Introgression mapping of genes for winter hardiness and frost tolerance transferred from Festuca arundinacea into Lolium multiflorum

Genes for winter hardiness and frost tolerance were introgressed from Festuca arundinacea into winter-sensitive Lolium multiflorum. Two partly fertile, pentaploid (2n = 5x = 35) F(1) hybrids F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) were generated and backcrossed twice onto L. multiflorum (2x). The backcross 1 (BC(1)) and backcross 2 (BC(2)) plants were preselected for high vigor and good fertility, and subsequently, a total of 83 BC(2) plants were selected for winter hardiness after 2 Polish winters and by simulated freezing tests. Genomic in situ hybridization (GISH) was performed on 6 winter-hardy plants selected after the first winter and shown to be significantly (P < 0.05) more frost tolerant than the L. multiflorum control. Among the analyzed BC(2) winter survivors, only diploid (2n = 2x = 14) plants were found. Five plants carried 13 intact L. multiflorum chromosomes and 1 L. multiflorum chromosome with a single introgressed F. arundinacea terminal chromosome segment. The sixth BC(2) winter survivor appeared to be Lolium without any Festuca introgression capable of detection by GISH. A combined GISH and fluorescence in situ hybridization analysis with rDNA probes of the most winter-hardy (after 2 winters) and frost-tolerant BC(2) plant revealed the location of an F. arundinacea introgression on the nonsatellite arm of L. multiflorum chromosome 2, the same chromosome location reported previously as a site for frost tolerance genes in the diploid and winter-hardy species Festuca pratensis.