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Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.

Approximately half of patients with cancer receive radiotherapy and, as cancer survivorship increases, the low rate of radiation-associated sarcomas is rising. Pharmacologic inhibition of p53 has been proposed as an approach to ameliorate acute injury of normal tissues from genotoxic therapies, but how this might impact the risk of therapy-induced cancer and normal tissue injuries remains unclear. We utilized mice that express a doxycycline (dox)-inducible p53 short hairpin RNA to reduce Trp53 expression temporarily during irradiation. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were subjected to RNA sequencing. Following single high-dose irradiation, 21% of animals with temporary p53 knockdown during irradiation developed a sarcoma in the radiation field compared with 2% of control animals. Following high-dose irradiation, p53 knockdown preserves muscle stem cells, and increases sarcoma development. Mice with severe acute radiation-induced injuries exhibit an increased risk of developing late persistent wounds, which were associated with sarcomagenesis. RNA sequencing revealed radiation-induced sarcomas upregulate genes related to translation, epithelial-mesenchymal transition (EMT), inflammation, and the cell cycle. Comparison of the transcriptomes of human and mouse sarcomas that arose in irradiated tissues revealed regulation of common gene programs, including elevated EMT pathway gene expression. These results suggest that blocking p53 during radiotherapy could minimize acute toxicity while exacerbating late effects including second cancers., Significance: Strategies to prevent or mitigate acute radiation toxicities include pharmacologic inhibition of p53 and other cell death pathways. Our data show that temporarily reducing p53 during irradiation increases late effects including sarcomagenesis., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Here, we present the draft genome sequences of Lactobacillus delbrueckii and Klebsiella pneumoniae , both isolated from the urinary bladder of an asymptomatic post-menopausal female patient with a diagnosis of recurrent urinary tract infections. These genomes will facilitate analyses of interbacterial interactions in the urinary microbiome., Competing Interests: The authors declare no conflict of interest.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Data regarding the microbiome of the gestational membranes are emerging and conflicting. Shifts in the microbial communities in the setting of labor, rupture of membranes, and intraamniotic infection are yet to be understood., Objective: This study aimed to characterize the microbiome of the gestational membranes of women in labor or with ruptured membranes, including those with and without intraamniotic infection., Study Design: Women with a singleton pregnancy at ≥28 weeks' gestation undergoing unscheduled cesarean delivery in the setting of labor or rupture of membranes were included. Demographic and clinical variables were collected. We defined suspected intraamniotic infection by standard clinical criteria; placentae and gestational membranes were also reviewed for histologic evidence of infection. Sterile swabs were collected from membranes at the time of delivery. Bacteria were cultured from the swabs, and the isolates were sequenced. DNA extraction and 16S sequencing of the swabs were also performed. Bacterial taxonomy was assigned to each sequence. Alpha diversity indices and beta-diversity metrics were calculated to test for differences in microbial community diversity and composition between uninfected and infected groups. Differential abundance of bacteria between infected and uninfected groups was tested at the class, family, and genus level., Results: Samples were collected from 34 participants. Clinical intraamniotic infection was diagnosed in 38% of participants, although 50% of placentae and membranes demonstrated histologic signs of infection. Of all samples, 68% grew bacteria on culture; this included 62% of the uninfected samples and 77% of the infected samples (P=.83). Multiple measures of alpha diversity were not significantly different between uninfected and infected groups. Similarly, analysis of beta diversity revealed that the microbial community was not significantly different between the uninfected and infected group. Several bacteria traditionally characterized as pathogenic, including Actinomyces and Streptococcus agalactiae, were identified in both infected and uninfected samples., Conclusion: The pathogenesis and clinical implications of intraamniotic infection remain poorly understood. Diverse bacteria are present in both infected and uninfected gestational membranes. A unique microbiologic signature may exist among the gestational membranes following labor or rupture of membranes, and further characterization of the pathogens specifically implicated in intraamniotic infection may allow for targeted therapy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study aims to investigate whether adding neoadjuvant radiotherapy (RT), anti-programmed cell death protein-1 (PD-1) antibody (anti-PD-1), or RT + anti-PD-1 to surgical resection improves disease-free survival for mice with soft tissue sarcomas (STS). We generated a high mutational load primary mouse model of STS by intramuscular injection of adenovirus expressing Cas9 and guide RNA targeting Trp53 and intramuscular injection of 3-methylcholanthrene (MCA) into the gastrocnemius muscle of wild-type mice (p53/MCA model). We randomized tumor-bearing mice to receive isotype control or anti-PD-1 antibody with or without radiotherapy (20 Gy), followed by hind limb amputation. We used micro-CT to detect lung metastases with high spatial resolution, which was confirmed by histology. We investigated whether sarcoma metastasis was regulated by immunosurveillance by lymphocytes or tumor cell-intrinsic mechanisms. Compared with surgery with isotype control antibody, the combination of anti-PD-1, radiotherapy, and surgery improved local recurrence-free survival (P = 0.035) and disease-free survival (P = 0.005), but not metastasis-free survival. Mice treated with radiotherapy, but not anti-PD-1, showed significantly improved local recurrence-free survival and metastasis-free survival over surgery alone (P = 0.043 and P = 0.007, respectively). The overall metastasis rate was low (∼12%) in the p53/MCA sarcoma model, which limited the power to detect further improvement in metastasis-free survival with addition of anti-PD-1 therapy. Tail vein injections of sarcoma cells into immunocompetent mice suggested that impaired metastasis was due to inability of sarcoma cells to grow in the lungs rather than a consequence of immunosurveillance. In conclusion, neoadjuvant radiotherapy improves metastasis-free survival after surgery in a primary model of STS., (©2022 American Association for Cancer Research.)

