Project description:The phenomenon of heterosis describes the increased agronomic performance of heterozygous F1-plants compared to their homozygous parental inbred plants. Heterosis is already manifested during the early stages of root development in maize. The goal of this study was to identify non-additive gene expression in primary roots of maize hybrids compared to the average expression levels of their parental inbred lines. To achieve this goal a two step strategy was selected. First, a microarray preselection of non-additively expressed candidate genes was performed. Subsequently, gene expression levels in a subset of genes were determined via high throughput qRT-PCR experiments. Initial microarray experiments identified 1941 non-redundant genes which displayed non-additive gene expression in at least one of the twelve analyzed hybrids compared to the midparent value of their parental inbred lines. Comparison of these 1941 genes with non-additively expressed genes identified in maize shoot apical meristems via the same experimental procedure in the same genotypes revealed significantly less overlap than expected by pure chance supporting. This supports the notion of organ specific patterns of non-additively expressed genes. qRT-PCR analyses of 64 of the 1941 non-additively expressed genes in four different hybrids revealed that the majority of non-additively expressed genes were expressed between the high and low parent expression values and only a small fraction of genes was expressed below low or above high parent levels. Subsequently, 22 of the 64 genes that displayed non-additive expression in all four hybrids were analyzed in twelve hybrids that were generated from four inbred lines. Among those genes a superoxide dismutase 2 was expressed significantly above the midparent value in all twelve hybrids and might thus play a protective role in antioxidative defense in the primary root of maize hybrids. These findings are consistent with the hypothesis that global expression trends but also the consistent differential expression of key genes might be relevant during the organ-specific manifestation of heterosis. Keywords: Comparative genomic hybridization

Project description:The phenomenon of heterosis describes the increased agronomic performance of heterozygous F1-plants compared to their homozygous parental inbred plants. Heterosis is already manifested during the early stages of root development in maize. The goal of this study was to identify non-additive gene expression in primary roots of maize hybrids compared to the average expression levels of their parental inbred lines. To achieve this goal a two step strategy was selected. First, a microarray preselection of non-additively expressed candidate genes was performed. Subsequently, gene expression levels in a subset of genes were determined via high throughput qRT-PCR experiments. Initial microarray experiments identified 1941 non-redundant genes which displayed non-additive gene expression in at least one of the twelve analyzed hybrids compared to the midparent value of their parental inbred lines. Comparison of these 1941 genes with non-additively expressed genes identified in maize shoot apical meristems via the same experimental procedure in the same genotypes revealed significantly less overlap than expected by pure chance supporting. This supports the notion of organ specific patterns of non-additively expressed genes. qRT-PCR analyses of 64 of the 1941 non-additively expressed genes in four different hybrids revealed that the majority of non-additively expressed genes were expressed between the high and low parent expression values and only a small fraction of genes was expressed below low or above high parent levels. Subsequently, 22 of the 64 genes that displayed non-additive expression in all four hybrids were analyzed in twelve hybrids that were generated from four inbred lines. Among those genes a superoxide dismutase 2 was expressed significantly above the midparent value in all twelve hybrids and might thus play a protective role in antioxidative defense in the primary root of maize hybrids. These findings are consistent with the hypothesis that global expression trends but also the consistent differential expression of key genes might be relevant during the organ-specific manifestation of heterosis. Keywords: Comparative genomic hybridization Comparison of four inbred lines and the resulting twelve reciprocal hybrids. Samples from all 16 genotypes were paired on 85 arrays following a hybridization scheme including a dye-swap according to Keller et al. (2005). A tailor-made design was developed for this purpose using simulated annealing that was optimized for estimating non-additive effects while at the same time allowing estimation of additive effects with comparable precision. Optimizing precision for estimation of non-additivity required the design to be unbalanced with respect to the number of genotype pairings and the number of replications per genotype. Hence, two RNA samples were hybridized to each of the 85 microarray chips. Each RNA sample represented an independent biological replicate

Project description:Small RNAs (sRNAs) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73, Mo17) and their hybrid. We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in siRNAs primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared to Arabidopsis or rice, we confirmed that like these simpler plant genomes, 24-nt siRNAs whose abundance differs between maize parents also show a trend of downregulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2) encoded by modifier of paramutation1 (mop1). We also discovered that 21-22nt siRNAs derived from a number of distinct retrotransposon families differentially accumulate between B73 and Mo17 as well as their hybrid. Thus, maize possesses a novel source of genetic variation for regulating both transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis. sRNA libraries were derived from RNA isolated from the seedling shoot apex and developing ear tissues from B73, Mo17, B73xMo17 and Mo17xB73. The shoot apex was chosen because it is enriched for meristematic tissue where cell proliferation occurs, rates of organ initiation are determined, and organ size is specified. The developing ear was examined because it is enriched in meristematic tissue and is undergoing rapid growth, and also because the mature ear shows the highest degree of heterosis. Total RNA was isolated and separated on a 15% TBE-Urea polyacrylamide gel. Using a 10-bp ladder, the sRNA fraction representing 10-40-bp was excised. sRNA libraries were prepared according to Lu et al. (2007) or manufacturer's instructitions (Illumina). A combination of Perl scripts and FASTX toolkit scripts were used to remove adapters, collapse identical sequences and count reads per sequence. Supplementary processed data text files contain the distinct sRNA sequences for all of the genotypes analyzed in that experiment. Abundance (reads per million) was calculated for each distinct sequence by dividing the number of reads of distinct sRNA in a library by the total number of sRNA reads for that library and multiplying this by 1 million. Genome builds: B73 genome, maizesequence.org release 4a.53 (October, 2009); Mo17 whole genome shotgun clones.

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Small RNA expression pattern in multiple inbred lines and their hybrids of maize embryo

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