Mendelian and Non-Mendelian Regulation of Gene Expression in Maize

Transcriptome variation plays an important role in affecting the phenotype of an organism. However, an understanding of the underlying mechanisms regulating transcriptome variation in segregating populations is still largely unknown. We sought to assess and map variation in transcript abundance in maize shoot apices in the intermated B73×Mo17 recombinant inbred line population. RNA-based sequencing (RNA-seq) allowed for the detection and quantification of the transcript abundance derived from 28,603 genes. For a majority of these genes, the population mean, coefficient of variation, and segregation patterns could be predicted by the parental expression levels. Expression quantitative trait loci (eQTL) mapping identified 30,774 eQTL including 96 trans-eQTL "hotspots," each of which regulates the expression of a large number of genes. Interestingly, genes regulated by a trans-eQTL hotspot tend to be enriched for a specific function or act in the same genetic pathway. Also, genomic structural variation appeared to contribute to cis-regulation of gene expression. Besides genes showing Mendelian inheritance in the RIL population, we also found genes whose expression level and variation in the progeny could not be predicted based on parental difference, indicating that non-Mendelian factors also contribute to expression variation. Specifically, we found 145 genes that show patterns of expression reminiscent of paramutation such that all the progeny had expression levels similar to one of the two parents. Furthermore, we identified another 210 genes that exhibited unexpected patterns of transcript presence/absence. Many of these genes are likely to be gene fragments resulting from transposition, and the presence/absence of their transcripts could influence expression levels of their ancestral syntenic genes. Overall, our results contribute to the identification of novel expression patterns and broaden the understanding of transcriptional variation in plants. © 2013 Lin et al

Genic and nongenic contributions to natural variation of quantitative traits in maize

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions

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

Genetic control of morphometric diversity in the maize shoot apical meristem

The maize shoot apical meristem (SAM) comprises a small pool of stem cells that generate all above-ground organs. Although mutational studies have identified genetic networks regulating SAM function, little is known about SAM morphological variation in natural populations. Here we report the use of high-throughput image processing to capture rich SAM size variation within a diverse maize inbred panel. We demonstrate correlations between seedling SAM size and agronomically important adult traits such as flowering time, stem size and leaf node number. Combining SAM phenotypes with 1.2 million single nucleotide polymorphisms (SNPs) via genome-wide association study reveals unexpected SAM morphology candidate genes. Analyses of candidate genes implicated in hormone transport, cell division and cell size confirm correlations between SAM morphology and trait-associated SNP alleles. Our data illustrate that the microscopic seedling SAM is predictive of adult phenotypes and that SAM morphometric variation is associated with genes not previously predicted to regulate SAM size

The Physical and Genetic Framework of the Maize B73 Genome

Maize is a major cereal crop and an important model system for basic biological research. Knowledge gained from maize research can also be used to genetically improve its grass relatives such as sorghum, wheat, and rice. The primary objective of the Maize Genome Sequencing Consortium (MGSC) was to generate a reference genome sequence that was integrated with both the physical and genetic maps. Using a previously published integrated genetic and physical map, combined with in-coming maize genomic sequence, new sequence-based genetic markers, and an optical map, we dynamically picked a minimum tiling path (MTP) of 16,910 bacterial artificial chromosome (BAC) and fosmid clones that were used by the MGSC to sequence the maize genome. The final MTP resulted in a significantly improved physical map that reduced the number of contigs from 721 to 435, incorporated a total of 8,315 mapped markers, and ordered and oriented the majority of FPC contigs. The new integrated physical and genetic map covered 2,120 Mb (93%) of the 2,300-Mb genome, of which 405 contigs were anchored to the genetic map, totaling 2,103.4 Mb (99.2% of the 2,120 Mb physical map). More importantly, 336 contigs, comprising 94.0% of the physical map (∼1,993 Mb), were ordered and oriented. Finally we used all available physical, sequence, genetic, and optical data to generate a golden path (AGP) of chromosome-based pseudomolecules, herein referred to as the B73 Reference Genome Sequence version 1 (B73 RefGen_v1)

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Integrating transposable elements in the 3D genome

Chromosome organisation is increasingly recognised as an essential component of genome regulation, cell fate and cell health. Within the realm of transposable elements (TEs) however, the spatial information of how genomes are folded is still only rarely integrated in experimental studies or accounted for in modelling. Whilst polymer physics is recognised as an important tool to understand the mechanisms of genome folding, in this commentary we discuss its potential applicability to aspects of TE biology. Based on recent works on the relationship between genome organisation and TE integration, we argue that existing polymer models may be extended to create a predictive framework for the study of TE integration patterns. We suggest that these models may offer orthogonal and generic insights into the integration profiles (or "topography") of TEs across organisms. In addition, we provide simple polymer physics arguments and preliminary molecular dynamics simulations of TEs inserting into heterogeneously flexible polymers. By considering this simple model, we show how polymer folding and local flexibility may generically affect TE integration patterns. The preliminary discussion reported in this commentary is aimed to lay the foundations for a large-scale analysis of TE integration dynamics and topography as a function of the three-dimensional host genome

Transformation and analysis of tobacco plant var Petit havana with T-urf13 gene under anther-specific TA29 promoter

T-urf13, a well-documented cms-associated gene from maize, has been shown to render methomyl sensitivity to heterologous systems like rice, yeast and bacteria when expressed constitutively. Since these transgenic plants were fertile, it was hypothesized that T-urf13 gene if expressed in anthers may result in male sterility that could be used for hybrid seed production. Hence, this work was aimed at analysing whether T-urf13 gene when expressed in anthers can result in male sterile plants or requires methomyl treatment to cause male sterility (controllable). This is the first report of transformation of tobacco with T-urf13 gene under anther-specific promoter (TA29) with or without mitochondrial targeting sequence. Most of the transgenic plants obtained were fertile; this was surprising as many male sterile plants were expected as T-urf13 gene is a cms associated gene. Our results suggest that it may not be possible to obtain male sterility by expressing URF13 in the anther by itself or by methomyl application