Genetic variability evaluation and cultivar identification of tetraploid annual ryegrass using SSR markers

Annual ryegrass (Lolium multiflorum) is a widely used cool-season turf and forage grass with high productivity and ornamental characteristics. However, the abundant intra-cultivar genetic variability usually hampers the application of conventional techniques for cultivar identification. The objectives of this study were to: (1) describe an efficient strategy for identification of six tetraploid annual ryegrass cultivars and (2) investigate the genetic diversity based on SSR markers. A total of 242 reliable bands were obtained from 29 SSR primer pairs with an average of 8.3 bands for each primer pair and the average value of polymorphic information content (PIC) was 0.304. The result of analysis of molecular variance (AMOVA) revealed that 81.99% of the genetic variation occurred in within-cultivars and 18.01% among-cultivars. The principal coordinate analysis (PCoA) showed that the first two principal axes explain 8.57% (PC1) and 6.05% (PC2) of total variation, respectively. By using multi-bulk strategy based on different filtering thresholds, the results suggested that bands frequency of 40% could be used as a reliable standard for cultivar identification in annual ryegrass. Under this threshold, 12 SSR primer pairs (00-04A, 02-06G, 02-08C, 03-05A, 04-05B, 10-09E, 12-01A, 13-02H, 13-12D, 14-06F, 15-01C and 17-10D) were detected for direct identification of six tetraploid annual ryegrass cultivars, which could be incorporated into conservation schemes to protect the intellectual property of breeders, ensure purity for consumers, as well as guarantee effective use of cultivars in future.