Tieming Ji

Maize inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.

Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.

Paternal Dominance of Trans-eQTL Influences Gene Expression Patterns in Maize Hybrids

A-Maize-ing Maize is one of our oldest and most important crops, having been domesticated approximately 9000 years ago in central Mexico. Schnable et al. (p. 1112; see the cover) present the results of sequencing the B73 inbred maize line. The findings elucidate how maize became diploid after an ancestral doubling of its chromosomes and reveals transposable element movement and activity and recombination. Vielle-Calzada et al. (p. 1078) have sequenced the Palomero Toluqueño (Palomero) landrace, a highland popcorn from Mexico, which, when compared to the B73 line, reveals multiple loci impacted by domestication. Swanson-Wagner et al. (p. 1118) exploit possession of the genome to analyze expression differences occurring between lines. The identification of single nucleotide polymorphisms and copy number variations among lines was used by Gore et al. (p. 1115) to generate a Haplotype map of maize. While chromosomal diversity in maize is high, it is likely that recombination is the major force affecting the levels of heterozygosity in maize. The availability of the maize genome will help to guide future agricultural and biofuel applications (see the Perspective by Feuillet and Eversole). Gene expression variation in maize hybrids is influenced by distant DNA sequences subject to paternal genomic imprinting. Heterosis refers to the superior performance of hybrid progeny relative to their inbred parents, but the mechanisms responsible are unknown. Hybrids between the maize inbred lines B73 and Mo17 exhibit heterosis regardless of cross direction. These reciprocal hybrids differ from each other phenotypically, and 30 to 50% of their genes are differentially expressed. We identified ~4000 expression quantitative trait loci (eQTL) that allowed us to identify markers linked to variation in expression. We found that over three-quarters of these eQTL act in trans (78%) and that 86% of these differentially regulate transcript accumulation in a manner consistent with gene expression in the hybrid being regulated exclusively by the paternally transmitted allele. This result suggests that widespread imprinting contributes to the regulation of gene expression in maize hybrids.

Gene expression of zebrafish embryos exposed to titanium dioxide nanoparticles and hydroxylated fullerenes

Increased release of engineered nanoparticles to the environment suggests a rising need for the monitoring and evaluation of potential toxicity. Zebrafish frequently have been used as a model species in human and aquatic toxicology studies. In this study, zebrafish embryos were microinjected in the otic vesicle with a sublethal dose of engineered nanoparticles (titanium dioxide/TiO(2) and hydroxylated fullerenes/C(60)(OH)(24)). A gene microarray analysis was performed on injected and control embryos to determine the potential for nanoparticles to change the expression of genes involved in cross talk of the nervous and immune systems. The exposure to TiO(2) and hydroxylated fullerenes caused shifts in gene regulation response patterns that were similar for downregulated genes but different for upregulated genes. Significant effects on gene regulation were observed on genes involved in circadian rhythm, kinase activity, vesicular transport and immune response. This is the first report of circadian rhythm gene deregulation by nanoparticles in aquatic animals, indicating the potential for broad physiological and behavioral effects controlled by the circadian system.

Characterization of global loss of imprinting in fetal overgrowth syndrome induced by assisted reproduction

Significance Large offspring syndrome (LOS) is a fetal overgrowth condition that mimics the human syndrome Beckwith–Wiedemann. These conditions have been observed with higher incidence in offspring conceived with the use of assisted reproductive technologies and are believed to be the result of misregulation of a set of genes that are expressed only from the maternally or paternally inherited chromosomes. These genes are known as imprinted genes. In our study, we demonstrate that the kidney, brain, muscle, and liver of LOS fetuses show misregulation of multiple imprinted genes when compared with controls. Furthermore, we show that the magnitude of overgrowth in LOS fetuses correlates with the number of misregulated imprinted genes. Our results may help create diagnostics for these fetal syndromes. Embryos generated with the use of assisted reproductive technologies (ART) can develop overgrowth syndromes. In ruminants, the condition is referred to as large offspring syndrome (LOS) and exhibits variable phenotypic abnormalities including overgrowth, enlarged tongue, and abdominal wall defects. These characteristics recapitulate those observed in the human loss-of-imprinting (LOI) overgrowth syndrome Beckwith–Wiedemann (BWS). We have recently shown LOI at the KCNQ1 locus in LOS, the most common epimutation in BWS. Although the first case of ART-induced LOS was reported in 1995, studies have not yet determined the extent of LOI in this condition. Here, we determined allele-specific expression of imprinted genes previously identified in human and/or mouse in day ∼105 Bos taurus indicus × Bos taurus taurus F1 hybrid control and LOS fetuses using RNAseq. Our analysis allowed us to determine the monoallelic expression of 20 genes in tissues of control fetuses. LOS fetuses displayed variable LOI compared with controls. Biallelic expression of imprinted genes in LOS was associated with tissue-specific hypomethylation of the normally methylated parental allele. In addition, a positive correlation was observed between body weight and the number of biallelically expressed imprinted genes in LOS fetuses. Furthermore, not only was there loss of allele-specific expression of imprinted genes in LOS, but also differential transcript amounts of these genes between control and overgrown fetuses. In summary, we characterized previously unidentified imprinted genes in bovines and identified misregulation of imprinting at multiple loci in LOS. We concluded that LOS is a multilocus LOI syndrome, as is BWS.

