Genetic relationship and diversity of cultivars and breeding lines of tetraploid Italian ryegrass (Lolium multiflorum Lam.) and its hybrids with Lolium-Festuca complex based on genome-wide allele frequency

Genetic relatedness and diversity of 62 cultivars and breeding lines of tetraploid Italian ryegrass (Lolium multiflorum Lam.; 39 accessions) and its interspecific hybrids, Festulolium (18 accessions), and hybrid ryegrass (Lolium × hybridum Hausskn.; 5 accessions), mainly from Japan, were revealed based on 2,824 genome-wide allele frequencies obtained by the genotyping by random amplicon sequencing-direct (GRAS-Di) method using bulk genomic DNA testing. Genomic composition of each accession was estimated according to the occurrence of 77,373 unique GRAS-Di sequences in the reference population consisting of diploid Italian ryegrass, meadow fescue (Festuca pratensis Huds.), and perennial ryegrass (Lolium perenne L.). The high correlation coefficient (0.98) between the fescue-specific reads ratio and the previously obtained f-ratio of genomic in situ hybridization in Festulolium cultivars suggests the usefulness of this simple method. Both cluster analysis based on Nei's standard genetic distance (D_ST) and principal component analysis (PCA) showed that groups were formed largely by species. However, the complex heritage of Lolium-Festuca (Festulolium) materials could not be determined by species registration or breeding history alone. Some Festulolium accessions were closely related to Italian ryegrass, whereas some defined as Italian ryegrass may actually be interspecific hybrids. The high genetic diversity of Festulolium compared to Italian ryegrass and hybrid ryegrass revealed by PCA seems due to the wide range of fescue-specific read ratios (0.04–33.0%). Tetraploid Italian ryegrass did not show clear structural differentiation, but some genetic relationships attributable to breeding history were demonstrated. Mean pairwise D_ST of tetraploid Italian ryegrass cultivars was significantly lower than that of diploids. Tetraploids and diploids could be separated by PCA plot. Although mean expected heterozygosities of tetraploid and diploid cultivars were not significantly different, the results suggest that the utilization of diploid genetic resources is effective in maintaining and increasing the genetic diversity of breeding populations of tetraploid Italian ryegrass.