--J. W. Higginbotham*, J. S. C. Smith, and O. S. Smith
*Also affiliated with Cold Spring Harbor Lab.
Before two-dimensional protein profiles are routinely used to describe inbred genotypes and to determine genetic associations between inbreds, the contribution of environmental factors needs to be understood to insure that results are reproducible and reliable.
This experiment was designed to examine the variation among protein profiles due to different environmental sources of kernels relative to the variation due to different genotypes. One lot of public inbred line B73 (seed source Johnston, 1986) and two lots of Pioneer Hi-Bred proprietary inbred line 207 (seed source Johnston, 1982 and 1986) were used.
Kernels from line B73 were placed on moist filter paper and incubated at 27-30 C until most of the kernels had germinated (27 hrs). Embryos were excised from eight germinated kernels, washed, dried, and weighed. Each embryo was placed in a spot well with 35-S methionine solution and labeled for 17 hrs. Subsequently, they were washed twice, dried, and reweighed. Total pre-label mass averaged 53mg per embryo, while total post-label mass averaged 92mg per embryo. Increase in mass was accompanied by elongation of all embryonic axes. Total elapsed time including imbibition and labeling was 45 hrs. Extraction of protein from the seedlings followed Damerval (Electrophoresis 7:52, 1986) and Higginbotham and Smith (in this Newsletter).
Kernels from the two lots of line 207 were treated as described for B73 except that total imbibition time was 43.5 hrs. This was due to slightly faster germination by 207. Embryos were labeled for 17 hrs. For lot 207-82, pre-label mass averaged 56mg per embryo, while post-label mass averaged 84mg per embryo. For lot 207-86, pre-label mass averaged 54mg per embryo, while post-label mass averaged 93mg per embryo.
The two-dimensional gel electrophoresis, fluorography, and computer analysis were performed at the QUEST facility of the Cold Spring Harbor Laboratory under the supervision of Heidi Sacco and Cecile Chang. Work with the QUEST facility was funded by Pioneer Hi-Bred International, Inc. through a cooperative agreement between Pioneer Hi-Bred and the Cold Spring Harbor Laboratory. A pH gradient of 4-8 was used in the first dimension, and a 12.5% SDS-polyacrylamide gel was used in the second dimension. Each of the eight gels that were run (2 B73, 3 207-82, and 3 207-86) were exposed to film for three periods of time. This generated a light, medium, and dark exposure for each gel.
The data set consisted of 1961 spots matched across all eight gels. Of these, nine spots had zero density values for 7 of the 8 gels and were deleted from the data set. Spot densities are in normalized units (ppm).
In order to determine the influence of environmental effects relative to genotypic effects, nested analysis of variance was performed on each spot (1952 nested analyses of variance). Two sets of spots were generated. The first set included those spots where at least 80% of their variation was partitioned between the two inbreds. The second set included those spots where at least 80% of their variation was partitioned between the two lots of 207. Based on this method of selecting spots, 14.7% (287 spots) showed large differences between the two lines, while less than 1.0% (16 spots) showed equally large differences between the two lots of 207. Other methods of spot selection and analysis gave similar results.
Relative to the influence of different genotypes on protein profiles,
the influence of different environmental sources appears to be very minimal.
The effect of different genotypes on the protein profiles is large and
easily extracted from the data set. It does not appear that major genotypic
effects are confounded to any extent with environmental effects. Those
spots which do show a putative environmental effect are also easily extracted.
Whether or not these conclusions are true for other inbreds and other environments
awaits further study.
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