Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses

Simple-sequence repeats (SSRs) comprising three tetranucleotide repeat sequences with two-base 'anchors', namely 5'-(AGAC) ₄GC, 5'-AC(GACA) ₄ and 5'-(GACA) ₄GT, were used in PCR reactions as primers to develop inter-SSR DNA fingerprints of the outbreeding grass species Lolium multiflorum, L. perenne, Festuca pratensis and F. arundinacea. Each species was represented by DNA samples from 3 to 6 varieties. In all four species distinctive species-specific DNA profiles were produced that were common across a number of varieties despite their diverse origin. While the fingerprints of the two ryegrasses, L. multiflorum and L. perenne, were the most similar, a number of inter-SSR DNA markers were generated that enabled them to be distinguished from each other. Some slight variations were found between varieties, which provided putative variety-specific markers for cultivar identification. In addition, variations in the DNA profiles of the genotypes of L. multiflorum and F. pratensis were examined, and the results showed that variety-specific fingerprints are integrated patterns made up from the profiles of individual genotypes. Amongst the primers used, AC(GACA) ₄ generated the best distinction between Lolium and Festuca individuals and provides an effective new tool for genome identification. A number of species-discriminating sequences, ranging in size between 550 bp and 1,600 bp, were cloned: three clones for F. pratensis, one clone for L. multiflorum and one clone for F. arundinacea. A F. pratensis fragment pFp 78H582 was sequenced. Southern hybridization confirmed the presence of this fragment in F. arundinacea (which contains one genome of F. pratensis), but no homology was found with L. multiflorum. However, a F. arundinacea clone amplified with (GACA) ₄GT, pFa 104H1350, was found to be unique to the E arundinacea genome.