Sequence Analysis and Conversion of Genomic RFLP Markers to STS and SSR Markers in Italian Ryegrass (Lolium multiflorum Lam.)

Abstract

To convert RFLP markers of Italian ryegrass to sequence-tagged site (STS) markers, we end-sequenced 93 previously mapped single or low-copy RFLP probes. Eighty-seven clones gave acceptable results from both forward and reverse directions, and 71 contigs were detected, while other clones could not be sequenced completely because their fragments were too long. BLAST search results showed that 16 clones matched sequences reported in rice and other plants. STS primers were designed for all the 93 clones by using Oligo software, and 66 primers amplified a single band with the expected size. Of the 2 SSR primers designed from the 2 clones containing (AT)_n or (TTA)_n repeats, 1 amplified an SSR. Fifty-seven (85%) of the 67 STS (and SSR) primer pairs could amplify products in perennial ryegrass, 47 (70%) in meadow fescue, and 55 (82%) in tall fescue—species closely related to Italian ryegrass. Forty percent of the STS primers detected within-cultivar length or presence /absence of polymorphisms.