Studies on global transcriptional regulator EBR1 and genome-wide gene expression in the fungal plant pathogen Fusarium graminearum
Zhao, C. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit; D. Tang, co-promotor(en): Theo van der Lee. - Wageningen : Wageningen University - ISBN 9789462575998 - 167
plant pathogenic fungi - gibberella zeae - transcription factors - gene expression - gene mapping - genomics - plantenziekteverwekkende schimmels - gibberella zeae - transcriptiefactoren - genexpressie - genkartering - genomica
Abstract of PhD thesis
Fusarium graminearum is a destructive plant pathogen that causes Fusarium head blight (FHB) on many crops, such as wheat, barley, rye and oat. In the first part of this thesis, we studied a transcription factor EBR1 that is required for radial growth and virulence in F. graminearum. Mutant ebr1 shows reduced apical dominance of the hyphal tip and loses its ability to penetrate the rachis of the spikelets. Subcellular localization analysis showed that EBR1 protein is exclusively localized in the nucleus of both conidia and hyphae. In the second part of thesis, by using RNA-Seq data, we revised 655 incorrectly predicted gene models and identified 231 genes with two or more alternative splice variants in F. graminearum. Furthermore, we analyzed the genome-wide gene expression pattern and found that genes locate in non-conserved regions of chromosomes showed relatively lower expression level. We further provided evidence showing that the non-conserved regions are full of gene relocations in F. graminearum.
Exploiting wild tomato genetic resources for resistance to Tomato Yellow Leaf Curl Virus
Caro Rios, C.M. - \ 2015
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462575936 - 187
solanum lycopersicum - tomatoes - wild relatives - plant genetic resources - disease resistance - tomato yellow leaf curl virus - gene mapping - solanum habrochaites - introgression - plant breeding - solanum lycopersicum - tomaten - wilde verwanten - genetische bronnen van plantensoorten - ziekteresistentie - tomatengeelkrulbladvirus - genkartering - solanum habrochaites - introgressie - plantenveredeling
Towards map-based cloning of partial resistance QTLs of barley to Puccinia hordei
Yeo, F.K.S. - \ 2014
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Rients Niks. - Wageningen : Wageningen University - ISBN 9789462570733 - 185
hordeum vulgare - gerst - ziekteresistentie - plantenziekteverwekkende schimmels - puccinia hordei - partiële resistentie - loci voor kwantitatief kenmerk - genkartering - plantenveredeling - hordeum vulgare - barley - disease resistance - plant pathogenic fungi - puccinia hordei - partial resistance - quantitative trait loci - gene mapping - plant breeding
Partial resistance of barley to Puccinia hordei and near-nonhost resistance to non-adapted rust fungi inherit polygenically. The two types of resistance seem to share some genes and have a similar prehaustorial mechanism of resistance, but partial resistance is less strong than near-nonhost resistance of barley. Partial resistance to adapted, “host”, rust fungi seems, therefore, like a weak form of nonhost resistance to non-adapted rust fungi. If partial resistance and nonhost resistance are indeed based on the same principles, one can understand nonhost resistance by studying partial resistance and vice versa. To study partial and nonhost resistance, as well as their association, the candidate gene(s) for resistance must be cloned and characterized for their action.
Five resistance quantitative trait loci (QTLs) for partial resistance (Rphq2, Rphq3, Rphq4, Rphq11 and Rphq16) and one nonhost resistance QTL (Rnhq) were selected to pursue map-based cloning. First, the effect of the QTLs was verified in near-isogenic lines (NILs). The NILs of Rphq2, Rphq3, Rphq4 and Rnhq (QTL-NILs) were available in L94 genetic background. L94 is extremely susceptible to Puccinia hordei, and, at seedling stage, somewhat susceptible to certain non-adapted rust fungi. The experimental barley line SusPtrit is also susceptible to P. hordei but, at seedling stage, also very susceptible to at least nine species of non-adapted rust fungi. In Chapter 3, we developed NILs in SusPtrit background for Rphq2, Rphq3, Rphq11, Rphq16 and two alleles of Rnhq, viz. L94 and Vada alleles. The effect of each QTL in L94 and SusPtrit genetic backgrounds was tested not only against different isolates of P. hordei but also against different species and isolates of non-adapted rust fungi. The QTL-NILs suggested that the effects of the partial resistance genes depended on rust species and rust isolates. Some introgressions conferred resistance to a broader spectrum of rust species and isolates than others, the broadest being the Rphq11-introgression. The NILs may overestimate the spectrum of effectiveness of the partial resistance genes because some NILs contain inadvertent donor genome in the background and the introgressed QTL region may contain several linked resistance genes, each with a narrow resistance spectrum. The introgression would then confer a resistance spectrum that is the combination of the spectra of several linked resistance genes. Allowing for the possibility of linkage of narrow-spectrum resistance genes, our study suggests that some genes may be involved in partial as well as nonhost resistance. Data also suggest that genetic background may play a role in the resistance conferred by the QTL-introgression.
The NILs also allow fine-mapping of the QTL as was done for Rphq2 in a previous study. In Chapter 4, we target to fine-map another two partial resistance QTLs of our interest, viz. Rphq11 and Rphq16. We, however, did not use the NILs for fine-mapping of Rphq11 and Rphq16. Instead, after validating the effect of Rphq11 and Rphq16 using the early breeding materials for developing NILs of Rphq11 and Rphq16, we developed fixed QTL-recombinants (i.e. homozygous recombinants at the Rphq11/Rphq16 QTL alleles, homozygous susceptible at the non-targeted QTL alleles). The genomic background of fixed QTL-recombinants was still segregating, but expected not to be relevant for the resistance level. Rphq11 was fine-mapped into a 0.2 cM genetic interval and a 1.4 cM genetic interval for Rphq16, before the NILs were ready. The strongest candidate gene for Rphq11 is a phospholipid hydroperoxide glutathione peroxidase (PHGPx). This gene corresponds to the new Rphq11 peak marker – WBE129, located within the refined 0.2 cM genetic intervals and was one of the candidate genes for Rphq11 identified through e-QTL mapping on Steptoe/Morex challenged with the same rust isolate. There was no clear candidate gene identified for Rphq16.
A QTL has to be fine-mapped into a sufficiently narrow genetic window to make physical mapping feasible. Rphq2 with a genetic window of 0.1 cM is ready for physical mapping. In Chapter 5, we have constructed two non-gridded Bacterial Artificial Chromosome (BAC) libraries of barley from Vada and SusPtrit. Based on the insert sizes of the BAC clones, the estimated genome coverage of the Vada BAC library is 2.6x and of the SusPtrit BAC library 3.7x. The genome coverage of Vada is comparable to the BAC library of Morex, HVVMRXALLhB and SusPtrit to HVVMRXALLeA. The estimation of genome coverage based on microsatellite markers indicates, however, Vada and SusPtrit BAC libraries to have 5.0x and 6.8x genome coverage, respectively. Based on genome insert size, the BAC library of Vada gives at least 93% probability of identifying a clone corresponding to any sequence of Vada and for the BAC library of SusPtrit a probability of 98% is expected. Together, the two BAC libraries give more than 99% probability of recovering any specific sequence from the barley genome. A tiling path of three BAC clones was constructed for Vada, which cover the Rphq2 genetic window. The physical window of Rphq2 in Vada BAC contig is approximately 195 Kbp. For SusPtrit, the three BAC clones forming the contig did not cover the entire genetic window of Rphq2. The physical length bridged by them is approximately 226 Kbp. The TriAnnot pipeline annotated 12 genes in both the Vada and the SusPtrit contig, but only four of the annotated genes are shared between Vada and SusPtrit. The candidate genes for Rphq2 might be a resistance factor in Vada or a susceptibility factor in SusPtrit. The peroxidases and kinases are good candidates to represent Rphq2. It is possible that one of the peroxidase or kinase gene members in the physical window of Rphq2 explains the resistance phenotype observed. Another possibility is that peroxidase or kinase gene members function as a complex QTL. A member of the Seven in absentia protein family (SINA) can be a candidate as well. The gene families to which previously cloned genes for partial resistance belong were not found to be represented in the Rphq2 region.
