Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of Lolium perenne, two cultivars of Lolium multiflorum, two cultivars of Festuca pratensis, two cultivars of Festuca arunidinacea and ten accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA-derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes.The efficiency of cross-specific amplification for LPSSR markers varied from 38% in F. pratensis to 93% in two cultivars of Festulolium and for LMSSR markers from 57% in F. pratensis to 87% in L. multiflorum. Of 40 amplified markers, fourteen (35%) produced species-difference alleles in the relation to cultivars used in the present study. Thirty-five LPSSR locus-derived alleles were found to be specific to Lolium species, 4 to F. pratensis and 6 to F. arundinacea. For LMSSR alleles, 8 were specific to Lolium species and 5 were only associated with L. multiflorum, and null alleles were detected for F. pratensis in all instances. These species-difference markers could clearly identify different accessions of In Festulolium hybrid breeding, it is desirable to be able to distinguish the DNA of the parental species, in order to monitor the extent of the introgressed 3 regions. Fluorescence in situ hybridisation (FISH) methodology using labelled genomic DNA (genomic in situ hybridisation: GISH) has been widely used to identify alien chromatin (Thomas et al. 1994, Humphreys et al. 1995, Humphreys and Pašakinskiené 1996. However, there are potential difficulties in the identification of small introgressed chromosome segments using this technique (Humphreys et al. 1998).The development of molecular genetic marker technologies provides alternative procedures for the assessment of genetic diversity, cultivar identification and the genomic composition of polyploid and hybrid taxa. Several Moreover, SSRs have been shown to provide a powerful means for discrimination between closely related genotypes in many plant species (Yang et al. 1994, Russell et al. 1997, Röder et al. 1998 The cycling regime for the PCR amplification of LMSSR markers consisted of an initial denaturation step of 10 min at 94°C, followed by denaturation for 1 min at 94°C followed by 30 s at 94°C, 30 s at 60°C, 2 min at 72°C; repeated for 35 cycles, and a final extension of 7 min at 72°C (Fujimori et al. in preparation).
Detection of SSR polymorphisms:The samples were prepared for polyacrylamide gel electrophoresis (PAGE) by addition of 2 μl gel loading buffer to the reaction mixture. Ten µl of amplification products were loaded on 10% (w/v) non-denaturing polyacrylamide gels for electrophoresis in 1×TBE. Each gel was run for 2.5 h at constant voltage (250V). The banding patterns were visualised following staining with ethidium ...