Identification of microRNAs and their mRNA targets during soybean nodule development: functional analysis of the role of miR393j‐3p in soybean nodulation

Plant microRNAs (miRNAs) play important regulatory roles in a number of developmental processes. The present work investigated the roles of miRNAs during nodule development in the crop legume soybean (Glycine max). Fifteen soybean small RNA libraries were sequenced from different stages of nodule development, including young nodules, mature nodules and senescent nodules. In order to identify the regulatory targets of the miRNAs, five parallel analysis of RNA ends (PARE) libraries were also sequenced from the same stages of nodule development. Sequencing identified 284 miRNAs, including 178 novel soybean miRNAs. Analysis of miRNA abundance identified 139 miRNAs whose expression was significantly regulated during nodule development, including 12 miRNAs whose expression changed > 10-fold. Analysis of the PARE libraries identified 533 miRNA targets, including three nodulation-related genes and eight nodule-specific genes. miR393j-3p was selected for detailed analysis as its expression was significantly regulated during nodule formation, and it targeted a nodulin gene, Early Nodulin 93 (ENOD93). Strong, ectopic expression of miR393j-3p, as well as RNAi silencing of ENOD93 expression, significantly reduced nodule formation. The data indicate that miR393j-3p regulation of ENOD93 mRNA abundance is a key control point for soybean nodule formation.

Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection

Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation.

Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing

ABSTRACT Genomic imprinting is an epigenetic mechanism that leads to parental-allele-specific gene expression. Approximately 150 imprinted genes have been identified in humans and mice but less than 30 have been described as imprinted in cattle. For the purpose of de novo identification of imprinted genes in bovine, we determined global monoallelic gene expression in brain, skeletal muscle, liver, kidney and placenta of day ∼105 Bos taurus indicus × Bos taurus taurus F1 conceptuses using RNA sequencing. To accomplish this, we developed a bioinformatics pipeline to identify parent-specific single nucleotide polymorphism alleles after filtering adenosine to inosine (A-to-I) RNA editing sites. We identified 53 genes subject to monoallelic expression. Twenty three are genes known to be imprinted in the cow and an additional 7 have previously been characterized as imprinted in human and/or mouse that have not been reported as imprinted in cattle. Of the remaining 23 genes, we found that 10 are uncharacterized or unannotated transcripts located in known imprinted clusters, whereas the other 13 genes are distributed throughout the bovine genome and are not close to any known imprinted clusters. To exclude potential cis-eQTL effects on allele expression, we corroborated the parental specificity of monoallelic expression in day 86 Bos taurus taurus × Bos taurus taurus conceptuses and identified 8 novel bovine imprinted genes. Further, we identified 671 candidate A-to-I RNA editing sites and describe random X-inactivation in day 15 bovine extraembryonic membranes. Our results expand the imprinted gene list in bovine and demonstrate that monoallelic gene expression can be the result of cis-eQTL effects.

Impacts of Salmonella enteritidis infection on liver transcriptome in broilers

Salmonella enterica serovar enteritidis is an enteric bacterium that can contaminate chicken eggs and meat, resulting in production losses and consumer illness. To provide insight into the systemic metabolic effects of S. enteritidis infection, liver samples were harvested 10‐days postinfection from broiler hens. Hepatic global gene expression levels were assessed using a chicken 44K Agilent microarray. Forty‐four genes were differentially expressed at a significance level of q value < 0.05. One hundred eighty‐three genes were differentially expressed at a suggestive significance level of q value < 0.1. A predominance of downregulation existed among significantly differentially expressed genes. Cell cycle and metabolism networks were created from the differentially expressed genes. Mitochondria‐mediated apoptosis, electron transport, peptidase activity, vein constriction, cell differentiation, IL‐2 signaling, Jak‐Stat signaling, B‐cell receptor signaling, GDP/GTP exchange, and protein recycling were among the functions of the differentially expressed genes that were down‐regulated in response to S. enteritidis. The effects of S. enteritidis infection on the liver transcriptome profiles of broilers reflect a predominance of downregulation of genes with cell cycle and metabolic functions. The most pronounced response was the downregulation of genes that function in metabolic pathways, inflammation, and mitochondria‐mediated apoptosis. These results provide insight into important systemic metabolic mechanisms that are active in the chicken liver in response to S. enteritidis infection at 10‐days postinfection. genesis 51:357–364, 2012.

Maternal Hyperleptinemia Is Associated with Male Offspring’s Altered Vascular Function and Structure in Mice

Children of mothers with gestational diabetes have greater risk of developing hypertension but little is known about the mechanisms by which this occurs. The objective of this study was to test the hypothesis that high maternal concentrations of leptin during pregnancy, which are present in mothers with gestational diabetes and/or obesity, alter blood pressure, vascular structure and vascular function in offspring. Wildtype (WT) offspring of hyperleptinemic, normoglycemic, Leprdb/+ dams were compared to genotype matched offspring of WT-control dams. Vascular function was assessed in male offspring at 6, and at 31 weeks of age after half the offspring had been fed a high fat, high sucrose diet (HFD) for 6 weeks. Blood pressure was increased by HFD but not affected by maternal hyperleptinemia. On a standard diet, offspring of hyperleptinemic dams had outwardly remodeled mesenteric arteries and an enhanced vasodilatory response to insulin. In offspring of WT but not Leprdb/+ dams, HFD induced vessel hypertrophy and enhanced vasodilatory responses to acetylcholine, while HFD reduced insulin responsiveness in offspring of hyperleptinemic dams. Offspring of hyperleptinemic dams had stiffer arteries regardless of diet. Therefore, while maternal hyperleptinemia was largely beneficial to offspring vascular health under a standard diet, it had detrimental effects in offspring fed HFD. These results suggest that circulating maternal leptin concentrations may interact with other factors in the pre- and post -natal environments to contribute to altered vascular function in offspring of diabetic pregnancies.