We propose to perform functional analysis of candidate genes through Agrobacterium-mediated stable transformation of the resistance allele into a susceptible genotype, such as SusPtrit. Unfortunately, SusPtrit is, as so many barley accessions, not amenable to Agrobacterium-mediated transformation. In Chapter 2, we developed a doubled haploid (DH) mapping population (n=122) by crossing SusPtrit with Golden Promise to develop a ‘Golden SusPtrit’, i.e., a barley line combining SusPtrit’s high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. Using the DH population, we identified nine genomic regions occupied by QTLs against four non-adapted rust fungi and P. hordei isolate 1.2.1 (Ph.1.2.1). From 12 DH lines that were most susceptible to the tested non-adapted rust fungi, we selected four DHs for an Agrobacterium-mediated transformation efficiency test. We obtained a DH line (SG062N) with transformation efficiency of 11 to 17 transformants per 100 immature embryos. The level of susceptibility of SG062N to non-adapted rust fungi is either similar to or higher than the level of susceptibility of SusPtrit. Against P. hordei, the latency period conferred by SG062N at seedling stage is as short as that conferred by SusPtrit. SG062N, designated ‘Golden SusPtrit’, will be a valuable experimental line that could replace SusPtrit in future nonhost and partial resistance studies, especially for stable transformation using candidate genes that may determine the differences in resistance levels against adapted and non-adapted rust fungi.
Insertional mutagenesis in the vascular wilt pathogen Verticillium dahliae
Santhanam, P. - \ 2014
Wageningen University. Promotor(en): Bart Thomma, co-promotor(en): Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789461738257 - 124
plantenziekteverwekkende schimmels - verticillium dahliae - verwelkingsziekten - insertiemutagenese - solanum lycopersicum - tomaten - modellen - pathogeniteit - genkartering - genomica - plant pathogenic fungi - verticillium dahliae - wilts - insertional mutagenesis - solanum lycopersicum - tomatoes - models - pathogenicity - gene mapping - genomics
Vascular wilt diseases caused by soil-borne pathogens are among the most
devastating plant diseases worldwide. The ascomycete fungus Verticillium dahliae
causes vascular wilt diseases in hundreds of dicotyledonous plant species, including
important crops such as eggplant, lettuce, olive, spinach and tomato. The resting
structures, microsclerotia, are triggered by root exudates to germinate and penetrate
the roots after which the fungus grows into the xylem vessels. The fungus colonizes
these vessels and interferes with the transportation of water and nutrients, resulting in
the development of symptoms such as stunting, wilting, chlorosis and vascular
browning. Verticillium wilt diseases are difficult to control due to the longevity of the
microsclerotia, the broad host range of the pathogen, the inability of fungicides to kill
the fungus once it has colonized the xylem vessels and the lack of natural resistance in
many plant species.
Chapter 1 is the introduction to this thesis that describes the identified
pathogenicity and virulence factors of V. dahliae and strategies to identify these
components. In spite of the economic importance of V. dahliae, relatively few
pathogenicity genes have been identified in this species. With the availability of
whole genome sequences and the development of functional genomics tools such as
random mutagenesis, targeted mutagenesis, transcriptomics, RNA interference
(RNAi) and comparative genomics, more strategies have become available to identify
novel pathogenicity and virulence genes.
Chapter 2 focuses on the identification of virulence and pathogenicity genes
of V. dahliae by screening of a library of random T-DNA insertion mutants. Using
Agrobacterium tumefaciens-mediated transformation, 900 T-DNA transformants with
random insertions were generated and screened for altered virulence on susceptible
tomato plants. This screening, followed by inverse PCR on selected transformants,
resulted in the identification of 55 potential pathogenicity and virulence genes. One of
the potential pathogenicity genes, VdNRS/ER, is a homolog of a nucleotide-rhamnose
synthase/epimerase-reductase (NRS/ER), which is presumably involved in the
biosynthesis of UDP-rhamnose. Using targeted mutagenesis, VdNRS/ER was deleted
from wild-type V. dahliae and the resulting deletion mutants were characterized.
VdNRS/ER deletion mutants exhibit unaltered vegetative growth and sporulation, but
the deletion mutants were no longer pathogenic on tomato and N. benthamiana and
showed impaired root attachment on tomato seedlings. These data suggest that UDPrhamnose
is required for pathogenesis of V. dahliae.
Chapter 3 describes the role of the V. dahliae homolog of Sge1, a
transcriptional regulator that was shown to play a role in pathogenicity and regulate
effector gene expression in Fusarium oxysporum. In this chapter it is demonstrated
that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual
conidiospores. It is furthermore shown that VdSge1 deletion strains have lost
pathogenicity on tomato. Since the VdSge1 deletion mutants are not able to infect and
colonize tomato plants, a tomato cell suspension culture was used to the study the
expression of Ave1, as well as nine other genes of which the expression is highly
induced in planta. This assay revealed that VdSge1 is not required for the induction of
the Ave1 effector that activates resistance mediated by Ve1 in tomato. Furthermore,
the expression of one other putative effector gene was not affected by VdSge1
deletion. However, VdSge1 was shown to be required for the expression of six
putative effector genes, whereas expression of the remaining two putative effectors
genes was negatively regulated. Thus, the data show that VdSge1 is required for V.
dahliae pathogenicity and differentially regulates effector gene expression.
Chapter 4 describes the functional characterization of the gene family
encoding necrosis- and ethylene-inducing-like proteins (NLPs) of V. dahliae. The
cytotoxic activity of NLP family members was determined using agroinfiltration into
tobacco leaves. This resulted in the identification of two out of the seven NLPs,
VdNLP1 and VdNLP2, that induced plant cell death. The genes encoding these
cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato.
Targeted deletion of VdNLP1 and VdNLP2 significantly reduced the virulence of V.
dahliae on tomato and Arabidopsis plants. In contrast, only deletion of VdNLP1
affected virulence on N. benthamiana whereas deletion of NLP2 did not. However,
subsequent transcriptional analysis revealed that VdNLP2 was not expressed in V.
dahliae during colonization of N. benthamiana. Moreover, VdNLP2 also affects
vegetative growth and conidiospore production. In conclusion, the expanded V.
dahliae NLP family shows differential cytotoxic activity between family members
and in planta induction of the cytotoxic NLP genes varies between plant hosts. In
addition, VdNLP2 plays a role in vegetative growth and conidiospore production in
addition to its contribution to virulence. Thus, evidence is provided for functional
diversification within the V. dahliae NLP family.
Finally in Chapter 5, the major findings of this thesis are discussed and
placed in a broader perspective.
Identification of Arabidopsis thaliana genes that can increase resistance towards phloem feeding insects
Chen, X. - \ 2013
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Ben Vosman. - [S.l.] : S.n. - ISBN 9789461737649 - 96
arabidopsis thaliana - insectenplagen - myzus persicae - plaagresistentie - genkartering - genexpressie - mutanten - plantenveredeling - turnip yellows virus - vectoren, ziekten - arabidopsis thaliana - insect pests - myzus persicae - pest resistance - gene mapping - gene expression - mutants - plant breeding - turnip yellows virus - disease vectors
Phloem feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, but also by vectoring plant viruses. During their evolution plants have developed a variety of defense traits to combat insects. These plant resistance traits can be antixenotic and/or antibiotic. Antixenosis is the first line of defense that prevents insects from landing and settling, while antibiosis reduces the population development of the colonizing insects.In this project we aimed at identifying genes that can increase resistance towards phloem feeding insects and also prevent, as far as possible, transmission of viruses. Acknowledging that changing the expression level or expression localization of genes might increase resistance, we screened an Arabidopsis thaliana activation tag gain-of-function mutant collection for increased resistance towards the green peach aphid (Myzus persicae). In these mutants, tagged genes are overexpressed by the strong 35S enhancer adjacent to the natural promoter that results in a dominant gain-of-function phenotype. The overexpression of a particular gene in such mutants may result in enhanced resistance to aphids and other phloem feeding insects.
To identify mutants with increased insect resistance efficient and reproducible screening methods needed to be developed first. Based on the hypothesis that there is a trade-off between plant fitness and plant resistance, we first screened a subset of 170 mutants that were previously selected based on their reduced growth to increase the chance of identifying mutants with increasedresistance. In this screening we usedchoice assays and selected one mutant that displays enhanced antixenosis based resistance towards aphids. Further characterization of this mutant revealed that that the antixenosis is phloem based and requires intact plants.
To evaluate aphid resistance of a larger number (>5000) of activation tag mutants, we established a high throughput screening system in which plant resistance against aphids is inferred from a reduced transmission of the circulative Turnip yellows virus(TuYV). This virus can only be transmitted into a plant after virus-infected aphids feed for a prolonged (> 10min) time from the phloem sap. In the initial screening 13 virus-free mutant lines were identified. The putative candidate mutant lines were re-evaluated and characterized, resulting in nine mutants on which aphids showed a reduced population development.
