Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside