The utility of RFLPs as molecular markers relies in part on their stability through successive generations even when exposed to different environmental conditions. In order to investigate the potential contribution of "environment" to RFLP stability, six established inbred lines were maintained under two environmental regimes for seven generations and then analyzed.
In 1978 seed from each of maize inbreds Oh51A, Oh43, Mo17, B73, W64A and A632 were collected and separated into two lots, one designated for Molokai, Hawaii and the other for London, Canada. Sibling (or isogenic) lines were maintained in their respective locations through self pollination for seven generations, and in the final generation a sample from each isogenic line was grown at the alternate location. It is implicit in this study that no apparent phenotypic variation was introduced during the pedigreed breeding, thereby confounding any observed variation in the DNA. To document the absence of phenotypic variation, three nursery plots, one at London in 1992 and two at London in 1993, included seed from both locations. Data were collected on node number, plant height and tassel branch number. No obvious differences were visible based on observations of field grown material at the London nursery.
Genomic DNA was isolated from six day etiolated plumules following a modified urea extraction protocol (Shure et al., Cell 35:225-233, 1983). DNA was digested using one of four restriction enzymes (EcoRI, HindIII, BamHI and BstI) according to manufacturer's instructions (Pharmacia). Fragments were separated by gel electrophoresis then capillary transferred to positively charged nylon membranes (Boehringer Mannheim, BM). Four gene specific sequences were random primer labelled using the digoxigenin system from BM. Detections were carried out as outlined by Engler-Blum et al. (Analyt. Biochem. 210:235-244, 1993) with modifications based on Maillet et al. (MNL 67:75, 1993). The sequences employed as probes were: Waxy (Wx), a 14 kb EcoRI fragment obtained from P. Dietrich; BI, a 1.9 kb EcoRI cDNA from V. Chandler; scMubG7-J, a 2.0 kb EcoRI/XbaI fragment, specific for the 5' region of a ubiquitin fusion protein gene; C1-5C, a 1.0 kb fragment specific for a polyubiquitin gene. The maize ubiquitin clones were provided by L. Liu. In total, sixteen clone enzyme combinations (CECs) were used to investigate all entries for each of the six genotypes.
There were no differences observed in either the number or size of fragments between the London and Hawaiian isogenic lines (all inbreds). We conclude that these RFLP markers remained stable over seven generations under the two different seed production locations. Though intervening generations were not examined, it seems unlikely that mutations arose and subsequently reverted to their previous state. Viable material is available for analysis; had there been any differences found they could have been traced to their origin.
All CECs revealed polymorphisms among at least two of the inbreds, while on average 4.2 different banding patterns were produced per CEC. The number of CECs used was not sufficient to determine accurately the distances among inbred lines as outlined by Smith et al. (MNL 65:66, 1991); however, it is interesting to note that the highest genetic similarity, as calculated according to Nei and Li (PNAS 76:5269-5273, 1979), was obtained between Mo17 and W64A (0.597), two inbreds that share a common parent (187-2).
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