Background: Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable., Results: Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers., Conclusions: This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a "washout" period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly. These results support that paternal preconception exposure to cannabis can influence offspring outcomes., (© 2022. The Author(s).)

Despite being in a golden age of bacterial epigenomics, little work has systematically examined the plasticity and functional impacts of the bacterial DNA methylome. Here, we leveraged single-molecule, real-time sequencing (SMRT-seq) to examine the m 6 A DNA methylome of two Salmonella enterica serovar Typhimurium strains: 14028s and a Δ metJ mutant with derepressed methionine metabolism, grown in Luria broth or medium that simulates the intracellular environment. We found that the methylome is remarkably static: >95% of adenosine bases retain their methylation status across conditions. Integration of methylation with transcriptomic data revealed limited correlation between changes in methylation and gene expression. Further, examination of the transcriptome in Δ yhdJ bacteria lacking the m 6 A methylase with the most dynamic methylation pattern in our data set revealed little evidence of YhdJ-mediated gene regulation. Curiously, despite G(m 6 A)TC motifs being particularly resistant to change across conditions, incorporating dam mutants into our analyses revealed two examples where changes in methylation and transcription may be linked across conditions. This includes the novel finding that the Δ metJ motility defect may be partially driven by hypermethylation of the chemotaxis gene tsr . Together, these data redefine the S. Typhimurium epigenome as a highly stable system that has rare but important roles in transcriptional regulation. Incorporating these lessons into future studies will be critical as we progress through the epigenomic era. IMPORTANCE While recent breakthroughs have enabled intense study of bacterial DNA modifications, limitations in current work have potentiated a surprisingly untested narrative that DNA methylation is a common mechanism of the bacterial response to environmental conditions. Essentially, whether epigenetic regulation of bacterial transcription is a common, generalizable phenomenon is a critical unanswered question that we address here. We found that most DNA methylation is static in Salmonella enterica serovar Typhimurium, even when the bacteria are grown under dramatically different conditions that cause broad changes in the transcriptome. Further, even when the methylation of individual bases change, these changes generally do not correlate with changes in gene expression. Finally, we demonstrate methods by which data can be stratified in order to identify coupled changes in methylation and gene expression.

