USDA-ARS and Montana State University

Isolation of repetitive DNA sequences specific to the genus Tripsacum

--Luther E. Talbert and Susan L. Moylan

Repetitive DNA sequences in plants tend to evolve rapidly, and as such, sequences specific to taxa have been isolated and used to document and monitor both artificial and natural interspecific hybrids. Perhaps the greatest use of repetitive DNA sequences as molecular markers has been with wheat and its relatives. Such markers have been used to monitor introgression of alien germplasm into wheat (e.g., Rayburn and Gill, 1987, Amer. J. Bot. 74:574-580) and to study the evolution of specific genomes in allopolyploid species (e.g., Dvorak et al., 1988, Genome 30:680-689; Talbert et al., 1991, Amer. J. Bot., in press). In the case of maize, previous work has shown that the transposable elements Mu and Spm are specific to the genus Zea (Talbert et al., 1989, J. Molec. Evol. 29:28-39), and this fact has been exploited to document an intergeneric hybrid involving Zea and Tripsacum (Talbert et al., 1990, Amer. J. Bot. 77:722-726). Given the long-standing interest in the use of Tripsacum germplasm as a source of beneficial genes for maize improvement, and the demonstrated utility of repetitive DNA sequences as molecular markers in several plant systems, it may be useful to isolate molecular probes for DNA sequences specific to Tripsacum. This report details the isolation of such molecular probes.

We cloned random TaqI fragments of Tripsacum dactyloides into the AccI site of pUC18 and transformed recombinant plasmids into the E. coli host NM522. Recombinant plasmids were isolated from bacterial cultures, digested with EcoRI and HindIII to liberate the inserts, and Southern blotted onto nylon membranes. Membranes were hybridized consecutively to nick-translated total DNA from Zea and Tripsacum, respectively. A total of three inserts were identified that showed a stronger signal with Tripsacum DNA than with Zea DNA. Subsequent hybridization of these inserts to EcoRI/HindIII-digested DNA from several Zea and Tripsacum species verified the Tripsacum-specificity of the inserts. Figure 1 shows that the inserts from plasmid pTrip9 (Panel A), pTrip25 (Panel B), and pTrip36 (Panel C) hybridize strongly to DNA from Tripsacum species (lanes d-g), but hybridize very little or not at all to Zea DNA (lanes a, b, and c).

Thus, three repetitive DNA sequences specific to the genus Tripsacum have been isolated. These sequences may complement the Zea-specific transposable elements Mu and Spm for documenting hybrids between Tripsacum and Zea, and monitoring introgression between the genera.

Figure 1. Southern blot of several Zea and Tripsacum species hybridized to Tripsacum-specific DNA probes. Lane a: Zea mays subsp. mays; b: Zea perennis; c: Zea diploperennis; d: Tripsacum latifolium; e: Tripsacum peruvianum; f: Tripsacum andersonii; g: Tripsacum dactyloides. Tripsacum DNAs were from J. F. Doebley, University of Minnesota. Panel A. Blot hybridized to pTrip9. Panel B. Blot hybridized to pTrip25. Panel C. Blot hybridized to pTrip36.

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