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Comparison of Transcript Profiles in Wild-Type and o2 Maize Endosperm in Different Genetic Backgrounds

Hongwu Jia, Monsanto, 3302 SE Convenience Blvd., Ankeny, IA 50021; Dan Nettleton, Department of Statistics, Iowa State Univ., Ames, IA, 50011; Gricelda Vazquez-Carrillo, Campo Experimental Valle de México, INIFAP, Texcoco, Mexico; Joan Peterson, Center for Crops Utilization Research, Iowa State University, Ames, IA, 50011; M. Paul Scott, 1407 Agronomy Hall, Ames, IA, 50011. This paper is a joint contribution from the Corn Insects and Crop Genetics Research Unit, USDA-ARS, and project no. 3781 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, INIFAP Campo Experimental Valle de México and Iowa State Univ. Department of Agronomy. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture

View larger version (28K): [in this window] [in a new window]   Figure 1. Comparison of kernel phenotypes in wild-type and o2 versions of eight inbred lines. Error bars indicate one standard deviation from the mean. Error bars are not shown for lysine content because each sample was measured once.

View larger version (27K): [in this window] [in a new window]   Figure 2. HPLC chromatograms of alcohol-extractable zein proteins from 14 DAP wild-type and o2 mutant endosperm. A: Comparison of HPLC chromatograms of B46 wild-type and B46o2 mutant. The protein levels of all four zein families are reduced significantly in B46o2 mutant. B: Comparison of HPLC chromatograms of M14 wild-type and M14o2 mutant. The ß-zein and some of the -zeins protein levels are reduced significantly, while the -zein levels are increased in M14o2 mutant.

View larger version (18K): [in this window] [in a new window]   Figure 3. Graphical approach to illustrating degree of differential expression in two genetic backgrounds. The degree of differential expression [log (wild-type/o2)] of transcripts likely to be differentially expressed (those with low P-values) is plotted as XY data, with each axis representing the degree of differential expression for an o2 vs. wild-type comparison in one genetic background. Ellipses indicate the regions where transcripts would be expected to cluster given the following hypotheses to explain differences in phenotypic severity in different genetic backgrounds. A. Hypothesis 1. The degree of differential expression hypothesis suggests one set of transcripts will be uniformly and differentially regulated, more so in one genetic background than the other. This difference in transcript levels causes the difference in phenotypic severity. B. Hypothesis 2. The genotype-specific differential expression hypothesis suggests certain transcripts will be differentially expressed in one genetic background while others will be differentially expressed in the other genetic background. The difference in phenotypic severity is determined by the specific transcripts that are differentially expressed in each genetic background.

View larger version (32K): [in this window] [in a new window]   Figure 4. Degree of differential expression [log (wild-type/o2)] in B46 (x axis) and M14 (y axis). Histograms on the right give the number of transcripts in each bins (defined as a 10 degree segment of the polar plot). Gaussian distributions were fit to each peak in the histogram, and the value of the peak of the fitted distribution is indicated with a line on each polar plot. A. Transcripts plotted are those identified as differentially expressed (P < 0.001) in the B46, M14 or pooled genotypes comparisons. B. Transcripts plotted are those identified as differentially expressed (P < 0.001) in the pooled genotypes comparison.

View larger version (17K): [in this window] [in a new window]   Figure 5. Results of transcript profiling. A. B46. B. M14. C: Eight genotypes pooled. Mean values for each transcript on the array is plotted. The signal intensity on the x axis is presented as a deviation from the mean. The red line is the LOWESS normalization curve. Zein transcripts are represented with red circles. The Opaque2 transcript is represented with a violet diamond. The b32 transcript is represented with an orange triangle with its point up. Pyruvate orthophosphate dikinase transcripts are represented with blue triangles with their points down. Cytosolic glyceraldehyde-3-phosphate dehydrogenase transcripts are represented with green #.

View larger version (12K): [in this window] [in a new window]   Figure 6. Comparison of the number of transcripts likely to be differentially expressed (wild-type vs. o2) for the indicated genotypes at a given probability (P value). The "pooled" line is for the comparison of eight wild-type genotypes pooled vs. a pool of their o2 counterparts.

View larger version (24K): [in this window] [in a new window]   Figure 7. Posterior probabilities of differential expression (PPDE) for specific transcripts estimated from our three transcript profiling experiments. These transcripts have been reported to be differentially expressed in previous investigations. PPDK, orthophosphate dikinase; G3PD, Glyceraldehyde-3-phosphate dehydrogenase; EF1a, Elongation factor 1 alpha; B32, ribosome inactivating protein.

View larger version (24K): [in this window] [in a new window]   Figure 8. Histogram of estimated posterior probabilities of differential expression for zein transcripts from three transcript profiling experiments.

View larger version (31K): [in this window] [in a new window]   Figure 9. Ethidium bromide stained agarose gels showing RT-PCR confirmation of microarray results and the estimated posterior probability of differential expression (PPDE) for that pair of transcripts. Negative images are presented so darker bands contain more DNA. In each PCR result, the band on the right is from o2 RNA and the band on the left is from wild-type RNA.