Urinary microbiome composition has been found to associate with health status and to change with age. Lactobacillus gasseri is one of the most frequently found lactic acid bacteria in the vaginal and urinary tracts of women. Here, we report a draft genome sequence of a urinary L. gasseri strain isolated from a healthy postmenopausal woman.

Objective: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing., Background: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes., Methods: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury., Results: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints., Conclusions: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Cannabis use alters sperm DNA methylation, but the potential reversibility of these changes is unknown. Semen samples from cannabis users and non-user controls were collected at baseline and again following a 77-day period of cannabis abstinence (one spermatogenic cycle). Users and controls did not significantly differ by demographics or semen analyses. Whole-genome bisulfite sequencing identified 163 CpG sites with significantly different DNA methylation in sperm between groups ( P  < 2.94 × 10 -9 ). Genes associated with altered CpG sites were enriched with those involved in development, including cardiogenesis and neurodevelopment. Many of the differences in sperm DNA methylation between groups were diminished after cannabis abstinence. These results indicate that sustained cannabis abstinence significantly reduces the number of sperm showing cannabis-associated alterations at genes important for early development., (© The Author(s) 2021. Published by Oxford University Press.)

Rationale & Objective: Patients with CKD are at elevated risk of metabolic acidosis due to impaired net acid excretion (NAE). Identifying early markers of acidosis may guide prevention in chronic kidney disease (CKD). This study compared NAE in participants with and without CKD, as well as the NAE, blood pressure (BP), and metabolomic response to bicarbonate supplementation., Study Design: Randomized order, cross-over study with controlled feeding., Setting & Participants: Participants consisted of 8 patients with CKD (estimated glomerular filtration rate 30-59mL/min/1.73m 2 or 60-70mL/min/1.73m 2 with albuminuria) and 6 patients without CKD. All participants had baseline serum bicarbonate concentrations between 20 and 28 mEq/L; they did not have diabetes mellitus and did not use alkali supplements at baseline., Intervention: Participants were fed a fixed-acid-load diet with bicarbonate supplementation (7 days) and with sodium chloride control (7 days) in a randomized order, cross-over fashion., Outcomes: Urine NAE, 24-hour ambulatory BP, and 24-hour urine and plasma metabolomic profiles were measured after each period., Results: During the control period, mean NAE was 28.3±10.2 mEq/d overall without differences across groups (P=0.5). Urine pH, ammonium, and citrate were significantly lower in CKD than in non-CKD (P<0.05 for each). Bicarbonate supplementation reduced NAE and urine ammonium in the CKD group, increased urine pH in both groups (but more in patients with CKD than in those without), and increased; urine citrate in the CKD group (P< 0.2 for interaction for each). Metabolomic analysis revealed several urine organic anions were increased with bicarbonate in CKD, including 3-indoleacetate, citrate/isocitrate, and glutarate. BP was not significantly changed., Limitations: Small sample size and short feeding duration., Conclusions: Compared to patients without CKD, those with CKD had lower acid excretion in the form of ammonium but also lower base excretion such as citrate and other organic anions, a potential compensation to preserve acid-base homeostasis. In CKD, acid excretion decreased further, but base excretion (eg, citrate) increased in response to alkali. Urine citrate should be evaluated as an early and responsive marker of impaired acid-base homeostasis., Funding: National Institute of Diabetes and Digestive and Kidney Diseases and the Duke O'Brien Center for Kidney Research., Trial Registration: Registered at ClinicalTrials.gov with study number NCT02427594., (Copyright © 2021. Published by Elsevier Inc.)

