ORIGINAL PAPER Identification of quantitative trait loci controlling partial clubroot resistance in new mapping populations of Arabidopsis thaliana Melanie �� Jubault �� Christine Lariagon �� Matthieu Simon �� Regine �� Delourme �� Maria J. Manzanares-Dauleux Received: 25 September 2007 / Accepted: 3 April 2008 / Published online: 22 April 2008 �� Springer-Verlag 2008 Abstract To date, mechanisms of partial quantitative resistance, under polygenic control, remain poorly under- stood, studies of the molecular basis of disease resistance have mainly focused on qualitative variation under oligo- genic control. However, oligogenic conferred resistance is rapidly overcome by the pathogen and knowledge of the relationship between qualitative and quantitative resistance is necessary to develop durably resistant cultivars. In this study, we exploited the Arabidopsis thaliana-Plasmodio- phora brassicae pathosystem to decipher the genetic architecture determining partial resistance. This soil-borne pathogen causes clubroot, one of the economically most important diseases of Brassica crops in the world. A quantitative trait locus (QTL) approach was carried out using two segregating populations (F2 and recombinant inbred lines) from crosses between the partially resistant accession Burren and the susceptible accession Columbia. Four additive QTLs (one moderate and three minor) con- trolling partial resistance to clubroot were identified, all the resistance alleles being derived from the partially resistant parent. In addition, four epistatic regions, which have no additive effect on resistance, were also found to be involved in partial resistance. An examination of candidate genes suggested that a potentially diverse array of mech- anisms is related to the different QTLs. By fine-mapping and cloning these regions, the mechanisms involved in partial resistance will be identified. Introduction Plants, like animals, are able to launch successful defense responses against invading micro-organisms. In order to limit pathogen growth, plants have evolved a sophisticated, multilayered system of passive and active defense mecha- nisms, leading to partial resistance (a compatible interaction) or to complete resistance (an incompatible interaction). Complete resistance is the most studied of these defense systems, it usually relies on molecules that specifically recognize, either directly or indirectly (known as the ���Guard��� hypothesis), a particular pathogen or strain of a given pathogen. These molecules are encoded by Resistance (R) genes, and each R protein initiates a defense response in the presence of a pathogen-derived elicitor protein that is termed the Avirulence (Avr) determinant (Hammond-Kosack and Parker 2003). The genetic rela- tionship between R and Avr proteins is commonly known as gene-for-gene resistance (Flor 1971). A great number of disease resistances, however, do not fit the gene-for-gene system, these include partial resis- tance of quantitative nature controlled by multiple genes. Despite the prevalence of quantitative variations in resis- tance levels in natural populations and crop plants (Young 1996), its molecular basis remain currently unknown. Indeed, it is not clear whether the genetic pathways that mediate quantitative and qualitative variations in resistance are the same or involve different genes. On the one hand, Communicated by C. Hackett. M. Jubault C. Lariagon R. Delourme M. J. Manzanares-Dauleux (&) Amelioration �� des Plantes et Biotechnologies Vegetales,���� UMR118 INRA-Agrocampus Rennes-Universite �� de Rennes 1, BP35327, 35653 Le Rheu Cedex, France e-mail: maria.manzanares@rennes.inra.fr M. Simon Station de Genetique �� �� et Amelioration �� des Plantes, Centre de Versailles, INRA, 78026 Versailles, France 123 Theor Appl Genet (2008) 117:191���202 DOI 10.1007/s00122-008-0765-8

QTLs and R genes have frequently been observed to co- localize (Gebhardt and Valkonen 2001 Kover et al. 2005 Perchepied et al. 2006 Wisser et al. 2005), suggesting that quantitative resistance could result from the action of weak R gene alleles and qualitative resistance from particularly strong alleles. Mutations in the rice Xa21 and the tomato I2 R-genes, involved in qualitative resistance to different kinds of pathogens, resulted in partial resistance pheno- types (Ori et al. 1997 Wang et al. 1998) and confirmed the previous hypothesis. On the other hand, genes involved in defense responses (such as the production of antimicrobial compounds, cell wall strengthening, callose formation, lignification, the oxidative burst) (Gebhardt and Valkonen 2001 Ramalingam et al. 2003 Trognitz et al. 2002 Wisser et al. 2005) or genes encoding metabolic enzymes (Taler et al. 2004) could also be conferring quantitative resistance. Clubroot, caused by the obligate biotroph protist Plas- modiophora brassicae Woron., is one of the economically most important diseases