Development of microsatellites for the cacao frosty pod rot pathogen,Moniliophthora roreri

Summary Moniliophithora roreri, causal agent of cacao frosty pod rot, is considered one of the most devastating pathogens of this crop. In this study,we report the development of and validation of 28 microsatellite loci from an enriched library. From these, ten loci were demonstrated tobe polymorphic in an Ecuadorian population composed of 27 isolates. The mean number of alleles per locus was 3.2, and the observedmean and expected heterozygosity were 0.03 and 0.43, respectively. The polymorphic microsatellites described herein may be used tostudy the genetic diversity as well as to comprehend other aspects of M. roreri population biology. 1 Introduction The hemibiotrophic basidiomycete Moniliophthora roreri (Cif.) H. C. Evans, Stalpers, Smason & Benny, infects only fruits ofneotropical trees from the genera Theobroma and Herrania.InTheobroma cacao L. fruit, the major component of chocolate, itcauses frosty pod rot disease, also known as moniliasis, an extremely destructive disease and currently the main yield-limitingfactor in several Latin American countries (Phillips-Mora 2003). Pod losses due to the disease are usually over 30%, but itmight reach 100% depending on factors such as disease management, favourable environmental conditions and time of intro-duction of the disease in a particular agroecological zone or site (Katip 1994; Krauss et al. 2003). It has been proposed thatM. roreri has its origin in the north-western region of Colombia and is now present in 11 Central and South Americancountries (Phillips-Mora et al. 2007). This disease poses a major threat to important cacao-producing areas in Brazil andCaribbean countries, such as Trinidad and Tobago, Dominican Republic and Haiti that are still free from the pathogen.It has been shown by AFLP/ISSR data that M. roreri is genetically variable and forms five genetic groups (Phillips-Mora etal. 2007), being two major ones: (i) the Bolivar group; comprising isolates from north of Santander in Colombia, all Peruvianand Venezuelan isolates, and 10 isolates from the periphery of Ecuador and (ii) the Co-West group, including, mainly, isolatesfrom western Colombia, central Ecuador and Central America. The other groups are all apparently endemic to Colombia(Co-East and Co-Central groups) or north-western Ecuador. The use of microsatellites or simple sequence repeats (SSRs) willallow broader inferences regarding the population biology of M. roreri. This knowledge is necessary to establish successfulintegrated management strategies for crop breeding. SSRs are considered suitable for genetic studies because they areco-dominantly inherited, allowing precise discrimination even of closely related individuals as well as allowing the heterozygotein diploid genomes to be distinguished. In addition, microsatellite analysis is inexpensive, highly reproducible and transfer-able across related species (Jarne and Lagoda 1996; Goul~ao and Oliveira 2001). SSRs have been the choice in populationgenetic studies of plant pathogens (Taylor et al. 1999), including pathogens of cacao such as M. perniciosa (Gramacho et al.2007), due to their high variability and statistical power (Reusch 2001). Like RAPDs, ISSRs are dominant markers, quick,cheap, easy to handle. Despite the lower reproducibility, ISSRs are applicable to screening information about the genetic vari-ation. However, limited statistical analysis and poor cross-experimental reproducibility represent some drawbacks of ISSRsmarkers, where the homozygous presence of a fragment is not distinguishable from its heterozygote. In this way, SSRs arevery powerful tools and should be more widely used for population studies of this fungus. The use of SSR allows researchersto address key questions about the population genetics of M. roreri, to cite a few practical applications recombination, mat-ing systems, host adaptation and population structure due to host adaptation. Thus, in this study, we characterized 28 micro-satellite loci for genetic analyses of populations of M. roreri that represent the first SSRs developed for this species.