Roman Briskine

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula

Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume–rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The population-scaled recombination rate is approximately one-third of the mutation rate, consistent with expectations for a species with a high selfing rate. Linkage disequilibrium, however, is not extensive, and therefore, the low recombination rate is likely not a major constraint to adaptation. Nucleotide diversity in 100-kb windows was negatively correlated with gene density, which is expected if diversity is shaped by selection acting against slightly deleterious mutations. Among putative coding regions, members of four gene families harbor significantly higher diversity than the genome-wide average. Three of these families are involved in resistance against pathogens; one of these families, the nodule-specific, cysteine-rich gene family, is specific to the galegoid legumes and is involved in control of rhizobial differentiation. The more than 3 million SNPs that we detected, approximately one-half of which are present in more than one accession, are a valuable resource for genome-wide association mapping of genes responsible for phenotypic diversity in legumes, especially traits associated with symbiosis and nodulation.

Epigenetic and Genetic Influences on DNA Methylation Variation in Maize Populations

This study examines the DNA methylation patterns of a diverse set of maize lines to show that many methylation variants are associated with local genetic variation, some of which may be due to specific transposon insertion variation. These results provide insight into how DNA methylation varies within a crop and highlight the complex nature of genetic and epigenetic influences on DNA methylation. DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

Maize Gene Atlas Developed by RNA Sequencing and Comparative Evaluation of Transcriptomes Based on RNA Sequencing and Microarrays

Transcriptome analysis is a valuable tool for identification and characterization of genes and pathways underlying plant growth and development. We previously published a microarray-based maize gene atlas from the analysis of 60 unique spatially and temporally separated tissues from 11 maize organs [1]. To enhance the coverage and resolution of the maize gene atlas, we have analyzed 18 selected tissues representing five organs using RNA sequencing (RNA-Seq). For a direct comparison of the two methodologies, the same RNA samples originally used for our microarray-based atlas were evaluated using RNA-Seq. Both technologies produced similar transcriptome profiles as evident from high Pearsons correlation statistics ranging from 0.70 to 0.83, and from nearly identical clustering of the tissues. RNA-Seq provided enhanced coverage of the transcriptome, with 82.1% of the filtered maize genes detected as expressed in at least one tissue by RNA-Seq compared to only 56.5% detected by microarrays. Further, from the set of 465 maize genes that have been historically well characterized by mutant analysis, 427 show significant expression in at least one tissue by RNA-Seq compared to 390 by microarray analysis. RNA-Seq provided higher resolution for identifying tissue-specific expression as well as for distinguishing the expression profiles of closely related paralogs as compared to microarray-derived profiles. Co-expression analysis derived from the microarray and RNA-Seq data revealed that broadly similar networks result from both platforms, and that co-expression estimates are stable even when constructed from mixed data including both RNA-Seq and microarray expression data. The RNA-Seq information provides a useful complement to the microarray-based maize gene atlas and helps to further understand the dynamics of transcription during maize development.

Candidate genes and genetic architecture of symbiotic and agronomic traits revealed by whole-genome, sequence-based association genetics in Medicago truncatula.

Genome-wide association study (GWAS) has revolutionized the search for the genetic basis of complex traits. To date, GWAS have generally relied on relatively sparse sampling of nucleotide diversity, which is likely to bias results by preferentially sampling high-frequency SNPs not in complete linkage disequilibrium (LD) with causative SNPs. To avoid these limitations we conducted GWAS with >6 million SNPs identified by sequencing the genomes of 226 accessions of the model legume Medicago truncatula. We used these data to identify candidate genes and the genetic architecture underlying phenotypic variation in plant height, trichome density, flowering time, and nodulation. The characteristics of candidate SNPs differed among traits, with candidates for flowering time and trichome density in distinct clusters of high linkage disequilibrium (LD) and the minor allele frequencies (MAF) of candidates underlying variation in flowering time and height significantly greater than MAF of candidates underlying variation in other traits. Candidate SNPs tagged several characterized genes including nodulation related genes SERK2, MtnodGRP3, MtMMPL1, NFP, CaML3, MtnodGRP3A and flowering time gene MtFD as well as uncharacterized genes that become candidates for further molecular characterization. By comparing sequence-based candidates to candidates identified by in silico 250K SNP arrays, we provide an empirical example of how reliance on even high-density reduced representation genomic makers can bias GWAS results. Depending on the trait, only 30–70% of the top 20 in silico array candidates were within 1 kb of sequence-based candidates. Moreover, the sequence-based candidates tagged by array candidates were heavily biased towards common variants; these comparisons underscore the need for caution when interpreting results from GWAS conducted with sparsely covered genomes.