Molecular analysis of two of these mutants revealed that the genes underlying the resistance were IRM1(Increased ResistancetoMyzus persicae1,At5g65040)and SKS13 (SKU5Similar13, At3g13400). In wild type plants,IRM1is strongly expressed in xylem and extremely low expressed in other plant tissue whereas SKS13 is exclusively expressed in pollen. We show that constitutive overexpression of these genes in all plant tissues confers enhanced resistance towards aphids. Analysis of aphid feeding behavior showed that the resistance conferred by IRM1and SKS13affect the aphids differently. On the IRM1 overexpressing mutant aphids encounter difficulties in reaching the phloem, indicating that resistance factors are located between the cell surface and the phloem. On the SKS13overexpressingmutant the phloem feeding of aphids is severely affected, indicating that resistance factors are phloem based. Further analysis strongly suggests the involvement of Reactive Oxygen Species (ROS) in the reduced aphid performance on the SKS13overexpressingmutant. We also show that the resistances are not aphid specific, as the performance of the cabbage aphid (Brevicoryne brassicae)is also affectedon both overexpressing mutants.
The results obtained in this thesis show that plant resistance to insects can be increased by expressing genes that are assigned for other biological functions. Characterization of the identified mutants revealed twogenes conferring enhanced aphid resistance via different mechanisms. These findings lead to a better understanding of plant-aphid interactions on the molecular level. Furthermore, such knowledge obtained from the model plant A.thalianashould be applied in crop plants, which can be achieved by transgenic and genetic studies in combination with newly developed techniques, such as RNAi and TILLING.
Analysis of Tomato spotted wilt virus effector-triggered immunity
Ronde, D. de - \ 2013
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - S.l. : s.n. - ISBN 9789461737212 - 190
tomatenbronsvlekkenvirus - plantenvirussen - ziekteresistentie - immuniteit - virulentie - genkartering - genetische merkers - genetische analyse - capsicum annuum - paprika's - tomato spotted wilt virus - plant viruses - disease resistance - immunity - virulence - gene mapping - genetic markers - genetic analysis - capsicum annuum - sweet peppers
ResistanceinCapsicumagainsttheTomatospottedwiltvirus(TSWV),typespeciesof the Tospovirusgenuswithinthe Bunyaviridaefamily,employsthe singledominant resistancegeneTsw.Thisresistance hasmeanwhilebeenbrokenbyresistance breaking (RB) TSWV isolates and is causing increasing problems in many different (Capsicumcultivating)countries.Theresearchdescribedhereaimedtoidentify andcharacterise theviralproteintriggeringTswresistanceandprovidefurther insightintothemechanismofTsw-mediatedresistance.Knowledgegainedfrom thegeneticandphenotypiccharacterisationofTsw-resistancebreakingisolateswas usedtodevelopdiagnosticmarkersfordetectionofTsw-breakingpathotypesin fieldcultivations.
TheNSsRNAsilencingsuppressor(RSS)proteinwasidentifiedastheavirulence determinant ofTsw-mediatedresistance(Chapter2).WhiletheNSsproteinfrom theTSWVresistanceinducer(RI)isolatewasactiveasRNAsilencingsuppressorand avirulencedeterminant,theNSsproteinfromtwodifferentTSWVRBisolateslacked bothfunctionsasevidencedfromtransientassays.Surprisingly,thecorresponding resistancebreakingvirusisolatesstillexhibitedRNAisuppressoractivity. Noneof the other viral proteins were able to aid in the transient recovery of RSS activity. Electrophoreticmobilityshift assays(EMSAs)usingplantextractscontaining transientlyexpressedNSsproteinsshowedashift ofsiRNAswithNSsRI,indicative forbinding,butnotwithNSsRB.InagreementwiththelocalleafRSSassaysusinga virusinfection,plantextractsofvirusinfectedleaveswereabletoshiftthesiRNAs, showing recovery of the RSS activityduring virus infection.
The linkage of RNAi suppression and avirulence in NSs was further investigated bymutationalanalysis(Chapter3).AlargesetofNSsmutantswasgeneratedusing alaninesubstitutions ofauthenticTSWVNSsaminoacidsandwastestedfortheir abilitytotriggerTsw-mediatedHRandabilitytosuppressRNAi.Theseassaysshowed thatthe N-terminaldomainofNSscarried mostimportantresiduesinvolvedwith bothactivities. However,singlemutationscouldbeintroducedthatdisruptedone function,whilemaintainingtheotheroneandviceversaindicatingthatRSSactivity andavirulencewerenotfunctionally linked.SwappingofdomainsbetweenNSsRI andNSsRB notonlyconfirmedtheimportanceoftheN-terminaldomainbutalso thespecificitywithintheTSWVspecies,sincedomainswapsbetweenNSsRIandNSs fromGRSV,arelatedbutdistinct Tospovirus,couldnottransfertheAvrphenotype toGRSV.MutationofaGW/WG-motifintheNterminalregionofNSsRI leadtoa lossofbothfunctionsandindicatedthatthismotif, knowntobeinvolvedinAGO1 interactionof other viral RSS, was of biological relevance for TSWV NSs.
Theputativeinteraction ofAGO1andNSswasinvestigatedbyusingdifferent approaches to co-immunoprecipitate (Co-IP) on transiently co-expressed tagged- AGO1and(His-)NSs(Chapter4).Initialindicationsforsuchinteraction were obtained,howeverfurthersupportforthisputativeinteraction willhavetocome fromcomplementaryexperiments,e.g. Yeast-2-hybrid (Y2H), FRET-FLIM or BiFC.
Severaladditional TSWVisolateswereanalysedthatbesidestheknownresistance inducing-and resistance breaking-phenotype showed a temperature-dependent phenotype(Chapter5).IsolatesclassifiedtothistypeexhibitedanRIphenotypeat standardgreenhouseconditions (~22°C)whileatelevatedtemperatures(≥28°C), butstillbelowtemperaturesthatinactivatedtheR-geneproduct(≥31°C),wereable tobreaktheresistance.Viruschallengingassaysatvariousconditionsindicatedthat inductionofTswresistanceatalower temperaturebythesesocalledtemperature dependentresistancebreakingisolates(TempRB)involveddenovosynthesisofthe avirulenceprotein,i.e.NSs,andthat proteinfoldingmight play arole. NSsproteins clonedandexpressedfromthisadditional newsetofTSWVresistanceinducing, resistancebreakingandtemperature dependentresistancebreakingisolates revealedvariableresultsregardless oftheircorrespondingvirusphenotype,when tested for their abilitytoinduceTsw-mediated HR andsuppress RNAi at normal greenhouseconditions(22°C).However,similarassaystoanalysetheiractivity attheelevatedtemperature(28°C)failedwhenusingAgrobacteriummediated transientassays.Sofar,themechanismoftemperature dependencyhasnotbeen clarified yetandneedsfurtherinvestigation.Usingtheinformationobtained,a diagnostictoolwasdevelopedtoscreenforthepotential presenceofresistance breakingisolatesofTSWVusingreversetranscription-polymerasechainreaction amplification(RT-PCR).Aprimersetwasdesignedtargetinganimportantcodon ataaposition79andshowedtobeabletodistinguishRB-isolatesfromRI-isolates. However,afewRB-isolatesstillescapedfromdetection indicatingthelimitedand conditionaluse of this tool.
In summary, NSs has been identified as Avr-determinant of Tsw-mediated resistance,butthisfunctionisnottightlylinkedtoitsRNAisuppressor-activity. Preliminarydataindicateaputativeinteraction betweenAGO1andNSs.Besides the typicalRIandRBphenotypes,athirdphenotypicclassofTSWVisolates has beenidentified thatexhibitsatemperaturedependencyontriggeringTsw- mediatedresistance andpossiblyinvolvesanalteredproteinfoldingofNSs.A diagnostic toolhasbeendevelopedtodetectresistancebreakingisolatesinthe fieldbasedonRT-PCR,butthistoolstillallowsforescapesofRBisolates.Theresults onNSsarediscussedinlightofitsroleaseffectorwithinthe‘Zig-zag-model’of planthostdefenceresponses.Finally,TSWVNSsisbriefly discussedandcompared totheanimal-infecting(NSs)paralogsoftheBunyaviridaefamily,alsoinlightof functional andstructuralhomologiesbetweenthesensorsofinnateimmunityin plant(R-genes)and animal (NLRs/TLRs) cell systems.
