ANALYSIS OF GENOME SPECIFIC SEQUENCES IN PHLEUM SPECIES - IDENTIFICATION AND USE FOR STUDY OF GENOMIC RELATIONSHIPS

Sau3AI ''shot gun'' cloning and colony hybridization with total genomic probes were used to isolate genome-specific sequences in Phleum species. The total DNA isolated from diploid species P. alpinum and P. bertolonii was partially digested with Snu3AI and cloned using pUC 19 as a vector to an E. coli strain DH5 alpha mcr. A partial genomic DNA library consisting of 3030 colonies for the genome of P. alpinum and one consisting of 3240 colonies for the genome of P. bertolonii were constructed. Twelve hundred and thirty colonies from the DNA library of P. alpinum and 1320 from that of P. bertolonii were respectively blotted to membrane filters and hybridized to the total genomic probes from these two species. Eight clones specific to P. alpinum and 13 specific to P. bertolonii were isolated through colony hybridization and further dot-blot hybridization. Most of these clones may carry highly or moderately repetitive sequences. Three sequences specific to P. alpinum and 3 specific to P. bertolonii were used as probes to hybridize the EcoRI-digested DNA samples from four species, P. alpinum, P. bertolonii, P. pratense and P. montanum, on Southern blot. The results from these hybridization experiments showed that all 3 P. bertolonii-specific probes and 2 of the 3 P. alpinum-specific probes hybridized to the DNA of P. pratense, thus confirming the conclusion of the close relationships between the cultivated timothy and its two wild relatives that was drawn in our previous study using the C-banding technique.