Choice of restriction enzyme digests of genomic DNA in profiling inbred lines
--J.S.C. Smith, O.S. Smith, D. Grant, S.L. Bowen, and R.A. Tenborg
The abilities of single and multiple individual digestions of genomic DNA to reveal polymorphisms using 6 restriction endonucleases on 13 inbred lines of maize that encompass a broad array of diversity in the U.S. Corn Belt were investigated. Genomic DNA of each line was extracted by CTAB (Saghai-Maroof et al., Proc. Natl. Acad. Sci. USA, 81:8014-8018, 1984). DNAs were individually digested by BamHI, BglII, EcoRI, EcoRV, HindIII, and KpnI. Electrophoresis, transfer to membrane, random-prime labelling, hybridization, and washes were performed as described in Smith et al. (Theor. Appl. Genet., 80:in press, 1990). Each genomic digest was probed separately with 100 clones that have been mapped and were, therefore, known to be well dispersed throughout the maize genome. Approximately equal numbers of probes from Brookhaven National Laboratory, the University of Missouri at Columbia, and Pioneer Hi-Bred International, Inc. were used.
KpnI digests were often unscorable because of blurred profiles or bands that migrated closely together; these digests were, therefore, excluded from further analysis. The mean values per probe for the percentage of all possible pairs of inbreds that revealed polymorphism (in parentheses) for single digestions of genomic DNA were as follows: BamHI(77%); BglII(80%); EcoRI(77%); EcoRV(77%); and HindIII(80%). Percentages of inbred pairs that were polymorphic for 2 individually digested and separately probed DNAs ranged from 77% to 86% (mean 81%). Percent inbred pairs polymorphic for 3 individually digested genomic DNAs ranged from 83% to 89% (mean 86%). The mean percentage of pairs that were polymorphic when 5 individual digestions were probed was 91%.
Use of a single restriction enzyme digest revealed, on average, 86% of the maximum variation that was revealed when 5 individual digestions of DNA were probed separately. The absolute percentages of polymorphism that are revealed will depend upon the degree of difference among the genotypes under study. Nevertheless, these data indicate that the use of additional probes rather than the use of additional restriction enzymes is a far more effective means of revealing variation. The employment of additional probes provides the further advantage of allowing the genome to be surveyed in greater detail. Therefore, it is proposed that for routine studies, profiles be acquired using a single restriction digest per probe. The particular restriction digestion of genomic DNA that is used for each probe can be determined so as to maximize the opportunity to reveal polymorphism and to reproducibly generate autoradiograms of high quality with variants expressing obviously discernible migration and molecular weight differences.
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