Unveiling and deploying durability of late blight resistance in potato : from natural stacking to cisgenic stacking
Kwang-Ryong Jo, - \ 2013
Wageningen University. Promotor(en): Richard Visser; Evert Jacobsen, co-promotor(en): Jack Vossen. - S.l. : s.n. - ISBN 9789085855798 - 168
solanum tuberosum - aardappelen - ziekteresistentie - phytophthora infestans - plantenziekteverwekkende schimmels - verdedigingsmechanismen - genkartering - transgene planten - solanum tuberosum - potatoes - disease resistance - phytophthora infestans - plant pathogenic fungi - defence mechanisms - gene mapping - transgenic plants
The potato, which receives an increased attention as a food crop, has long been in threats from the oomycete Phytophthora infestans, the causal agent of late blight. This disease still remains the most important constraint in potato producing regions of the world. It might cause the complete destruction of the foliage and tubers of potato if meteorological conditions are conducive to the onset and spread of late blight epidemics. Although fungicides applications provide sufficient levels of late blight control, they impose high input costs to the farmer, are detrimental to human and environment and increase the capacity of the pathogen to develop resistance to the active ingredients of fungicides applied. The increased genetic diversity in P. infestanspopulations due to sexual recombination between two mating types in many parts of the world and the emergence of fungicide resistant strains poses the necessity to develop potatoes that possess high levels of durable resistance as an alternative to the use of fungicides. Clones MaR8 and MaR9 from the Mastenbroek differential set, used to assess virulence towards Rgenes, have been known for their strong resistance to P. infestans. This also holds for cultivar Sarpo Mira which has retained resistance in the field over several years without fungicide applications. Uncovering genetic basis of such, partly naturally-formed, late blight resistance is the prerequisite for the implementation of durable resistance in a breeding scheme. In this study, MaR8, MaR9and cv. Sarpo Mira were used as plant materials for unveiling durability of late blight resistance in potato. First, F1 mapping populations from crosses between these resistant materials with susceptible parents were assessed for late blight resistance in field trials and in detached leaf assays (DLA) after inoculation with an incompatible P. infestans isolate IPO-C. A 1:1 segregation of resistance and susceptibility was observed in the MaR8derived-F1 population in field trials, but not in detached leaf assays. NBS profiling and Rgene cluster directed profiling (CDP), followed by marker landing in the newly sequenced potato genome, referred to as “anchored scaffold approach”, led to the mapping of R8at a new locus on chromosome IX rather than on chromosome XI, the previously suggested chromosomal position (Chapter 2). The Rgene mediated resistance reaction in potato is a consequence of an (in)direct interaction between the pathogen Avrand host Rgene product that leads to a hypersensitive cell death (HR). We screened a wide collection of RXLR effectors of P. infestansfor eliciting cell death in the differential potato MaR8 by agroinfiltration (Chapter 3). R8-specific cell death to one effector PITG_07558, termed AVR8, co-segregated with the R8-mediated resistance to P. infestansisolate IPO-C in a F1 population. From the notion that Avr8is identical to effector AvrSmira2that was previously found to associate with field resistance in cultivar Sarpo Mira, we performed genetic mapping studies in a Sarpo Mira-based F1 population and indeed Rpi-Smira2localized in the R8locus. To investigate the geographical and phylogenetic origin of R8in the Solanumgene pool, we conducted functional screens for AVR8 responsiveness in 98 wild genotypes (72 accessions of 40 species) of Solanumsection Petota. We identified twelve AVR8 responding Solanum accessions originating both from Central and South America. Interestingly, our study involving late blight resistance from the differential plant MaR9described that it is near the R8 locus on chromosome IX (Chapter 4). An integrated approach combining 1. a Rgene ”de-stacking” approach using Rgene specific marker analysis and effector responses, 2. the whole plant climate cell assay, and 3. CDP profiling enabled a clear picture for the presence of two closely linked genes, termed R9aand R9b. It was shown that R9alocates in a Tm-22 cluster of NB-LRR genes and, most likely will be a member of the Tm-22 Rgene family (Chapter 4). The identified fully co-segregating Tm-2 likeCDP markers were used to select the R9agene-containing BAC clone, demonstrating the possibility of BAC landing by marker saturation in the targeted chromosomal regions (Chapter 5). For cloning R9agene, a bacterial artificial chromosome (BAC) library derived from the differential plant MaR9, was screened with co-segregating Rgene CDP markers whereby two overlapping BAC clones carrying CDP markers were obtained. Sequence annotation of the complete insert of these BAC clones identified the presence of two complete Rgene analogs (RGA9.1 andRGA9.2) of the NB-LRR class in one BAC clone. Two RGAs, including their natural regulatory transcriptional elements, were subcloned by long-range PCR into a binary vector for plant transformation. After transformation, it was found that RGA9.1was able to complement the susceptible phenotype in cultivar Desiree. RGA9.1, now designated R9a,encodes a CC-NB-LRR protein of the Tm2 family, where the LRR consensus is only loosely fitted. Agroinfiltration-based effector screens for identifying the Avrgenes matching the R9agene was performed, leading to the discovery of Avrblb2 homologs which trigger R9amediated hypersensitivity in Nicotiana benthamiana (Chapter 5).Resistance profiling with 54 P. infestans isolates showed that MaR9 and S.xedinense accessions had similar resistance spectra as the Rpi-blb2containing cultivar Bionica. Transformation of potato with resistance genes and antibiotic resistance markers encounters consumers’ criticism. These criticisms are considerably less if only resistance genes from crossable species, and no antibiotic resistance selection marker is used. Genes deriving from crossable species are referred to as cisgenes. For the production of cisgenic potatoes with a broader resistance spectrum and potential durability, Agrobacterium-mediated marker free transformation and PCR selection of transformants was performed. This way four potato cultivars (Atlantic, Bintje, Potae9 and Doip1) were successfully transformed with a construct containing two cisgenic Rpigenes (Rpi-vnt1from Solanum venturiiand Rpi-sto1from Solanum stoloniferum) (Chapter 6). Resistance assays in untransformed varieties with five P. infestansisolates showed that cvs. Potae9 and Doip1 were already resistant to certain isolates. Single Rpigene containing transgenic plants for all 4 varieties were obtained and used as references. Marker free transformation with a construct containing two Rpi genes (cisgenesis) was compared to kanamycin assisted selection (transgenesis) in terms of regeneration and transformation frequency, vector backbone integration, and T-DNA copy number. In addition, the different time tracks to harvest regenerated shoots for the selection of PCR positive regenerants for one or both Rpi-genes were studied. Through further analyses involving phenotypic evaluations in the greenhouse, agroinfiltration of avirulence (Avr) genes and detached leaf assays, totally eight cisgenic plants were selected. Two cisgenic plants of cv. Altantic and four of cv. Bintje, were selected that showed broad spectrum late blight resistance due to the activity of both Rpigenes. Based on characterization of two cisgenic transformants of cv. Potae9, it was demonstrated that the existing late blight resistance spectrum has been broadened by adding the two Rpigenes. Finally, results from this study are discussed in terms of genetic and molecular mechanism of durability and cisgenic deployment to address the challenges of the durable resistant potato variety development (Chapter 7). We pursue possible options for durability in the nature of the Rgenes or their cognate Avrgenes. The comparative analysis of several features of available R-AVR pairs shows that major components for producing durability are the copy number variation in the P. infestansgenome and abundance of the Avrgene in different isolates. As a counterpart of such an Avrgene, potato Rgenes that display broad spectrum resistance and often have abundant functional homologs among various wild Solanumspecies could be optional for Rgene combinations providing durability. Multiple years’ on-site-monitoring of resistance spectrum in natural Rgene stacks demonstrates that stacking of several broad spectrum Rpigenes or even “defeated” Rgenes could sum up to high levels of resistance potentially capable to provide durability to commercial potato cultivars. Our data about acquirement of complementary resistance spectrum by cisgenic introduction of two broad spectrum resistance genes into cultivars support a first step into that direction.
Characterization of major resistance genes to Tomato Yellow Leaf Curl Virus
Verlaan, M.G. - \ 2013
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Richard Kormelink. - [S.l.] : s.n. - ISBN 9789461735614 - 158
solanum lycopersicum - tomaten - tomatengeelkrulbladvirus - ziekteresistentie - plantenveredeling - genkartering - introgressie - uitschakelen van genexpressie - solanum lycopersicum - tomatoes - tomato yellow leaf curl virus - disease resistance - plant breeding - gene mapping - introgression - gene silencing
Tomato yellow leaf curl disease, a devastating disease of tomato, is caused by a complex of begomoviruses generally referred to as Tomato yellow leaf curl virus (TYLCV). Almost all breeding for TYLCV resistance has been based on the introgression of the Ty-1 and Ty-3 resistance loci derived from Solanum chilense LA1969 and LA1932/LA2779 respectively. The aim of this thesis was to fine map, clone and characterize these two TYLCV resistance genes.
