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Small RNA sequencing in maize hybrids and inbreds

ABSTRACT: 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.

ORGANISM(S): Zea mays

PROVIDER: GSE37411 | GEO | 2012/05/26

SECONDARY ACCESSION(S): PRJNA159733

REPOSITORIES: GEO

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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.

Project description:Heterosis, also known as hybrid vigor, has been extensively utilized to increase productivity in crop, yet the underlying molecular mechanisms remain largely elusive. Recent studies have reported that in addition to mRNA transcription, epigenetic variations in DNA methylation, small RNAs and histone modifications also contribute to heterosis. However, the operative mode of post-transcriptional regulation on gene expression such as RNA m6A methylation and translational efficiency in heterosis has never been explored. In this study, we generated transcriptome-wide profiles of mRNA abundance, m6A methylation, and translational efficiency from the maize (Zea mays) F1 hybrid B73×Mo17 and its two parental lines B73 and Mo17 to ascertain contributions of each regulatory layer to heterosis at the seedling stage. We documented that although the global abundance and the distribution configuration of m6A maintained unchanged, a greater number of genes have gained m6A modification in hybrid compared to parent lines. m6A modification and translational efficiency exhibited greater variations between hybrid and parents as compared with observed variation of mRNA abundance. In hybrid, the vast majority of genes with m6A modification exhibited non-additive expression pattern, the percentage of which was exceedingly higher than that of differential genes at mRNA abundance and translation efficiency levels. Non-additive genes involved in different biological processes were hierarchically coordinated by discrete combinations of three regulatory layers. These findings suggest that transcriptional and post-transcriptional regulations on gene expression adopt divergent approaches to participate in the formation of heterosis in hybrid. Overall, the integrated multi-omics analysis provides a valuable portfolio for interpreting transcriptional and post-transcriptional regulation on gene expression in maize hybrid, and pave new avenues for exploring molecular mechanisms underlying hybrid vigor.

Project description:Combining dissimilar parents often leads to increased vigor in the hybrid offspring. “Heterosis” describes both this behavior and its underlying Mendelian and non-Mendelian interactions [1], although its molecular basis remains largely unknown. Recent comparisons of small RNA (sRNA) profiles from parents and their heterotic progeny identified correlations between interparental 24-nucleotide (24-nt) RNA variation and non-additive 24-nt RNA changes in the resulting hybrid [2,3]. 24-nt RNAs guide de novo cytosine methylation, and several proteins are required for their biogenesis, including a Snf2-like ATPase: required to maintain repression1 (RMR1) [4]. We found height variation between heterotic hybrids +/- RMR1 activity, implicating a role for RMR1 in heterosis. Based on the published correlations mentioned above [2,3], we hypothesized that RMR1-loss reduces parental sRNAs, altering their relative ratios and changing the sRNA profiles in the resulting hybrid from those of a standard hybrid (from +RMR1 parents). To probe this hypothesis, we profiled sRNAs from parents and hybrids +/- RMR1 function, limiting the parental diversity to only portions of chromosomes 6 and 9. Our analysis will address how RMR1 loss changes hybrid sRNAs in the presence and absence of underlying genetic variation and help to determine how this loss results in different phenotypic outcomes from heterotic crosses. 1. Shull (1948) Genetics 2. Groszmann et al (2011) PNAS 3. Barber et al (2012) PNAS 4. Hale et al (2007) PLoS Biology We used an interchange chromosome (T6-9 043-1) introgressed into B73 (~95% B73) to create a local region of genetic diversity when crossed as a female parent to 100% B73 individual. Small RNAs from immature cobs were sequenced from both of these parents and their resulting hybrid (2 samples each). Additionally, a similar cross where the T6-9 containing female parent lacked RMR1 activity (rmr1-1 / rmr1-1) was used to generate a counterpart hybrid whose small RNAs were also profiled (2 samples) from immature cobs. Finally, small RNAs from 2 immature cobs of an unrelated rmr1 heterozygote (again highly introgressed into B73) were sequenced as well.

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Small RNA sequencing in maize hybrids and inbreds

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