Regulation of Small RNA Accumulation in the Maize Shoot Apex

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial–abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs

Intragenic Meiotic Crossovers Generate Novel Alleles with Transgressive Expression Levels

Meiotic recombination is an evolutionary force that generates new genetic diversity upon which selection can act. Whereas multiple studies have assessed genome-wide patterns of recombination and specific cases of intragenic recombination, few studies have assessed intragenic recombination genome-wide in higher eukaryotes. We identified recombination events within or near genes in a population of maize recombinant inbred lines (RILs) using RNA-sequencing data. Our results are consistent with case studies that have shown that intragenic crossovers cluster at the 5\u27 ends of some genes. Further, we identified cases of intragenic crossovers that generate transgressive transcript accumulation patterns, that is, recombinant alleles displayed higher or lower levels of expression than did nonrecombinant alleles in any of ~100 RILs, implicating intragenic recombination in the generation of new variants upon which selection can act. Thousands of apparent gene conversion events were identified, allowing us to estimate the genome-wide rate of gene conversion at SNP sites (4.9 X 10-5). The density of syntenic genes (i.e., those conserved at the same genomic locations since the divergence of maize and sorghum) exhibits a substantial correlation with crossover frequency, whereas the density of nonsyntenic genes (i.e., those which have transposed or been lost subsequent to the divergence of maize and sorghum) shows little correlation, suggesting that crossovers occur at higher rates in syntenic genes than in nonsyntenic genes. Increased rates of crossovers in syntenic genes could be either a consequence of the evolutionary conservation of synteny or a biological process that helps to maintain synteny

In Vitro Deletion Mapping of the Viral Strand Replication Origin of Pseudomonas Bacteriophage Pf3

The origin of viral strand replication of the filamentous bacteriophage Pf3 has been characterized in Escherichia coli by in vitro deletion mapping techniques. The origin region was functionally identified by its ability to convey replicative properties to a recombinant plasmid in a polA host in which the replication origin of the vector plasmid is not functional. The origin of Pf3 viral strand replication is contained within a DNA sequence of 139 bp. This sequence covers almost completely one of the intergenic regions of the Pf3 genome, and it specifies both replication initiation and termination functions. Although no nucleotide sequence homology is present between the Pf3 origin of viral strand replication and that of the E. coli filamentous phages Ff (M13, fl, and fd) and IKe, their map positions and functional properties are very similar

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors. CONCLUSIONS We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping

Microdissection of Shoot Meristem Functional Domains

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND: Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS: By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors. CONCLUSIONS: We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping