbungen und schwerwiegende Deformationen auf, Pflanzen sind gestaucht und Blätter deformiert. Von den verschiedenen Viren, die weltweit für den Befall von Zucchini und anderen Cucurbitaceaen verantwortlich sind, gehören ZYMV und WMV-2 zu den bedeutendsten. Die konventionelle Züchtung konnte zwar bereits virustolerante Sorten hervorbringen, diese können jedoch Mischinfektionen oder der Befall von aggressiven Stämmen der o. g. Viren auf dem Felde nicht standhalten. Im Rahmen dieser Arbeit wurde daher ein transgene Strategie gewählt, um mittels ?Post Transkriptionales Gen Silencing? Resistenzen gegen ZYMV und WMV-2 in Zucchini zu induzieren. Für die Herstellung eines ?Inverted Repeat?-Konstrukts wurden hochkonservierte Sequenzbereiche aus den Hüllproteinen des ZYMV und des WMV-2 verwendet. Das Konstrukt wurde unter Kontrolle des 35S Promotors in einen binären Vektor kloniert. Als Ausgangsmaterial für die Agrobacterium-vermittelte Transformation von Zucchini wurde somatisch embryogener Kallus ausgewählt. Nach 13 bis 20 Wochen Inkubation auf hormonhaltigem Medium konnte an verschiedenen Explantaten bei allen 3 verwendeten Zucchini Genotypen embryogener Kallus induziert werden. Je nach verwendetem Explantat und Genotypen wurde eine Effizienz der Embryogenese von 5 bis 100% festgestellt. Die Reifung der somatischen Embryonen und ihre Konversion zu Pflanzen zeigten sich als stark abhängig vom Alter des embryogenen Kallus. Die Reifung und Konversion von frisch induziertem embryogenem Material war sehr effizient. Nach einer 2 jährigen Erhaltungsphase unter in vitro ?Bedingungen war eine Reifung und Konversion jedoch nicht mehr möglich. Die Agrobacterium-vermittelte Transformation von embryogenem Kallus wurde mit Hilfe der transienten Expression des GUS-Gens etabliert. Die höchste transiente Expression konnte bei Anwendung folgender Parameter erreicht werden: Verwendung des supervirulenten Agrobacterium-Stamms ATHV, optische Dichte von 0,85 der Bakteriensuspension, Waschbehandlung des embryogenen Materials vor der Cokultur, die Applikation von 1 mM Acetosyringon während der Cokultur und einer Vorkulturphase von 5-9 Tagen vor der Cokultur. Die Optimierung der Selektion von transformierten Zellen wurde mit Hilfe des GFP-Reportergens durchgeführt. Für den Genotypen CX3006 zeigte eine Kanamycin-Konzentration von 300 mg/l die größte Zahl von GFP-positiven Bereichen, jedoch war die Selektion mit Paramomycin am effizientesten, da nur eine Konzentration von 150 mg/l benötigt wurde. Für den Genotypen Dundoo erwies sich Paramomycin in einer Konzentration von 200 mg/l als effizientestes Selektionsmittel. Obwohl gezeigt werden konnte, dass die Selektion von transformierten embryogenen Zellen möglich ist, konnte während dieser Arbeit keine transgenen Zucchinipflanzen regeneriert werden, da für die Transformationsexperimente bedauerlicherweise nur auf altes embryogenes Kallusmaterial zurückgegriffen werden konnte, das während der langen Erhaltungskulturphase die Kompetenz zur Regeneration verloren hat. Die Funktionalität des ?Inverted Repeat?-Konstrukts wurde in der Modellpflanze Nicotiana benthamiana nachgewiesen. Transgene N. benthamiana-Pflanzen wurde mit PCR, Southern Blot Analyse untersucht und die Aufspaltung in der T1-Generation überprüft. Bei allen untersuchten Linien konnte PTGS in Infiltrationsexperimenten gezeigt werden. Hierbei wurden GFP-Sensorkonstrukte allein bzw-. in Kombination mit einem P19-Konstrukt infiltriert. In allen Linien konnte nach Infiltration der Sensorkonstrukte keine GFP-Fluoreszenz beobachtet werden, hingegen führte eine kombinierte Infiltration der Sensorkonstrukte mit dem P19 Supressor bei allen Linien zu einer starken GFP-Fluoreszenz. Mit dieser Arbeit konnte die Vorraussetzung für die Herstellung von virusresistentem Zucchinimaterial geschaffen werden. Mit Hilfe des etablierten Protokoll für die Agrobacterium-vermittelte Transformation von embryogenen Zucchinizellen und dem funktionellen ?Inverted Repeat?-Konstrukt besteht die Möglichkeit, in Zukunft ausgehend von frisch induziertem embryogenem Kallus transgene virusresistente Zucchinipflanzen erzeugt zu erzeugen

