Cory D. Hirsch

RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

Significance RNA-directed DNA methylation (RdDM) provides a system for targeting DNA methylation to asymmetric CHH (H = A, C, or T) sites. This RdDM activity is often considered a mechanism for transcriptional silencing of transposons. However, many of the RdDM targets in the maize genome are located near genes or regulatory elements. We find that the regions of elevated CHH methylation, termed mCHH islands, are the boundaries between highly methylated (CG, CHG), silenced chromatin and more active chromatin. Analysis of RdDM mutants suggests that the function of the boundary is to promote and reinforce silencing of the transposable elements located near genes rather than to protect the euchromatic state of the genes. The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.

Spud DB: A resource for mining sequences, genotypes, and phenotypes to accelerate potato breeding

Potato is the worlds third most important crop, and is becoming increasingly important in developing countries. Cultivated potato is a highly heterozygous tetraploid (2n = 4x = 48) and suffers from significant inbreeding depression when selfed. As potato can be vegetatively propagated, breeding has been based primarily on phenotypic selection in F1 populations. However, recent advances in genome sequencing and genotyping methods have resulted in the development of large genomic, genetic, and phenotypic datasets that will enable more efficient and rapid breeding approaches. We have developed Spud DB (http://potato.plantbiology.msu.edu/) for the community to access the potato genome sequence and associated annotation datasets, along with phenotypic and genotypic data from a diversity panel of 250 potato clones. The Breeders Assistant is a web tool to retrieve pertinent phenotypic and genotypic data in a user‐guided manner, and query polymorphic markers such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) to identify custom sets of markers for a gene or region of interest. To browse and query the potato genome, a genome browser with 94 tracks of genome annotation, sequence variants, and expression abundance has been deployed. Spud DB also provides a comprehensive search page to data mine the potato genome through tools that query sequence identifiers, functional annotation, gene ontology (GO), InterPro domains, and basic local alignment search tool (BLAST) databases. Collectively, this resource links potato genomic data with phenotypic and genotypic data from a large collection of potato lines for use by the potato community, especially breeders and geneticists.

Lineage-Specific Adaptive Evolution of the Centromeric Protein CENH3 in Diploid and Allotetraploid Oryza Species

Centromeres in eukaryotic species are defined by the presence of a centromere-specific histone H3 variant, CENH3. CENH3 plays a key role in recruiting other centromeric proteins; thus, it is the central component in kinetochore formation and centromere function. The CENH3 proteins in several plant and animal species were found to be under positive selection, which was hypothesized to respond to the rapid changing of the repetitive DNA sequences associated with the centromeres. Here, we report the expression and evolution of the CenH3 genes in two allotetraploid rice species as well as their representative diploid progenitor species. Both copies of the CenH3 genes were transcribed in the two allotetraploid species and showed a nonpreferential expression pattern. Contrasting positive and stabilizing selection of the CenH3 genes was associated with different diploid Oryza species. This lineage-specific adaptive evolution of CENH3 was maintained in the two allotetraploid species. Thus, we demonstrate that the allopolyploidization events did not alter the expression or evolutionary patterns of the CenH3 genes in the Oryza species.

Organization and evolution of subtelomeric satellite repeats in the potato genome.

Subtelomeric domains immediately adjacent to telomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes. A common feature associated with subtelomeric regions in different eukaryotes is the presence of long arrays of tandemly repeated satellite sequences. However, studies on molecular organization and evolution of subtelomeric repeats are rare. We isolated two subtelomeric repeats, CL14 and CL34, from potato (Solanum tuberosum). The CL14 and CL34 repeats are organized as independent long arrays, up to 1-3 Mb, of 182 bp and 339 bp monomers, respectively. The CL14 and CL34 repeat arrays are directly connected with the telomeric repeats at some chromosomal ends. The CL14 repeat was detected at the subtelomeric regions among highly diverged Solanum species, including tomato (Solanum lycopersicum). In contrast, CL34 was only found in potato and its closely related species. Interestingly, the CL34 repeat array was always proximal to the telomeres when both CL14 and CL34 were found at the same chromosomal end. In addition, the CL34 repeat family showed more sequence variability among monomers compared with the CL14 repeat family. We conclude that the CL34 repeat family emerged recently from the subtelomeric regions of potato chromosomes and is rapidly evolving. These results provide further evidence that subtelomeric domains are among the most dynamic regions in eukaryotic genomes.

