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Title:   Author  item
                                            Goodwin, Stephen

                                                         - *Steve*item
                                            VAN DER LEE, T - PLANT RESEARCH INTLitem
                                            Cavaletto, Jessicaitem
                                            HEKKERT, B - PLANT RESEARCH INTLitem
                                            Crane, Charlesitem
                                            KEMA, G - PLANT RESEARCH INTL
  Submitted to: Fungal Genetics and Biology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/19/2006
                                    Publication  Date: 10/30/2006
                                    Citation: Goodwin, S.B., Van Der Lee, T., Cavaletto, J.R., Hekkert, B., Crane, C.F., Kema, G.H. 2006. Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the Septoria tritici blotch pathogen Mycosphaerella graminicola. Fungal Genetics and Biology. 44:398-414.Interpretive Summary: Mycosphaerella graminicola, the cause of Septoria tritici blotch of wheat, is an economically important fungus that is being developed as a new model system for cereal diseases. The existing genetic map of this organism was based on older marker types that are not easily transferable among laboratories. Microsatellites are the marker of choice for modern genetic studies because they are highly polymorphic, very repeatable, and easily transferred among laboratories. However, only eight microsatellites had been identified in M. graminicola and none had been mapped genetically. To rectify this situation, a database of 30,137 Expressed Sequence Tag (EST) sequences from M. graminicola was scanned with an automated software pipeline for di- and trinucleotide microsatellites. The bioinformatic analysis identified 109 possible microsatellite loci and developed primers for 99 of them that were tested for amplification and polymorphism by polymerase chain reaction. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 21 plus two previously published microsatellite loci were integrated into the existing genetic map on 12 of the 22 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also was tested successfully with DNA from the related banana pathogen M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be used by geneticists and plant pathologists for high-throughput genetic analyses of this important plant pathogen and its close relatives.
                                Technical Abstract: A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned for di- and trinucleotide units repeated tandemly six or more times, with an automated software pipeline.  The bioinformatics analysis identified 109 possible SSR loci and developed flanking primers for 99 of them that were tested for amplification and polymorphism on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population.  Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested.  Among these 51 loci, 23 were polymorphic between the parents of the mapping population.  Twenty-one of these as well as two previously published microsatellite loci were integrated into the existing genetic map on 12 of the 22 linkage groups.  Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested.  A subset of the primer pairs also was tested successfully with DNA from the related banana pathogen M. fijiensis.  The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of this important plant pathogen.


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