The Ty-1 gene has been used in tomato breeding already for almost 20 years. Its exact genetic location was however unknown which made precise marker assisted breeding difficult. Here we have analysed the recombination behavior of the chromosomal region where Ty-1 is introgressed by applying newly developed molecular markers in two F2 populations obtained from two commercial Ty-1 carrying hybrids. A big S. chilense introgression was detected in both populations that coveralmost the whole short arm and a part of the long arm of chromosome 6. In this introgression recombination suppression was detected and Fluorescence in situ Hybridization (FISH) analysis revealed two chromosomal rearrangements between S. lycopersicum and S. chilense LA1969. These rearrangements are most likely the cause of the observed recombination suppression. Using disease tests on progeny of informative recombinants Ty-1 was mapped to a region of approximately 600 kb which partly overlapped with the mapped region for Ty-3, which led to the indication that Ty-1 and Ty-3 could be allelic. Altogether these results nicely demonstrate the usefulness of FISH as a powerful tool to aid in the accurate mapping of genes that are introgressed from wild species into cultivated tomato (Chapter 2).
To further fine map and ultimately clone Ty-1 and Ty-3 more plants were screened for recombination events and consequently recombinant inbred lines were generated. By developing new markers in combination with disease tests both genes were fine mapped to a very small, almost similar genomic region (approximately 70 kb). Using a Tobacco Rattle Virus-Virus Induced Gene Silencing approach, the resistance genes were finally identified. It was shown that Ty-1 and Ty-3 are allelic and that they code for a RNA-dependent RNA polymerase (RDR) belonging to the RDRɣ type which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRαtype, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRɣ type. With the identification of Ty-1/Ty-3, a completely new class of resistance genes was unveiled (Chapter 3).
The Ty-1/Ty-3 allele is characterized by a 4 amino acid insertion at the 5-prime part of the protein and by a catalytic DFDGD motif. The allelic variation of this gene was examined using cDNA from five S. chilense derived lines and using draft assemblies of whole genome sequences from more than 50 tomato cultivars, landraces and related wild species. Tobacco Rattle Virus induced gene silencing was used to silence Ty-1/Ty-3 and altogether showed that resistance was compromised in three out of five S. chilense derived lines tested. One line with resistance derived from S. chilense LA1971 remained resistance after silencing of Ty-1/Ty-3. For another line (8783, derived from LA1932) only 4 out of 13 plants showed symptoms after silencing,but silencing in this line was inefficient because only one out of three PDS controls showed photobleaching. Comparison of the two typical features of the Ty-1/Ty-3 gene showed no sequence variation amongst S. chilense derived lines. The catalytic domain was found to be conserved among all tomato lines and species analysed, while the characteristic 4 amino acid insertion was also observed in three species closely related to S. chilense, e.g.Solanum corneliomulleri, Solanum peruvianum and Solanum huaylasense. This indicated that most S. chilense accessions most likely carry a functional TYLCV resistance locus on chromosome 6, allelic to Ty-1/Ty-3, and Solanum species related to S. chilense could possibly be useful for future TYLCV resistance breeding (Chapter 4).
The Ty-1 gene encoded an RDR and for this reason most likely conferred resistance involving amplification of the siRNA signal. In the last experimental chapter (Chapter 5) this hypothesis was tested. It was shown that upon TYLCV challenging of resistant Ty-1 and Ty-3 lines low virus titers were detected concomitant with the production of relatively high levels of siRNAs. In contrast to the situation in susceptible tomato Moneymaker where high virus titers were observed, but the amount of siRNAs produced lower compared to those in Ty-1 and Ty-3. Analysis of the spatial genomic siRNA distribution showed a consistent and subtle enrichment for siRNAs derived from the CP (V1) and C3 gene in Ty-1 and Ty-3 lines compared with Moneymaker. In tomato plants containing the Ty-2 resistance gene, included as a control and not an RDR, the virus was hardly detectable but the siRNA profile similar to the one observed in TYLCV-challenged susceptible tomato Moneymaker. Furthermore, genome methylation analysis revealed a relative hypermethylation of the TYLCV CP (V1) promoter region in genomic DNA collected from Ty-1 in comparison to susceptible tomato Moneymaker.
Altogether this thesis describes the mapping, cloning and characterization of the TYLCV resistance genes Ty-1 and Ty-3. Future breeding efforts for TYLCV resistance can now exploit in-gene markers and the insights obtained can possibly direct future research and breeding efforts on plant virus resistance (Chapter 6).
Resistance and susceptibility to late blight in Solanum: gene mapping, cloning and stacking
Verzaux, E.C. - \ 2010
Wageningen University. Promotor(en): Richard Visser; Evert Jacobsen. - [S.l. : S.n. - ISBN 9789085856313 - 144
phytophthora infestans - solanum - wilde verwanten - ziekteresistentie - genkartering - dna-klonering - genetische kartering - plantenveredeling - resistentieveredeling - phytophthora infestans - solanum - wild relatives - disease resistance - gene mapping - dna cloning - genetic mapping - plant breeding - resistance breeding
The potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin.
The genetic and molecular basis of natural variation for plant growth and related traits in Arabidopsis thaliana
Pieper, B. - \ 2009
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): M. Reymond. - [S.l.] : S.n. - ISBN 9789085854685 - 131
arabidopsis thaliana - moleculaire genetica - groei - loci voor kwantitatief kenmerk - bloei - genetische variatie - genkartering - plantengenetica - arabidopsis thaliana - molecular genetics - growth - quantitative trait loci - flowering - genetic variation - gene mapping - plant genetics
Plant growth is a complex quantitative trait of which the (eco)physiological aspects have been researched in great detail. In contrast, little is known about the genetic basis and molecular basis of plant growth. Most of what is known today has been discovered by using reverse genetic approaches that target single genes. Forward genetics using naturally occurring variation provides the possibility to investigate genetic effects within the context of the genetic background. This thesis describes the results of a study that aimed at elucidating the genetic basis and the underlying molecular basis of natural genetic variation for plant growth and related traits in Arabidopsis thaliana. A set of over 30 growth related traits has been quantified in two Arabidopsis RIL populations, derived from crosses between the accessions Landsberg erecta (Ler) x Kashmir-2 and Landsberg erecta x Shakdara (Sha) respectively. The projected rosette area (PRA) and Feret diameter of each plant was measured repeatedly during its growth using digital image analysis and. A logistic model and a linear model were fitted to the PRA data for quantification of plant growth rate. The dimensions of the largest rosette leaves, plant hight and chlorophyll content index were measured. Furthermore, flowering time and leaf numbers were recorded. A selection of 15 traits was used to perform a multi-trait QTL analysis using mixed-model methodology. A total 19 QTL were detected of which some art similar locations in both populations. QTL important for flowering time were also found to explain nearly half of the genetic variance for plant growth rate. These QTL mainly mapped to similar locations in both populations indicating a common genetic basis for the affected traits in the studied accessions. However, population specific QTL with no effect on flowering time accounted for the remaining genetic variance explained for plant growth. These QTL were chosen for follow-up studies in order to elucidate the underlying molecular basis. The detected QTL effects were validated in a selected set of near isogenic lines (NILs). These NILs will provide the opportunity to fine-map the QTL in order to identify the genes, and in particular the DNA polymorphisms responsible for these effects. For a single QTL fine-mapping could be advanced efficiently, currently down to 151 genes, due to particularly penetrant effects on plant morphology. QTL that also affected flowering time were not the main interest of these studies because they mapped to positions of genes that were already described for their role in the regulation of this trait. However, although the relation between flowering time and plant growth of such QTL had been described in literature, no distinction between pleiotropy and closely linked QTL could be made so far. A QTL that effected flowering time and plant growth rate, among other traits, located on the bottom end of chromosome 5 has been fine-mapped and cloned using NILs with introgressions of Sha in the genetic background of Ler. Fine-mapping achieved a resolution at which it could be proven that the cluster of MAF2-MAF5 (MADS affecting flowering) genes was underlying the detected QTL effects on flowering time. The effects on plant growth were fine-mapped to a slightly larger region this included an additional 3 genes. Although it was known that these genes affect flowering time, their role in natural variation for this trait had not been directly shown before. Sequence analysis of the cluster showed that Sha carried a fusion consisting of the 5’ portion of MAF2 and the 3’ portion of MAF3. An intact MAF3 gene was not found in this accession. Recently it was shown that such MAF2/MAF3 fusions are relatively common among Arabidopsis accessions but the loss of MAF3 makes Sha unique. Genomic complementation indicated that neither MAF2, MAF3 nor MAF4 can explain the QTL effects by itself. The current hypothesis is therefore that both MAF2 and MAF3 are required in the regulation of flowering time to reconstitute the phenotypical value of Ler in a NIL.