Several factors in Western society, including widespread use of antibiotics, chronic inflammation, and loss of complex eukaryotic symbionts such as helminths, have a dramatic impact on the ecosystem of the gut, affecting the microbiota hosted there. In addition, reductions in dietary fiber are profoundly impactful on the microbiota, causing extensive destruction of the niche space that supports the normally diverse microbial community in the gut. Abundant evidence now supports the view that, following dramatic alterations in the gut ecosystem, microorganisms undergo rapid change via Darwinian evolution. Such evolutionary change creates functionally distinct bacteria that may potentially have properties of pathogens but yet are difficult to distinguish from their benign predecessors., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Background: APOE4 has been hypothesized to increase Alzheimer's disease risk by increasing neuroinflammation, though the specific neuroinflammatory pathways involved are unclear., Objective: Characterize cerebrospinal fluid (CSF) proteomic changes related to APOE4 copy number., Methods: We analyzed targeted proteomic data from ADNI CSF samples using a linear regression model adjusting for age, sex, and APOE4 copy number, and additional linear models also adjusting for AD clinical status or for CSF Aβ, tau, or p-tau levels. False discovery rate was used to correct for multiple comparisons correction., Results: Increasing APOE4 copy number was associated with a significant decrease in a CRP peptide level across all five models (q < 0.05 for each), and with significant increases in ALDOA, CH3L1 (YKL-40), and FABPH peptide levels (q < 0.05 for each) except when controlling for AD clinical status or neurodegeneration biomarkers (i.e., CSF tau or p-tau). In all models except the one controlling for CSF Aβ levels, though not statistically significant, there was a consistent inverse direction of association between APOE4 copy number and the levels of all 24 peptides from all 8 different complement proteins measured. The odds of this happening by chance for 24 unrelated peptides would be less than 1 in 16 million., Conclusion: Increasing APOE4 copy number was associated with decreased CSF CRP levels across all models, and increased CSF ALDOA, CH3L1, and FABH levels when controlling for CSF Aβ levels. Increased APOE4 copy number may also be associated with decreased CSF complement pathway protein levels, a hypothesis for investigation in future studies.

Invasive aspergillosis (IA) is a major cause of critical illness in immunocompromised (IC) patients. However, current fungal tests are limited. Disease-specific gene expression patterns in circulating host cells show promise as novel diagnostics, however it is unknown whether such a 'signature' exists for IA and the effect of iatrogenic immunosuppression on any such biomarkers. Male BALB/c mice were separated into 6 experimental groups based on Aspergillus fumigatus inhalational exposure and IC status (no immunosuppression, cyclophosphamide, and corticosteroids). Mice were sacrificed 4 days postinfection. Whole blood was assayed for transcriptomic responses in peripheral white blood cells via microarray. An elastic net regularized logistic regression was employed to develop classifiers of IA based on gene expression. Aspergillus infection triggers a powerful response in non-IC hosts with 2718 genes differentially expressed between IA and controls. We generated a 146-gene classifier able to discriminate between non-IC infected and uninfected mice with an AUC of 1. However, immunosuppressive medications exhibited a confounding effect on this transcriptomic classifier. After controlling for the genomic effects of immunosuppression, we were able to generate a 187-gene classifier with an AUC of 0.92 in the absence of immunosuppression, 1 with cyclophosphamide, and 0.9 with steroids. The host transcriptomic response to IA is robust and conserved. Pharmacologic perturbation of the host immune response has powerful effects on classifier performance and must be considered when developing such novel diagnostics. When appropriately designed, host-derived peripheral blood transcriptomic responses demonstrate the ability to accurately diagnose Aspergillus infection, even in the presence of immunosuppression., (Copyright © 2020 Elsevier Inc. All rights reserved.)

For most eukaryotes, sexual reproduction is a fundamental process that requires meiosis. In turn, meiosis typically depends on a reciprocal exchange of DNA between each pair of homologous chromosomes, known as a crossover (CO), to ensure proper chromosome segregation. The frequency and distribution of COs are regulated by intrinsic and extrinsic environmental factors, but much more is known about the molecular mechanisms governing the former compared to the latter. Here we show that elevated temperature induces meiotic hyper-recombination in Arabidopsis thaliana and we use genetic analysis with mutants in different recombination pathways to demonstrate that the extra COs are derived from the major Type I interference sensitive pathway. We also show that heat-induced COs are not the result of an increase in DNA double-strand breaks and that the hyper-recombinant phenotype is likely specific to thermal stress rather than a more generalized stress response. Taken together, these findings provide initial mechanistic insight into how environmental cues modulate plant meiotic recombination and may also offer practical applications., Competing Interests: The authors have declared that no competing interests exist.