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

Comparative analyses of the maize reference B73 genome assembly and the newly assembled PH207 genome and their transcriptomes provide insights into variation between heterotic groups of elite maize. Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.

Transposable element influences on gene expression in plants

Transposable elements (TEs) comprise a major portion of many plant genomes and bursts of TE movements cause novel genomic variation within species. In order to maintain proper gene function, plant genomes have evolved a variety of mechanisms to tolerate the presence of TEs within or near genes. Here, we review our understanding of the interactions between TEs and gene expression in plants by assessing three ways that transposons can influence gene expression. First, there is growing evidence that TE insertions within introns or untranslated regions of genes are often tolerated and have minimal impact on expression level or splicing. However, there are examples in which TE insertions within genes can result in aberrant or novel transcripts. Second, TEs can provide novel alternative promoters, which can lead to new expression patterns or original coding potential of an alternate transcript. Third, TE insertions near genes can influence regulation of gene expression through a variety of mechanisms. For example, TEs may provide novel cis-acting regulatory sites behaving as enhancers or insert within existing enhancers to influence transcript production. Alternatively, TEs may change chromatin modifications in regions near genes, which in turn can influence gene expression levels. Together, the interactions of genes and TEs provide abundant evidence for the role of TEs in changing basic functions within plant genomes beyond acting as latent genomic elements or as simple insertional mutagens. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Conservation and Purifying Selection of Transcribed Genes Located in a Rice Centromere

Comparative DNA sequence analysis of orthologous centromeres from three rice species revealed the presence of seven conserved genes. Surprisingly, all seven genes are under strong purifying selection despite being harbored in a region that is free of detectable chromosomal exchanges (crossing-over), a phenomenon suggestive of strong functional constraints on these genes. Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8 genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements.

Reduced representation approaches to interrogate genome diversity in large repetitive plant genomes

Technology and software improvements in the last decade now provide methodologies to access the genome sequence of not only a single accession, but also multiple accessions of plant species. This provides a means to interrogate species diversity at the genome level. Ample diversity among accessions in a collection of species can be found, including single-nucleotide polymorphisms, insertions and deletions, copy number variation and presence/absence variation. For species with small, non-repetitive rich genomes, re-sequencing of query accessions is robust, highly informative, and economically feasible. However, for species with moderate to large sized repetitive-rich genomes, technical and economic barriers prevent en masse genome re-sequencing of accessions. Multiple approaches to access a focused subset of loci in species with larger genomes have been developed, including reduced representation sequencing, exome capture and transcriptome sequencing. Collectively, these approaches have enabled interrogation of diversity on a genome scale for large plant genomes, including crop species important to worldwide food security.

Structure and evolution of plant centromeres.

Investigations of centromeric DNA and proteins and centromere structures in plants have lagged behind those conducted with yeasts and animals; however, many attractive results have been obtained from plants during this decade. In particular, intensive investigations have been conducted in Arabidopsis and Gramineae species. We will review our understanding of centromeric components, centromere structures, and the evolution of these attributes of centromeres among plants using data mainly from Arabidopsis and Gramineae species.

Centromeres: Sequences, Structure, and Biology

Although technological advances have continued to change the speed, cost, and number of plant genomes sequenced (see Flagel and Blackman 2012, this volume), parts of genomes remain to be sequenced and explored. Even the best-sequenced plant genomes, including Arabidopsis thaliana and rice, are missing 7–8% of their total genomic information (Kaul et al. 2000; Goff et al. 2002; Yu et al. 2002). One chromosomal region not often sequenced in genome projects is the centromere. Centromeres of almost all higher eukaryotes contain large stretches (up to several megabases) of tandemly repeated arrays of satellite DNA and retrotransposons. Such long arrays of highly homogenized repetitive DNA sequences cannot readily be cloned, sequenced, and assembled using the currently available cloning and sequencing technologies.