The Molecular Signature for Local Adaptation in the Seagrass <i>Posidonia oceanica</i>

In the last century, seagrass ecosystems have suffered a worldwide decline ascribed to multiple environmental stressors, among which the reduction of light available for the photosynthesis and the increase in temperature represent the strongest constraints for their growth and survival. Despite conservation, this decline is at present still continuing. In order to understand the genetic adaptive response to light and temperature in the seagrass Posidonia oceanica, two different strategies have been pursued: a genome scan approach along a latitudinal and a bathymetric gradient and a differential gene expression analysis along the bathymetric gradient, where light and temperature were the two main selective factors. For the genome scan approach 6 populations (Delimara - Malta, Lacco Ameno - Island of Ischia, Italy, Marettimo Island- Italy, Meloria - Italy, Piombino - Italy and Stareso - Corsica, France) were sampled along the bathymetric gradient at two different depths (-5m and -25m). The same populations were used for the latitudinal gradient analysis by grouping them on the basis of their geographic location (Southern group: Delimara, Lacco Ameno and Marettimo; Northern group: Meloria, Piombino and Stareso). No genes under selection were identified in the genome scan along the bathymetric gradient. Three putative genes under selection were identified in the genome scan along the latitudinal gradient and were involved in the photosynthesis and in the translation process. For assessing differential gene expression, a transcriptome sequencing of plants sampled at two different depths and different times of the day in the Stareso meadow was performed by RNAseq technology. The analysis highlighted the capability of plants living in shallow waters to cope with environmental stresses imposed by high light and high temperature. Transcriptome data generated from this study increased the resources available in P. oceanica and will be very useful for further investigations of the adaptation of in this plant

Monitoring important soil-borne plant pathogens in Swedish crop production using real-time PCR

The global demand for food will increase considerably in the nearest future and among the major constraints to agricultural productivity are biotic stresses caused by microorganisms. In this thesis, the causal agents of four of the most important soil-borne diseases threatening the Swedish production of oilseed rape, sugar beets and red clover were selected as targets for developing diagnostic assays using real-time PCR. The disease risk assessment of clubroot in oilseed rape was improved by developing a real-time PCR assay for quantification of the causal pathogen Plasmodiophora brassicae directly in soil samples. Broad disease risk categories including a threshold level for growing resistant cultivars were established. A real-time PCR assay for quantification of Aphanomyces cochlioides, the oomycete causing root rot in sugar beets, was also developed in the present study. The potential use of this assay as a tool in disease risk assessment was demonstrated for fields with high risk of infection. Real-time PCR was used to monitor the plant- and airborne inoculum of Sclerotinia sclerotiorum, the causal fungus of Sclerotinia stem rot, in spring oilseed rape. We found that determining the presence of S. sclerotiorum on petals was not useful for stem rot risk assessment since (i) the inoculum incidence on petals varied during flowering, (ii) there was no clear relationship between petal infection and stem rot incidence and (iii) spore release and flowering were not synchronized at one of the field experimental sites. Real-time PCR detection of the incidence of S. sclerotiorum DNA on leaves revealing the field-borne inoculum and quantification of the airborne inoculum are likely more reliable tools for predicting the potential risk of disease. The pathogen complex causing red clover root rot was monitored over three years in two field experiments. Fusarium avenaceum, Phoma spp. and Cylindrocarpon destructans were detected in red clover roots early in the seeding year using real-time PCR and the levels of pathogen DNA generally increased during the following years. Significant linear relationships were found between the amount of pathogen DNA and disease severity index

