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Proceedings
THE DEVELOPMENT OF MICROSATELLITE MARKERS IN LOLIUM PERENNE
C. Taylor, P.B. Holm and B. Boelt
Department of Plant Biology Danish Institute of Agricultural Sciences Research Centre Flakkebjerg Slagelse 4200 Denmark
Microsatellite markers posses a number of positive attributes when compared to other molecular marker systems. Analysis is via the polymerase chain reaction (PCR); they are co-dominant and show Mendelian inheritance and they are highly polymorphic. Consequently, they are becoming the molecular markers of choice for plant genome analysis. We report the generation of four L. perenne genomic libraries enriched for (CA)n, (CT)n, (AAC)N and (GAA)N simple sequence repeats, respectively, using biotinylated oligonucleotides complementary to core SSR sequences and capture via DynalbeadsTM . The libraries were screened twice using a synthetic oligonucleotide corresponding to the repeat motif enriched for revealing a total of 104 (21.7%) CA-containing clones, 32 (6.7%) CT-containing clones, 40 (8.3%) AAC-containing clones and 26 (5.4%) GAA-containing clones. Six positive clones from each library were isolated and sequenced. One CA-positive and two CT-positive clones could not be sequenced. Sequence analysis of the remaining clones indicated that all contained a repeat of the type expected. Perfect (18.75%), imperfect (50.0%) and compound repeats (31.25%) were identified with the number of repeats ranging from 13 to 51. Five clones derived from the GAA-enriched library and two clones derived from the CT-enriched library were duplicates. Flanking primers were derived from 16 (66.7%) of the 24 clones analysed. Of these, 12 (50%) amplified a product in the expected size range and 6 have been used to assay SSR polymorphism in individuals from two L. perenne varieties and one ecotype. The levels of polymorphism observed was high with more alleles tending to be observed in the ecotype. The usefulness of microsatellite markers is demonstrated by the ability to differentiate between putative clonal material and the ability to categorically determine the hybrid status of seeds derived from controlled crosses. Microsatellite markers should therefore provide breeders and researchers with a powerful new tool for analyses including varietal identification, hybrid/clonal status, parental selection as well as genetic linkage mapping. Interestingly, a number of the microsatellite markers analysed also amplified products in L. multiflorum and L. westerwoldicum indicating that markers isolated from L. perenne may have wider application.