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 sequ ences with two-base 'anchors', namely 5'-(AGAC)(4)GC, 5'-AC(GACA)(4) and 5' -(GACA)(4)GT, were used in PCR reactions as primers to develop inter-SSR DN A fingerprints of the outbreeding grass species Lolium multiflorum, L. pere nne, Festruca 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 va rieties despite their diverse origin. While the fingerprints of the two rye grasses, 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. multifloru m and F. pratensis were examined, and the results showed that variety-speci fic fingerprints are integrated patterns made up from the profiles of indiv idual genotypes. Amongst the primers used, AC(GACA), generated the best dis tinction between Lolium and Festruca individuals and provides an effective new tool for genome identification. A number of species-discriminating sequ ences, ranging in size between 550 bp and 1,600 bp, were cloned: three clon es for F: pratensis, one clone for L. multiflorum and one clone for F. arun dinacea. A F. pratensis fragment pFp 78H582 was sequenced. Southern hybridi zation confirmed the presence of this fragment in F. arundinacea (which con tains one genome of F. pratensis), but no homology was found with L. multif lorum. However, a F: arundinacea clone amplified with (GACA)(4)GT, pFa 104H 1350, was found to be unique to the F: arundinacea genome.