Genomic Signature of Adaptation to Climate in Medicago truncatula

Local adaptation and adaptive clines are pervasive in natural plant populations, yet the effects of these types of adaptation on genomic diversity are not well understood. With a data set of 202 accessions of Medicago truncatula genotyped at almost 2 million single nucleotide polymorphisms, we used mixed linear models to identify candidate loci responsible for adaptation to three climatic gradients—annual mean temperature (AMT), precipitation in the wettest month (PWM), and isothermality (ITH)—representing the major axes of climate variation across the species’ range. Loci with the strongest association to these climate gradients tagged genome regions with high sequence similarity to genes with functional roles in thermal tolerance, drought tolerance, or resistance to herbivores of pathogens. Genotypes at these candidate loci also predicted the performance of an independent sample of plant accessions grown in climate-controlled conditions. Compared to a genome-wide sample of randomly drawn reference SNPs, candidates for two climate gradients, AMT and PWM, were significantly enriched for genic regions, and genome segments flanking genic AMT and PWM candidates harbored less nucleotide diversity, elevated differentiation between haplotypes carrying alternate alleles, and an overrepresentation of the most common haplotypes. These patterns of diversity are consistent with a history of soft selective sweeps acting on loci underlying adaptation to climate, but not with a history of long-term balancing selection.

Reshaping of the maize transcriptome by domestication

Through domestication, humans have substantially altered the morphology of Zea mays ssp. parviglumis (teosinte) into the currently recognizable maize. This system serves as a model for studying adaptation, genome evolution, and the genetics and evolution of complex traits. To examine how domestication has reshaped the transcriptome of maize seedlings, we used expression profiling of 18,242 genes for 38 diverse maize genotypes and 24 teosinte genotypes. We detected evidence for more than 600 genes having significantly different expression levels in maize compared with teosinte. Moreover, more than 1,100 genes showed significantly altered coexpression profiles, reflective of substantial rewiring of the transcriptome since domestication. The genes with altered expression show a significant enrichment for genes previously identified through population genetic analyses as likely targets of selection during maize domestication and improvement; 46 genes previously identified as putative targets of selection also exhibit altered expression levels and coexpression relationships. We also identified 45 genes with altered, primarily higher, expression in inbred relative to outcrossed teosinte. These genes are enriched for functions related to biotic stress and may reflect responses to the effects of inbreeding. This study not only documents alterations in the maize transcriptome following domestication, identifying several genes that may have contributed to the evolution of maize, but highlights the complementary information that can be gained by combining gene expression with population genetic analyses.

Population Genomics of the Facultatively Mutualistic Bacteria Sinorhizobium meliloti and S. medicae

The symbiosis between rhizobial bacteria and legume plants has served as a model for investigating the genetics of nitrogen fixation and the evolution of facultative mutualism. We used deep sequence coverage (>100×) to characterize genomic diversity at the nucleotide level among 12 Sinorhizobium medicae and 32 S. meliloti strains. Although these species are closely related and share host plants, based on the ratio of shared polymorphisms to fixed differences we found that horizontal gene transfer (HGT) between these species was confined almost exclusively to plasmid genes. Three multi-genic regions that show the strongest evidence of HGT harbor genes directly involved in establishing or maintaining the mutualism with host plants. In both species, nucleotide diversity is 1.5–2.5 times greater on the plasmids than chromosomes. Interestingly, nucleotide diversity in S. meliloti but not S. medicae is highly structured along the chromosome – with mean diversity (θπ) on one half of the chromosome five times greater than mean diversity on the other half. Based on the ratio of plasmid to chromosome diversity, this appears to be due to severely reduced diversity on the chromosome half with less diversity, which is consistent with extensive hitchhiking along with a selective sweep. Frequency-spectrum based tests identified 82 genes with a signature of adaptive evolution in one species or another but none of the genes were identified in both species. Based upon available functional information, several genes identified as targets of selection are likely to alter the symbiosis with the host plant, making them attractive targets for further functional characterization.

