Gene expression analyses in maize inbreds and hybrids with varying levels of heterosis

Abstract

Background: Heterosis is the superior performance of F1 hybrid progeny relative to the parental phenotypes. Maize exhibits heterosis for a wide range of traits, however the magnitude of heterosis is highly variable depending on the choice of parents and the trait(s) measured. We have used expression profiling to determine whether the level, or types, of non-additive gene expression vary in maize hybrids with different levels of genetic diversity or heterosis.

Results: We observed that the distributions of better parent heterosis among a series of 25 maize hybrids generally do not exhibit significant correlations between different traits. Expression profiling analyses for six of these hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation. The majority of differentially expressed genes in each of the six different hybrids exhibited additive expression patterns, and approximately 25% exhibited statistically significant non-additive expression profiles. Among the non-additive profiles, approximately 80% exhibited hybrid expression levels between the parental levels, approximately 20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range.

Conclusion: We have found that maize inbred genetic diversity is correlated with transcriptional variation. However, sampling of seedling tissues indicated that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines. These findings suggest that heterosis is probably not a consequence of higher levels of additive or non-additive expression, but may be related to transcriptional variation between parents. The lack of correlation between better parent heterosis levels for different traits suggests that transcriptional diversity at specific sets of genes may influence heterosis for different traits.

Figure 1

Schematic diagram of potential patterns of hybrid gene expression. This hypothetical gene exhibits higher expression in parent 2 than in parent 1. Five different potential patterns of hybrid expression (A-E) are diagrammed. The hybrid could exhibit (A) below-low parent expression (BLP); (B) low parent-like expression (LP); (C) mid-parent expression; (D) high parent-like expression (HP); or (E) above high parent expression (AHP). Only mid-parent expression is classified as additive. The BLP, LP-like, HP-like and AHP expression patterns would all be examples of non-additive expression.

Figure 2

Heterosis for non-yield traits. The percent BPH is shown for all traits and all hybrids scored in this study. The numerical BPH values are available in Additional file 2. Red bars represent BPH for hybrids generated between SS and NSS inbreds, blue bars represent BPH for hybrids generated within SS and NSS inbreds, and grey bars represent BPH for hybrids derived from an inbred line with mixed origin (F2).

Figure 3

Relationship between parental genetic diversity and hybrid heterosis among traits and hybrids. The percentage better parent heterosis (BPH) for each hybrid is plotted against the genetic distance between parents. The 25 hybrids were scored based on percentage BPH for five traits (plant final height, days to flowering, weight of 50 seeds, 11-day height and 11-day biomass). Traits measured on field-grown plants are shown in (A) and traits measured on greenhouse-grown plants are shown in (B). Average percent BPH is shown based on two field replicates (A) and three greenhouse replicates (B). Spots representing crosses between stiff stalk (SS) and non-stiff stalk (NSS) groups are shown in red, and spots representing crosses within either group are shown in blue. The Pearson's R correlation value and p-value of the regression are shown for each trait. The six hybrids that were used for expression profiling are labelled in each of the five plots.

Figure 4

Relationship between parental genetic diversity and differential gene expression. The number of differentially expressed genes identified for each inbred-hybrid group based on stringent statistical criteria is plotted against the genetic distance between parents. Spots representing crosses between stiff stalk (SS) and non-stiff stalk (NSS) groups are shown in red, and spots representing crosses within either group are shown in blue. The Pearson's R correlation value and p-value of the regression are shown.

Figure 5

Validation of differential expression using MassArray and 70-mer platforms. The magnitude of differential expression between inbred lines based on the Affymetrix data was compared to the magnitude of differential expression detected using the MassArray platform and 70-mer microarray platform. The subset of the genes identified as differentially expressed on the Affymetrix platform (FDR < 0.05, and additional quality control filters; see Methods) was used for these analyses. The color coding of the data points indicates the inbred genotype comparison. (A) The same inbred RNA samples used for Affymetrix microarray analyses were mixed in a pairwise 1:1 ratio and subjected to MassArray relative allelic quantification [25]. The correlation between the MassArray proportions and the proportions calculated from the Affymetrix dataset (inbred 1 signal divided by the sum of the two inbred signals) are shown. Each spot represents the proportion of one allele per inbred-inbred comparison. The B73 and Mo17 sequence SNPs were used to design the assays, thus this comparison is most highly represented in this analysis. (B) Many genes that were determined to be differentially expressed in the Affymetrix dataset were also present on the 70-mer microarray platform. The correlation of the inbred expression fold-differences on the 70-mer oligonucleotide microarray and the Affymetrix microarray are shown. Each spot represents the fold-differences of one gene per inbred-inbred comparison. The 70-mer microarray data validated the directionality of the Affymetrix microarray patterns in 84–91% of the differentially expressed profiles (see main text).

Figure 6

Distribution of d/a values for Affymetrix differentially expressed genes. Distributions of d/a ratios for differentially expressed genes based on Affymetrix microarray data. (A) d/a type I values indicate the hybrid expression levels relative to the low-parent and high-parent levels. The distributions are very similar for the six different hybrids. Hybrid expression patterns center approximately around the mid-parent level with very flat distributions outside of the parental range. (B) d/a type II values indicate the hybrid expression levels relative to the maternal-parent and paternal-parent levels. Again, all six hybrids exhibit similar distributions peaking around mid-parent levels, indicating no maternal or paternal expression biases. (C) The distributions of d/a type II values for the subset of differentially expressed genes that exhibited non-additive hybrid expression profiles. The distributions indicate that the non-additive patterns for most genes are still within the parental range, and are oftentimes observed near the mid-parent (additive) values.

Figure 7

Distribution of d/a values for 70-mer array differentially expressed genes. Distributions of d/a (type I) ratios for differentially expressed genes based on the 70-mer oligonucleotide microarray data. (A) The d/a distributions for all differentially expressed genes. The distributions of the four hybrids are very similar to one another and peak at approximately zero, as was observed in Affymetrix microarray data. (B) The d/a distributions for the subset of differentially expressed genes that are also represented with features on the Affymetrix platform. The distributions are similar to those in (A). In both (A) and (B), the proportion of DE genes with d/a values above 3.0 or below -3.0 are all plotted as a single data point. The proportion of d/a values above 3.0 and below -3.0 for hybrid B84 × B73 plotted beyond the range of the displays and are not shown.

Figure 8

Distribution of DE, AHP and BLP genes among gene ontologies. (A) The distributions of GO terms assigned for the entire Affymetrix microarray, the differentially expressed genes, and the subsets of additive and non-additive differentially expressed genes are compared. (B) The distributions of GO terms assigned for the entire Affymetrix microarray, the AHP subset and the BLP subset are compared. In both (A) and (B), the GO terms are ordered on the graph from highest frequency on the microarray (left) to lowest frequency on the microarray (right) within the Biological process, Cellular component, and Molecular function categories, respectively.