Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratoryprepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in .10 5 -fold amplification of the target genomes with ,6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. C LASSICAL population genetics, coupled with advances in coalescent modeling, has been foundational to studies of the evolutionary histories and ecological forces that shape natural populations (Rosenberg and Nordborg 2002;Hume et al. 2003;Wakeley 2004). However, detecting fine scale processes using population genetics and coalescent analyses is limited by the amount of available sequence data per sample. Datasets with substantially greater genetic information per sample, such as genomic data from population-level sampling, would be optimal to study biological processes at all relevant scales. The promise of population genomics for many microbial species is tempered, however, by the difficulty of isolating and preparing microbial genomes for nextgeneration sequencing. Currently, sequencing microbial genomes requires laboratory culture to isolate them from other organisms with which they are naturally associated to obtain the appropriate samples for sequencing-sufficient numbers of the target genome with limited contaminating DNA (Mardis 2008).Methodological issues in obtaining populations of genomes from microbial species occur both because the target microbial genomes often constitutes only a miniscule fraction of the DNA in complex, field-derived samples and because many important microbial specie...
Vegetative phase change in is mediated by a decrease in the level of and , resulting in an increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Changes in chromatin structure are required for the downregulation of and , but whether chromatin structure contributes to their initial elevated expression is unknown. We found that mutations in components of the SWR1 complex (, ) and in genes encoding H2A.Z ( and ) reduce the expression of and , and accelerate vegetative phase change, indicating that H2A.Z promotes juvenile vegetative identity. However, and did not accelerate the temporal decline in miR156, and the downregulation of and was not accompanied by significant change in the level of H2A.Z at these loci. We conclude that H2A.Z contributes to the high expression of early in shoot development, but does not regulate the timing of vegetative phase change. Our results also suggest that H2A.Z promotes the expression of by facilitating the deposition of H3K4me3, rather than by decreasing nucleosome occupancy.
Phenotypic plasticity--the capacity of a single genotype to produce different phenotypes in response to varying environmental conditions--is widespread. Yet, whether, and how, plasticity impacts evolutionary diversification is unclear. According to a widely discussed hypothesis, plasticity promotes rapid evolution because genes expressed differentially across different environments (i.e., genes with "biased" expression) experience relaxed genetic constraint and thereby accumulate variation faster than do genes with unbiased expression. Indeed, empirical studies confirm that biased genes evolve faster than unbiased genes in the same genome. An alternative hypothesis holds, however, that the relaxed constraint and faster evolutionary rates of biased genes may be a precondition for, rather than a consequence of, plasticity's evolution. Here, we evaluated these alternative hypotheses by characterizing evolutionary rates of biased and unbiased genes in two species of frogs that exhibit a striking form of phenotypic plasticity. We also characterized orthologs of these genes in four species of frogs that had diverged from the two plastic species before the plasticity evolved. We found that the faster evolutionary rates of biased genes predated the evolution of the plasticity. Furthermore, biased genes showed greater expression variance than did unbiased genes, suggesting that they may be more dispensable. Phenotypic plasticity may therefore evolve when dispensable genes are co-opted for novel function in environmentally induced phenotypes. Thus, relaxed genetic constraint may be a cause--not a consequence--of the evolution of phenotypic plasticity, and thereby contribute to the evolution of novel traits.
Age-dependent changes in plant defense against herbivores are widespread, but why these changes exist remains a mystery. We explored this question by examining a suite of traits required for the interaction between swollen thorn acacias (genus Vachellia) and ants of the genus Pseudomyrmex. In this system, plants provide ants with refuge and food in the form of swollen stipular spines, protein-lipid–rich “Beltian” bodies, and sugar-secreting extrafloral nectaries—the “swollen thorn syndrome.” We show that this syndrome develops at a predictable time in shoot development and is tightly associated with the temporal decline in the microRNAs miR156 and miR157 and a corresponding increase in their targets—the SPL transcription factors. Growth under reduced light intensity delays both the decline in miR156/157 and the development of the swollen thorn syndrome, supporting the conclusion that these traits are controlled by the miR156-SPL pathway. Production of extrafloral nectaries by Vachellia sp. that do not house ants is also correlated with a decline in miR156/157, suggesting that this syndrome evolved by co-opting a preexisting age-dependent program. Along with genetic evidence from other model systems, these findings support the hypothesis that the age-dependent development of the swollen thorn syndrome is a consequence of genetic regulation rather than a passive developmental pattern arising from developmental constraints on when these traits can develop.
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