of Brassica crops in the world. This soil-borne pathogen causes the hypertrophy (abnormal cell enlargement) and hyperplasia (uncontrolled cell divi- sion) of infected roots into characteristic clubs. These obstruct nutrient and water transport, stunt the growth of the plant and consequently reduce crop yield and quality. Since the pathogen survives as resting spores for a long period (up to 15 years) in the soil, control of the disease by agricultural practices and/or chemical treatments is difficult and/or expensive. Thus, the development of resistant cul- tivars is currently the most efficient way to control clubroot in all Brassica crops. Both qualitative and quantitative clubroot resistances were identified in different Brassica- ceae species including the three most commonly cultivated species: Brassica napus, Brassica rapa and Brassica ol- eracea (Hirai 2006). However, the type of clubroot resistance introduced into commercial cultivars is usually monogenic or oligogenic and rapidly overcome. Successful strategies for breeding clubroot resistant cultivars will depend on the relationship between the different types of resistance (race-specific or race non-specific, qualitative or quantitative) and the impact of their association on size and genetic composition of pathogen populations. Thus, knowledge of clubroot resistance gene functions and associated mechanisms is required for the development of durable host-plant resistance. However, although numerous studies on the genetic control of clubroot resistance in Brassicas have been carried out (Hirai 2006), clubroot resistance genes or QTLs have not been isolated and their potential function remains currently unidentified. The wild Brassicaceae Arabidopsis thaliana is also a host for clubroot (Koch et al. 1991). This model plant provides several advantages for cloning and characteriz- ing plant disease resistance genes. Indeed, the multitude of publicly available molecular tools, including the complete genome sequence for Columbia (Arabidopsis Genome Initiative 2000) and partial genome sequences for numerous other accessions (Nordborg et al. 2005), means that the cloning of disease resistance genes can progress more quickly in Arabidopsis than in other plant species. Furthermore, P. brassicae does not present host specificity in Brassicaceae (i.e. the same isolate can infect different species). Consequently, the pathosystem P. brassicae ��� A. thaliana appears to be a good model for the analysis of the molecular mechanisms involved in Brassicaceae clubroot resistance. Knowledge acquired on the model plant could then be rapidly integrated and transferred to cultivated crops. Up to now, research on clubroot using Arabidopsis as a model host system was mainly focused on the potential involvement of several metabolic pathways in the pathogenesis of the disease, such as hormonal regulation by auxin (Grsic et al. 1999 Ludwig-Muller et al. 1999 Neuhaus et al. 2000) or cytokinins (Devos et al. 2006 Siemens et al. 2006) and trehalose synthesis (Brodmann et al. 2002). With the exception of the identification of RPB1, a gene located on chromosome 1 involved in complete clubroot resistance (Fuchs and Sacristan �� 1996), very little information is currently available on the genetic control of clubroot resistance in Arabidopsis. The observation of accessions of worldwide origin has revealed that there is natural variation in the responses of A. thaliana to clubroot infection (Alix et al. 2007 Fuchs and Sacristan �� 1996 Siemens et al. 2002). Alix et al. (2007) identified the accession Burren (Bur-0) as partially resistant to the P. brassicae isolate eH. This finding makes it possible to use segregating populations to genetically dissect quantitative trait loci (QTLs) control- ling these defense mechanisms and gain insight into the molecular basis of quantitative partial resistance and its possible relationship to qualitative resistance. This type of strategy has led to the identification of genetic factors required for Arabidopsis resistance or susceptibility to other bacterial and fungal pathogens (Denby et al. 2004 Kover et al. 2005 Kover and Cheverud 2007 Llorente et al. 2005 Perchepied et al. 2006). Consequently, to investigate the genetic basis of partial clubroot resistance in A. thaliana, a F2 population was generated by crossing the susceptible accession Columbia (Col-0) and the partially resistant accession Bur-0. A recombinant inbred line (RIL) population produced from crosses between the same parents (Bur-0 9 Col-0) was also available at INRA Versailles. Using these two populations, we identified several QTLs (both additive and epistatic) conferring partial clubroot resistance in A. thaliana. The complete genomic sequence of Arabidopsis was then scanned for putative candidate genes underlying the QTLs and their role in partial resistance is discussed. 192 Theor Appl Genet (2008) 117:191���202 123