Analysis of genome-specific sequences inPhleum species: Identification and use for study of genomic relationships

Abstract

Sau3AI "shot gun" cloning and colony hybridization with total genomic probes were used to isolate genome-specific sequences inPhleum species. The total DNA isolated from diploid speciesP. alpinum andP. bertolonii was partially digested withSau3AI and cloned using pUC19 as a vector to anE. coli strain DH5αmcr. A partial genomic DNA library consisting of 3030 colonies for the genome ofP. alpinum and one consisting of 3240 colonies for the genome ofP. bertolonii were constructed. Twelve hundred and thirty colonies from the DNA library ofP. alpinum and 1320 from that ofP. bertolonii were respectively blotted to membrane filters and hybridized to the total genomic probes from these two species. Eight clones specific toP. alpinum and 13 specific toP. 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 toP. alpinum and 3 specific toP. bertolonii were used as probes to hybridize theEcoRI-digested DNA samples from four species,P. alpinum,P. bertolonii,P. pratense andP. montanum, on Southern blot. The results from these hybridization experiments showed that all 3P. bertolonii-specific probes and 2 of the 3P. alpinum-specific probes hybridized to the DNA ofP. 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.

Isolation and characterization of simple sequence repeat markers in the hexaploid forage grass timothy (Phleum pratense L.)

“…The latter view seems to be generally accepted. On the other hand, Cai and Bullen (1994) pointed out that the degree of differentiation between P. alpinum and P. bertolonii is relatively low, judging from genome-specific sequences. This view seems to agree with the results of earlier studies by Nordenskiold (1945), who concluded on the basis of interspecific hybridization and cytological observation of hybrids that P. bertolonii might be partly homologous to P. alpinum.…”

“…It has a moderately large genome size (e.g., 1C=4,067 Mb; Bennett and Smith 1991), but the genomic constitution of this species (autopolyploid vs allopolyploid) is unclear (Muntzing and Prakken 1940;Leven 1941;Myers 1944;Nordenskiold 1945;Stebbins 1950;Wilton and Klebesadel 1973;Cai and Bullen 1991). There have been very few molecular studies of timothy (Cai and Bullen 1994;Ogawa et al 2001), because this species is a hexaploid with a large genome size and, moreover, it is crosspollinated.…”

An unusual Group 2 LEA gene family in citrus responsive to low temperature

“…Plaques showing positive signals to the CA probe but not to the control probe were collected and subjected to a second round screening using the same procedure. Phage collected from the second round screening were further purified using dot-blot hybridization analysis [7]. Clones confirmed in the dot-blot hybridization analysis were subsequently rescued by in vivo excision from lambda and maintained as a Bluescript plasmid.…”

Identification of genes involved in a water stress response in timothy and mapping of orthologous loci in perennial ryegrass

“…Timothy is valued for its winter hardiness, good palatability, and moderate nutritional feed value and thus is a major forage crop in northern latitudes. Its genomic constitution is complex (AAAABB) with the A-genome originating from the tetraploid form of P. alpinum and the B-genome coming from the diploid P. bertolonii (Cai and Bullen 1994). Unfortunately, the genetic and genomic tools available for this species are limited.…”