The Genetics of Pork Quality
Wijk, H.J. van - \ 2006
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Henk Bovenhuis. - [S.l.] : S.n. - ISBN 9789085045045 - 176
varkens - varkensvlees - vleeskwaliteit - karkassamenstelling - genetica - genetische parameters - loci voor kwantitatief kenmerk - genoomanalyse - genkartering - merkers - pigs - pigmeat - meat quality - carcass composition - genetics - genetic parameters - quantitative trait loci - genome analysis - gene mapping - markers
This thesis describes the genetics of carcass composition and pork quality traits. A large population of commercial finishers was extensively phenotyped for growth, carcass composition and meat quality traits. Genetic parameters were estimated based on those measurements. The population was genotyped using 73 microsatellite markers covering approximately 50 percent of the genome. The covered genome regions were pre-selected based on published QTL for carcass composition and meat quality traits that were mainly obtained from divergent crosses. Significant evidence for QTL was obtained. Although with chromosome-wise significance values of 5% the obtained evidence was not very strong despite the large population size. Additional half-sib families and markers were typed to validate and further investigate the map position of QTL identified in regions on chromosomes 2, 4, 11, 13 and 14. A variance component (VC) analysis method using linkage and linkage disequilibrium was applied to further characterize and refine the map position of the QTL. The results were compared with results obtained by the classic regression analysis method. The VC analysis results reveal the considerable contribution of the dam haplotypes to the variance of meat quality traits. An accurate positioning of the QTL however was not yet possible with the marker density so far. Therefore, new microsatellites were developed by in silico analysis of BAC-end sequences (BES) of BACs on the porcine physical map and genomic shotgun sequences. This resulted in the identification of thousands of new markers covering the porcine genome with over 200 new markers in the region of interest on SSC4. The ~200 markers were tested and resulted in ~60 markers that were informative and used in an effort to further fine mapping of the QTL on SSC4.
Mixed model methodology for the identification of genetical factors underlying trait variation in plants
Malosetti, M. - \ 2006
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Fred van Eeuwijk. - [S.l.] : S.n. - ISBN 9789085043515 - 128
planten - loci voor kwantitatief kenmerk - genetische merkers - genkartering - plantenveredeling - veredelingsprogramma's - modellen - genotype-milieu interactie - plants - quantitative trait loci - genetic markers - gene mapping - plant breeding - breeding programmes - models - genotype environment interaction
The advent of DNA-marker technology has made the detection of quantitative trait loci (QTLs) a routine activity in plant breeding. While standard procedures are available for QTL mapping, more flexible strategies are required to analyse the complex data typically produced by plant breeding programs. This thesis presents a general and flexible mixed model QTL mapping approach. The philosophy is to include genotypic information derived from molecular markers as explanatory variables to model complex phenotypic responses, while exploiting the flexibility of mixed models to account for complex variance-covariance structures in the data. Attractive generalisations of our QTL models incorporate explicit information on genotypes and environments, for example, as obtained from crop growth models, thereby opening the way to eco-physiological QTL models.The application of the methodology to cases commonly found in plant breeding is illustrated throughout the various chapters of the thesis. In chapter 2, a QTL model is presented for probably the most typical experimental set up in plant breeding, the multi-environment trial. The QTL model describes the genetic basis of genotype by environment interaction for a single trait and tests for environment-specific QTL effects (QTL by environment interaction). After detection of QTLs underlying genotype by environment interaction, the model is extended to make QTL expression dependent on environmental variables. In a reanalysis of yield data stemming from the North American Barley Genome Mapping Project (NABGMP), a QTL for yield was detected on barley chromosome 2H whose effect was proportional to the temperature range at heading time. In chapter 3, the single trait multi-environment model is elaborated to the multi-trait multi-environment situation. We show with another reanalysis of data from the NABGMP how questions related to pleiotropy and genetic linkage as causing genetic correlations between traits can be addressed. Both pleiotropic and linked QTLs were found to explain genetic correlations between heading date and yield. In chapter 4, the linear mixed model is generalised to a non-linear mixed model to describe parameters of a growth curve as a function of QTL effects. The approach is illustrated by an example on potato leaf senescence. QTLs were identified that affect growth trajectories in different ways, thereby contributing to a better understanding of the genetic control of complex traits over time. In chapter 5, we show that not only designed bi-parental cross populations can be naturally handled by mixed model QTL formulations, but also non-designed populations within association mapping approaches. With another example in potato, we analyse historical disease resistance data produced over 25 years of potato variety trial testing in combination with targeted molecular marker techniques to detect interesting markers for breeders. Pedigree information was used to improve the modelling of genetic variances and correlations between genotypes. An association mapping approach using mixed models incorporating pedigree information performed better than commonly used association mapping strategies. Significant associations were detected that proved to be consistent when tested in a confirmatory data set. Finally, chapter 6 consists in a discussion of the mixed model approach as a general framework for QTL mapping in plant breeding.
Over verschillen en overeenkomsten
Groenen, M.A.M. - \ 2005
Wageningen : Wageningen Universiteit - 35
vee - genomen - dna - genen - genkartering - dna-sequencing - genetische variatie - moleculaire genetica - spraak - livestock - genomes - dna - genes - gene mapping - dna sequencing - genetic variation - molecular genetics - speech
Genoomonderzoek bij landbouwhuisdieren levert een belangrijke bijdrage aan een verhoogd fundamenteel wetenschappelijk inzicht in het biologisch functioneren van dier en mens en maakt het in toenemende mate mogelijk om binnen de fokkerij dieren te selecteren op basis van bekende variaties in het erfelijk materiaal (DNA)
Herkenning van de elicitor Ecp2 van Cladosporium fulvum in tomaat en niet-waard planten
Kock, M. de - \ 2005
Gewasbescherming 36 (2005)3. - ISSN 0166-6495 - p. 132 - 134.
plantenziekteverwekkers - plantenziekteverwekkende schimmels - ziekteresistentie - genetisch bepaalde resistentie - chromosomen - genen - genkartering - genlokalisatie - loci - solanum - passalora fulva - plant pathogens - plant pathogenic fungi - disease resistance - genetic resistance - chromosomes - genes - gene mapping - gene location - loci - solanum - passalora fulva
Op 4 oktober 2004 promoveerde Maarten de Kock aan de Wageningen Universiteit op het proefschrift getiteld 'Recognition of the Cladosporium fulvum Ecp2 elicitor in tomato and non-host plants'. De resultaten van dit promotieonderzoek vormen een wetenschappelijke basis voor verdere bestudering van de co-evolutie tussen planten en pathogenen en maken een voorzichtige voorspelling van de duurzaamheid van resistentiegenen mogelijk
Different genetic bases of immune responses in laying hens
Siwek, M.Z. - \ 2005
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Jan van der Poel; Henk Parmentier. - [S.l.] : S.n. - ISBN 9789085041535 - 153
hennen - pluimvee - immuniteitsreactie - genetische factoren - lijnverschillen - selectief fokken - kwantitatieve kenmerken - loci - genkartering - hens - poultry - immune response - genetic factors - line differences - selective breeding - quantitative traits - loci - gene mapping
Chicken genome mapping - Constructing part of a road map for mining this bird's DNA
Aerts, J. - \ 2005
Wageningen University. Promotor(en): Martien Groenen. - Wageningen : - ISBN 9789085042082 - 187
kippen - genoomanalyse - genkartering - genetische kartering - dna-sequencing - koppeling - genetica - fowls - genome analysis - gene mapping - genetic mapping - dna sequencing - linkage - genetics
The aim of the research presented in this thesis was to aid in the international chicken genome mapping effort. To this purpose, a significant contribution was made to the construction of the chicken whole-genome BAC-based physical map (presented in Chapter A). An important aspect of this construction was the integration of the genetic and physical maps (see Chapters 2 and 3) in order to anchor clone-contigs to a chromosomal location.
In addition to the construction of the physical map, two different SNP resources were analyzed. First, it is shown in Chapter 5 that cione-based sequencing is a valid SNP discovery strategy. Second, an anaLysis of public SNP repositories was presented, focussing on intra-genic SNP discovery in human (Chapter 6) which pinpointed some issues related to mining public SNP databases. In Chapter 7, the extent of linkage disequiiibrium -~ reflecting the history of recombinations - was assessed in two regions of the chicken genome. This information is useful for setting up linkage and association studies. Finally, a software tool was described (Chapter 8) that was implemented to automate the analysis of sequencing data, which is especially important in major mapping experiments as described in this thesis.