Genomic Distribution of Maize Facultative Heterochromatin Marked by Trimethylation of H3K27

Chromatin modifications contribute to the regulation of gene expression. The genome-wide distribution of a specific chromatin modification, trimethylation of Lys-27 of histone H3, was profiled in five tissues of maize. There is evidence that this chromatin modification plays an important role in regulating tissue-specific expression for a number of maize genes, including many transcription factors and imprinted genes. Trimethylation of histone H3 Lys-27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize (Zea mays) plants of two genetic backgrounds, B73 and Mo17. There were more substantial differences in the genome-wide profile of H3K27me3 between different tissues than between the two genotypes. The tissue-specific patterns of H3K27me3 were often associated with differences in gene expression among the tissues and most of the imprinted genes that are expressed solely from the paternal allele in endosperm are targets of H3K27me3. A comparison of the H3K27me3 targets in rice (Oryza sativa), maize, and Arabidopsis thaliana provided evidence for conservation of the H3K27me3 targets among plant species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived from whole-genome duplication, suggesting the potential for subfunctionalization of chromatin regulation of paralogs. Genomic profiling of H3K27me3 in loss-of-function mutant lines for Maize Enhancer of zeste-like2 (Mez2) and Mez3, two of the three putative H3K27me3 methyltransferases present in the maize genome, suggested partial redundancy of this gene family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 required Mez2 and/or Mez3, and there was limited evidence for functional consequences of H3K27me3 at these targets.

Fine-scale population recombination rates, hotspots, and correlates of recombination in the Medicago truncatula genome

Recombination rates vary across the genome and in many species show significant relationships with several genomic features, including distance to the centromere, gene density, and GC content. Studies of fine-scale recombination rates have also revealed that in several species, there are recombination hotspots, that is, short regions with recombination rates 10–100 greater than those in surrounding regions. In this study, we analyzed whole-genome resequence data from 26 accessions of the model legume Medicago truncatula to gain insight into the genomic features that are related to high- and low-recombination rates and recombination hotspots at 1 kb scales. We found that high-recombination regions (1-kb windows among those in the highest 5% of the distribution) on all three chromosomes were significantly closer to the centromere, had higher gene density, and lower GC content than low-recombination windows. High-recombination windows are also significantly overrepresented among some gene functional categories—most strongly NB–ARC and LRR genes, both of which are important in plant defense against pathogens. Similar to high-recombination windows, recombination hotspots (1-kb windows with significantly higher recombination than the surrounding region) are significantly nearer to the centromere than nonhotspot windows. By contrast, we detected no difference in gene density or GC content between hotspot and nonhotspot windows. Using linear model wavelet analysis to examine the relationship between recombination and genomic features across multiple spatial scales, we find a significant negative correlation with distance to the centromere across scales up to 512 kb, whereas gene density and GC content show significantly positive and negative correlations, respectively, only up to 64 kb. Correlations between recombination and genomic features, particularly gene density and polymorphism, suggest that they are scale dependent and need to be assessed at scales relevant to the evolution of those features.

Phylogenetic Signal Variation in the Genomes of Medicago (Fabaceae)

Genome-scale data offer the opportunity to clarify phylogenetic relationships that are difficult to resolve with few loci, but they can also identify genomic regions with evolutionary history distinct from that of the species history. We collected whole-genome sequence data from 29 taxa in the legume genus Medicago, then aligned these sequences to the Medicago truncatula reference genome to confidently identify 87 596 variable homologous sites. We used this data set to estimate phylogenetic relationships among Medicago species, to investigate the number of sites needed to provide robust phylogenetic estimates and to identify specific genomic regions supporting topologies in conflict with the genome-wide phylogeny. Our full genomic data set resolves relationships within the genus that were previously intractable. Subsampling the data reveals considerable variation in phylogenetic signal and power in smaller subsets of the data. Even when sampling 5000 sites, no random sample of the data supports a topology identical to that of the genome-wide phylogeny. Phylogenetic relationships estimated from 500-site sliding windows revealed genome regions supporting several alternative species relationships among recently diverged taxa, consistent with the expected effects of deep coalescence or introgression in the recent history of Medicago.