Déterminisme génétique de la résistance du tournesol au Phoma

L'interaction "génotype-isolat" pour la résistance partielle au Phoma macdonaldii chez le tournesol a été déterminée en utilisant 28 génotypes inoculés par sept isolats de ce pathogène dans des conditions contrôlées. Deux lignées AS613 et PAC2 ont été isolées, qui présentent une résistance spécifique à l’isolat MP8. Nos résultats montrent également l’existence d’une résistance non-spécifique chez les génotypes testés. Pour déterminer l'hérédité de la résistance partielle à cette maladie, nous avons effectué des croisements entre cinq génotypes sélectionnés parmi les 28 de la première expérimentation présentant une variabilité importante pour la résistance partielle à des isolats du Phoma. Les génotypes parentaux et leurs hybrides F1 ont été inoculés par deux isolats sélectionnés (MP8 et PM10). Les aptitudes générales et spécifiques à la combinaison pour la résistance aux deux isolats sont significatives, indiquant que les effets additif et dominant des gènes sont importants dans le contrôle génétique de la résistance partielle au Phoma. Nous avons développé une nouvelle carte de liaison du tournesol en utilisant 100 individus F2 issus du croisement entre M6-54-1 et ENSAT-B4, identifiés par nos expérimentations précédentes. Les régions génomiques contrôlant la résistance partielle aux isolats de P. macdonaldi ont été identifiés. Un QTL localisé sur le groupe de liaison (LG) 8 est commun aux trois isolats testés. Des QTLs spécifiques sont également identifiés pour chaque isolat. Dans une autre expérimentation et en utilisant la carte récemment enrichie de notre laboratoire, des QTLs contrôlant la résistance partielle au Phoma ont été identifiés par l’étude d’une population de 99 LRs issue du croisement entre PAC2 et RHA266 inoculé par deux isolats (MP8 et MP10). Parmi les 10 QTLs détectés, quatre situés sur LG 5 et LG15 sont commun pour les deux isolats, les autres sont spécifiques de chaque isolat. La variabilité génétique de 60 mutants a été étudiée en utilisant les marqueurs AFLP. Des marqueurs potentiellement associés à la résistance partielle au Phoma ont été identifiés. le profil de 12 gènes potentiellement associés à la résistance partielle du tournesol à P. macdonaldii a été testé sur le mutant M6-54-1 et la lignée originelle AS613, inoculés par trois isolats de P. macdonaldii en utilisant la RT-PCR quantitative. Les gènes codant pour la phenylalanine ammonia-lyase 2 (PAL2) et la thaumatine ont montré des différences significatives au niveau de l'expression, entre les interactions compatibles et partiellement compatibles. Une corrélation directe entre les niveaux d'expression de gènes codant pour le facteur de transcription HD- Zip et la protéine phosphatase avec le symptôme de la maladie a été observée chez la lignée originelle et son mutant inoculé par l’isolat, MA6. ABSTRACT : Genotype-isolate interaction for resistance to Phoma black stem in sunflower were investigated using 28 genotypes, inoculated by seven isolates of Phoma macdonaldii in controlled conditions. Two specific resistant lines (AS613 and PAC2) to MP8 isolate were identified. Our results showed that there was also a wide range of isolate- onspecificpartial resistance among the genotypes tested. In order to determine the inheritance of partial resistance to the disease, five genotypes of 28 with wide range of genetic variability for partial resistance were crossed. Parental genotypes and their F1 hybrids were inoculated by one of the two selected isolates (MP8 and PM10). General and specific combining ability effects for resistance to Phoma black stem were highly significant for each of the two isolates, indicating that additive and dominant gene effects were important in controlling partial resistance to Phoma black stem. A new linkage map was developed using 100 F2 plants derived from the cross between M6- 54-1 and ENSAT-B4 selected according to the results of the above-mentioned experiments, and the gnomic regions controlling partial resistance to three P. macdonaldii were identified. One QTL located on linkage group (LG) 8 was common to the three isolates tested. Other QTLs were specific for each isolate separately. QTLs controlling quantitative resistance to Phoma black stem was also investigated using our new reference map (Poormohammad kiani et al., 2007) with 99 RILs derived from the cross between PAC2 and RHA266 inoculated by the two isolates (MP8 and MP10). Through ten detected QTLs, four of them were common between two isolates on LG5 and LG15 whereas the others were specific for each isolate. Genetic variability among a population of 60 mutant lines was studied using AFLP markers and putative molecular markers associated with partial resistance to Phoma black stem was identified. The profile of 12 genes potentially associated with partial resistance of sunflower to P. macdonaldii was investigated in resistant mutant line M6-54-1 and its original line AS613, which were selected according to the results of our previous experiments using quantitative real-time RT-PCR. Plants were inoculated by three P. macdonaldii isolates. Genes encoding phenylalanine ammonia-lyase 2 (PAL2) and thaumatin-like protein showed significant differences in expression between partial compatible and compatible interactions. A straightforward correlation between expression levels of genes encoding HD-Zip transcription factor and protein phosphatase with disease symptom was observed in original line and its mutant inoculated by MA6 isolat