The construction of an ultra-dense genetic linkage map of potato
Os, H. van - \ 2005
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Herman van Eck. - s.n. - ISBN 9789085042211 - 96
solanum tuberosum - aardappelen - genkartering - genetische merkers - gegevensanalyse - kruisingen - solanum tuberosum - potatoes - gene mapping - genetic markers - data analysis - crosses
An ultra-dense genetic linkage map with nearly 10,000 AFLP loci was constructed from a heterozygous diploid potato population. It is among the densest meiotic maps ever constructed. A fast marker ordering algorithm was used in combination with genotyping error-detection software to obtain "skeleton bin maps". A "bin" is a position on the genetic map with a unique segregation pattern and separated from adjacent bins by a single recombination event. Subsequently all marker loci were assigned to the bins on the map by maximum likelihood based on their original segregation pattern. From the markers that were heterozygous in either the maternal or paternal parent, 98% could be fit in the bins. Of the markers that were heterozygous in both parents (bridge markers), only 79% could be fit. In both parental maps the twelve chromosomes could be identified. In addition, the paternal map includes a small unassigned linkage group with a severe segregation distortion. Recombination frequencies or marker positions are non-randomly distributed across the map. Putative centromeric regions showed extensive marker clustering while putative recombination hot spots resulted in large intervals up to 15 cM without markers. The markers derived from EcoRI/MseI and SacI/MseI primer combinations clustered more frequently than those derived from PstI/MseI primer combinations. The distribution of recombination events per chromatid indicated an overall absence of the occurrence of 0 chiasmata and excluded absolute chiasma interference within arms. The ultra-high density map has been used for anchoring of BAC-contigs for a sequence ready potato physical map and gene cloning.
Mapping of yield, yield stability, yield adaptability and other traits in barley using linkage disequilibrium mapping and linkage analysis
Kraakman, A.T.W. - \ 2005
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Fred van Eeuwijk. - Wageningen : s.n. - ISBN 9789085042051 - 136
hordeum vulgare - gerst - genkartering - verstoord koppelingsevenwicht - genetische merkers - kwantitatieve kenmerken - genotype-milieu interactie - gerstevergelingsvirus - roestziekten - ziekteresistentie - hordeum vulgare - barley - gene mapping - linkage disequilibrium - genetic markers - quantitative traits - genotype environment interaction - barley yellow dwarf virus - rust diseases - disease resistance
Plants is mostly done through linkage analysis. A segregating mapping population Identification and mappping of Quantitative Trait Loci (QTLs) in is created from a bi-parental cross and linkages between trait values and mapped markers reveal the positions ofQTLs. Inthisstudyweexploredlinkagedisequilibrium(LD)mappingof traits in a set of modernbarleycultivars. LDbetweenmolecularmarkerswasfoundup to a distance of 10 centimorgan,whichislargecomparedtootherspecies.Thelarge distancemightbeinducedby LDincreasingfactorssuchasinbreedingandthefactthatthepopulationismostlikelybasedon arathersmallset offoundinggenotypes. Associationsbetweenmarkersandtraitswerefoundforyield,yieldstability,leafrustresistance(LR),barleyyellowdwarfvirusresistance(BYD), plantheight,anddaystoheading.Trait-associatedmarkersfromLDanalysiswerelocatedinregionswherealreadyQTLsforthetraitconsideredhadbeenreportedfromstudiesbasedonbi-parentalcrosses. In addition, newQTLswerefoundforyield,yieldstability, LRandBYD.WeexpectthatLDmappingwillbecomeavaluableextension toconventionalQTLanalysisin plantbreeding.Specialattentionwasgivento traitsdescribinggenotype×environmentinteractions.Statisticalmodelswereusedtodefinemeasuresforyieldadaptabilityandyieldstabilitywithoutincludingenvironmentalfactorsdirectlyinthemodels.Adaptabilitywasdefinedastheresponsivenessofthegenotypetotheenvironment,andstabilitywasdefinedastheunexplaineddeviationfromthestatisticalmodel. LDmappinginbarleycultivarsresultedinmarker-traitassociations foryieldstability. In addition, linkage analyses in fourdoubled-haploidpopulationsresultedindetectionofmanyQTLsforadaptability, butonlya single QTL forstability.Weconcludedthatadaptabilitymeasuresweregeneticallybetterdefinedthanstabilitymeasuresandthatselectionforadaptabilityshouldbepossible.
Identification, characterization and high-resolution mapping of resistance genes to Phytophthora infestans in potato
Park, T.H. - \ 2005
Wageningen University. Promotor(en): Richard Visser; Evert Jacobsen. - Wageningen : - ISBN 9789085041894 - 136
solanum tuberosum - aardappelen - phytophthora infestans - plantenziekteverwekkende schimmels - ziekteresistentie - genen - genkartering - genetische modificatie - plantenveredeling - solanum tuberosum - potatoes - phytophthora infestans - plant pathogenic fungi - disease resistance - genes - gene mapping - genetic engineering - plant breeding
Potato ( Solanum tuberosum L.) is one of the most important crops in the world. The oomycete Phytophthora infestans (Mont. de Bary) is the causal agent of late blight which is the most devastating disease of the cultivated potato. It causes economic losses of several billion US dollars in crop production and protection annually worldwide. To control this disease, the application of enormous amounts of chemicals is required, which is expensive and environmentally unsafe. Also, late blight is increasingly more difficult to control because the pathogen is developing tolerance to the chemicals and new strains of the pathogen are evolving. Therefore, introduction of resistance genes from wild Solanum species into potato cultivars is considered as the most promising and environmental-safe approach to achieve durable late blight resistance.
Late blight resistance exists in a wide range of Solanum wild species of which 75 % have a diploid constitution (2n=2x=24) although the majority of the cultivated potato is tetraploid (2n=4x=48). Therefore, several different approaches such as the use of 2n gametes, colchicine treatment, intraspecific hybridization, somatic hybridization, embryo rescue and gene transformation are used to overcome the difficulties caused by different ploidy levels. Of these approaches, gene transformation is the most controlled and time-efficient method to introduce resistance into the cultivated potato. However, gene identification and gene isolation are the first prerequisites for this approach.
During the last century, most effort for late blight resistance was focused on the wild species S. demissum which contains at least 11 race-specific resistance genes ( R1 - R11 ). However, these resistances were rapidly overcome by new virulent pathogens which evolved. Therefore, new sources of late blight resistance are required and several resistances from different wild species such as S. microdontum , S. bulbocastanum , S. berthaultiiandS . pinnatisectumwere characterized. In the present research, we investigated both foliage and tuber blight resistances in different mapping populations derived from different sources of Solanum species.
The pathogen P. infestans can infect both foliage and tuber of potato. To study tuber blight resistance, a wounded tuber assay was developed and the correlation between tuber and foliage blight resistance in four different mapping populations CE, SHRH, RH94-076 and RH4X-103, in which foliage blight resistances were identified, was examined (Chapter 2). Tuber blight resistance was found to be correlated with foliage blight resistance in three populations CE, SHRH and RH94-076, while it was independent in the RH4X-103 population. Further investigation to explore late blight resistances using molecular markers and resistance assays on both foliage and tuber with four different isolates was focused on the RH4X-103 population. Three specific resistance genes in foliage, tuber or both were segregating. The tuber blight resistance locus was closely linked to marker GP179 and co-segregating with the R1 -derived marker 76_2s on chromosome 5. The R1 gene functioned both as a foliage and tuber blight R gene, whereas the Rpi-abpt and R3a genes acted only on foliage.
S. bulbocastanumis one of the most promising wildSolanumspecies possessing late blight resistance. ABPT material, which is a quadruple hybrid created by bridge crosses using the fourSolanumspecies;S. a caule,S. b ulbocastanum,S. p hurejaandS. t uberosum,to overcome incompatibility betweenS. bulbocastanumandS. tuberosumwere used to introduce late blight resistance. A tetraploid mapping population (RH4X-103) containing the late blight resistance geneRpi-abptwas created by a 4x-2x cross and was originally derived from ABPT material (Chapter 3). Resistance assay and AFLP marker analysis allowed localization of theRpi-abptlocus on chromosome 4. The origin of the resistance was confirmed by the analysis ofRpi-abptlocus-linked AFLP markers and the resistance assay of ABPT-related wild species accessions. An extended population of 1383 offspring was screened to construct a high-resolution map where the Rpi-abpt locus was consequently localized in a 0.5 cM interval between flanking AFLP markers and was co-segregating with one AFLP marker. The translated protein sequence of the co-segregating AFLP marker appeared to be highly homologues to several disease resistance proteins.
The investigation of the race-specific R2-like gene was performed in a diploid mapping population (Chapter 4) primarily used for mapping nematode resistance. R2-like was phenotypically indistinguishable from the S. demissum -derived R2 gene although S. demissum is not directly involved in the pedigree of the population andR2-likeis expected to originate from another species. The R2-like locus was placed in a 0.4 cM interval, flanked by two AFLP markers on chromosome 4 using the extended population of 1586 offspring. Four AFLP markers were also identified to be co-segregating with the R2-like locus.
A further late blight R gene ( Rpi-blb3)on chromosome 4 was identified in an intraspecific hybrid population derived from S. bulbocastanum (Chapter 5). The Rpi-blb3 locus was localized in a 0.9 cM interval between flanking AFLP markers and co-segregated with one AFLP marker in a population of 1396 offspring. According to the results of AFLP marker allele similarity tested by all co-segregating markers with four different R genes Rpi-abpt , R2-like , Rpi-blb3 and R2 , the four R genes appeared to be genetically very close and could be members of the same R gene cluster although the four R genes showed different race-specificities and origins.
Additional interesting scientific results came from the 4x-2x population (RH4X-103) used for late blight resistance. In this population, the male parent produced unreduced 2n pollen by FDR (First Division Restitution), allowing the localization of centromeres by using half-tetrad analysis (Chapter 6). 130 male parent-derived AFLP markers were analyzed and the genetic linkage group and location of 95 AFLP markers was determined by comparing them with the reference markers of the ultra high density (UHD) map of potato. Depending on the heterozygosity (simplex) or homozygosity (nulliplex or duplex) of the AFLP markers of the offspring, the position of centromeres and the genetic distance of the AFLP markers from the centromere were determined.In FDR 2n pollen, all the parental heterozygous loci from centromere to first crossover are expected to be heterozygous (Aa) resulting in simplex genotype (Aaaa) of their offspring, when they are combined with homozygous recessive gametes (aa) from the female parent. In contrast, a single crossover between the locus and centromere releases 50 % homozygous and 50 % heterozygous gametes in FDR.The centromeres of 10 chromosomes were accurately localized.The centromere positions were compared with those, which were putatively identified in the UHD map by the fact that markers are dense due to suppression of recombination, and proved to be identical. The centromere position of two chromosomes could not precisely be determined by the half-tetrad analysis approach due to the absence of the 100 % heterozygous markers. However, their positions could be inferredbased on the combination of the results between the increasing or decreasing rate of heterozygosity in the half-tetrad analysis and marker density in the UHD map.Additional investigation of three chromosomes with sufficient numbers of markers proved that only one crossover occurs per chromosome arm, as a result of interference of recombination via centromere and telomere.
In conclusion, three differentRgenes againstP. infestansin foliage were identified and the loci were accurately determined. These R loci are genetically very close and could be allelic. Our research for tuber blight resistance showed thatRgenes are not always active in both foliage and tuber. The results of the research described in this thesis demonstrate a great potential for further research. This will be directed towards the isolation of the R genes which can be used to introduce resistance into cultivated potato and to explore tuber blight resistance using a genetic transformation approach.
Pulmonary Hypertension Syndrome in Chicken: Peeking Under QTL Peaks
Rabie, T. - \ 2004
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Richard Crooijmans. - Wageningen : S.n. - ISBN 9789085041320 - 147
vleeskuikens - kippen - ascites - hypertensie - kwantitatieve kenmerken - genkartering - genen - genetica - broilers - fowls - ascites - hypertension - quantitative traits - gene mapping - genes - genetics
Pulmonary hypertension syndrome (PHS) is characterized a cascade of events resulting in cardiac anomalies including an enlarged heart, right ventricular hypertrophy, variable liver changes, and accumulation of fluid in the abdominal cavity (ascites). PHS is not a disease; it is a condition in which excess amount of ascitic fluids (a combination of lymph and blood plasma which has leaked from the liver) accumulate in the abdominal cavity which has prompted the common name"water belly"Several genetic and environmental factors such as altitude, temperature, lighting, ventilation, and nutritional factors seem to influence the development of the PHS. The work described in this dissertation is a first step towards the identification of genetic factors controlling PHS.An experimental population was specifically generated with the aim to map QTL for a number of different economically important traits including PHS. The population is based on a so-called full-sib/half-sib design based on three generations (G 1 , G 2 and G 3 ) derived from a cross between two genetically different outcross broiler dam lines originated from the White Plymouth Rock breed. Chapter 2 describes the initial mapping of a several QTL for traits related to PHS using a total genome scan. The total genome scan was performed on ten full sib families consisting of 20 G 1 and 456 G 2 birds which were typed with 420 microsatellite markers covering 24 autosomal chromosomes. Phenotypic observations were collected on 4202 G 3 birds and a full sib across family regression interval mapping approach was used to identify the QTL. Statistical evidence for QTL was found on chicken chromosomes (GGA) 2, 4 and 6. Suggestive linkage was found on chromosomes 5, 8, 10, 27, and 28. Phenotypic measurements mostly were related to the condition of the heart (%RV, %TV, RATIO), appearance of the internal organs, ascites and survival. The most significant QTL that were identified are located on GGA2 for the traits, right and total ventricular weight as percentage of body weight (%RV and %TV). A related trait, the ratio of right ventricular weight as percentage to total ventricular weight (RATIO), reached the suggestive threshold on this chromosome.The observed test statistics were not very high, which was to be expected in a G 2 cross between two related breeds. The subsequent confirmation of the observed QTL in the next generations therefore, is an essential step before attempting to further narrow down the identified QTL intervals. The validation of the QTL on chromosomes 8, 10 and 28 and the confirmation of those on GGA8 and GGA10 is described in Chapter 3 .Validation was performed in generations G6 to G8 of the experimental cross. The population consisted of 19 full sib families (a combination of G 6 /G 7 and G 7 /G 8 ). In total 14 microsatellite markers were analyzed and twelve PHS-related traits measured on more than 1500 individuals. Significant results were found for the traits body weight at 2 and 5 weeks of age, right ventricular weight, RATIO (RV:TV) and total mortality. The most significant QTL were located on chromosome 8 for traits body weight at 5 wk of age ( ADL0278-MCW0351 ), right ventricular weight ( ADL0278-MCW0351 ) and RATIO (RV:TV) ( ROS0075-ADL0278 ) and on chromosome 10 for traits body weight at 2 wk of age ( MCW0035-ADL0102 ) and total mortality ( ADL0158-LEI0112 ). The QTL on GGA28 could not be confirmed. The validation and confirmation of the significant QTL on GGA2 and GGA4 are described in Chapter 4 . This chapter also describes the further fine mapping of these two QTL using a combined linkage disequilibrium/linkage analysis approach (LDLA). Validation and fine mapping was performed on an advanced intercross line (AIL) created by random intercrossing in each generation from G 3 onwards until G 8 . In total 47 microsatellite markers were used, located within approximately 25 Mb and 16 Mb on GGA2 and GGA4, respectively. QTL originally detected in G 2 /G 3 generations were confirmed on the two chromosomes, for the traits RATIO on GGA2 and for BW5 AS on GGA4. Additional QTLs were detected for the traitpCO 2 on GGA2, and for BW3 AS on GGA4. These two traits had not previously been measured in generation 3 of the original study. The combined LDLA approach enabled a 3-4 fold reduction of the size of the QTL interval.The improvement of the physical map for GGA4 and the generation of a detailed comparative map between this chicken chromosome and human chromosomes 4 and X are described in Chapter 5. The mapping resolution of the physical map for GGA4 was improved by a combination of radiation hybrid (RH) mapping and BAC mapping. The ChickRH6 hybrid panel was used to construct an RH map of GGA4. Eleven microsatellites known to be located on GGA4 were included as anchors to the genetic linkage map for this chromosome. Based on the known conserved synteny between GGA4 and human chromosomes 4 and X, sequences were identified for the orthologous chicken genes from these human chromosomes by BLAST analysis. These sequences were subsequently used for the development of STS markers to be typed on the RH panel. Using a logarithm of the odds (LOD) threshold of 5.0, nine-linkage groups could be constructed which were aligned with the genetic linkage map of this chromosome. The resulting RH map consisted of the 11 microsatellite markers and 50 genes. To further increase the number of genes on the map and to provide additional anchor points for the physical BAC map of this chromosome BAC clones were identified for 22 microsatellites and 99 genes.The combined RH and BAC mapping approach resulted in the mapping of 61 genes on chicken GGA4 considerably increasing the resolution of the chicken-human comparative map for this chromosome. This enhanced comparative mapping resolution enabled the identification of multiple rearrangements between chicken chromosome 4 and human chromosomes 4q and Xp.Finally, the results of this thesis are discussed within a broader context in Chapter 6and in particular in relation to the recently published draft sequence of the chicken genome and the polymorphism map consisting of 2.8 million single nucleotide polymorphisms.