A forward genetic screen is one of the best methods for revealing the function of genes. In plants, this technique is highly efficient, as it is relatively easy to grow and screen hundreds or thousands of individuals. The cost efficiency and ease of data production afforded by next-generation sequencing have created new opportunities for rapid mapping of induced mutations. Current mapping tools are often not user friendly, are complicated, or require extensive preparation steps. To simplify the process of mapping new mutations, we developed a pipeline that takes next-generation sequencing fastq files as input, calls on several well-established and freely available genome-analysis tools, and outputs the most likely causal DNA changes. The pipeline has been validated in Arabidopsis thaliana (Arabidopsis) and can be readily applied to other species, with the possibility of mapping either dominant or recessive mutations., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Meiotic recombination ensures the fertility of gametes and creates novel genetic combinations. Although meiotic crossover (CO) frequency is under homeostatic control, CO frequency is also plastic in nature and can respond to environmental conditions. Most investigations have focused on temperature and recombination, but other external and internal stimuli also have important roles in modulating CO frequency. Even less is understood about the molecular mechanisms that underly these phenomenon, but recent work has begun to advance our knowledge in this field. In this review, we identify and explore potential mechanisms including changes in: the synaptonemal complex, chromatin state, DNA methylation, and RNA splicing., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Genetic variation is the fundamental medium of evolution. In allopolyploids, which are the product of hybridization and whole genome duplication, if homologous chromosomes always pair, then all descendants of a single diploid F1 hybrid lineage will be genetically identical. Contrarily, genetic variation among initially isogenic lineages is augmented when homeologous chromosomes pair; this added variation may contribute to phenotypic evolution. Mimulus sookensis is a naturally occurring, small-flowered allotetraploid derived from the large-flowered Mimulus guttatus and small-flowered Mimulus nasutus. Because diploid F1 hybrids between M. guttatus and M. nasutus have large flowers, phenotypic evolution post-polyploidization is implied in M. sookensis. Here, we present genetic and phenotypic analyses of synthetic neoallotetraploid Mimulus derived from a cross between M. guttatus and M. nasutus. Genetic marker data from S2 and BC1N progeny suggest that chromosomes regularly pair with their homologous counterpart. By measuring the phenotype of synthetic neoallotetraploids, we demonstrate that polyploidization per se does not induce the small flowers of M. sookensis. Moreover, phenotypic measurements of synthetic allotetraploid F2s and S4 families suggest that rare homeologous recombination events have a negligible phenotypic effect in the first few generations. In total, the results are consistent with either exceedingly rare homeologous pairing and recombination or spontaneous fragment loss. The low levels of fragment loss and phenotypic variation in neoallotetraploids suggest that homeologous recombination after polyploidization is not a major mechanism of phenotypic evolution in M. sookensis. Rather, it may be that spontaneous mutations or epigenetic changes after allopolyploidization have driven phenotypic evolution in M. sookensis.

Polyploidy (whole-genome duplication) has contributed significantly to angiosperm evolution and diversification. To date, it has been found that most polyploids are the result of multiple formation events, which may contribute to genetic diversity and affect interfertility among polyploid lineages of independent origin. A recently discovered allotetraploid derivative of Mimulus guttatus and M. nasutus, Mimulus sookensis, is found throughout the valleys of western Oregon and Vancouver Island. Here, we analyse the patterns of nucleotide diversity at three chloroplast and six nuclear loci in M. guttatus, M. nasutus and M. sookensis, to gain insight into the formation of M. sookensis. By analysing the patterns of genetic variation seen in the diploid progenitors in comparison with the variation seen in M. sookensis, we are able to show that M. sookensis has recurrently formed. We also observed that most M. sookensis individuals are fixed heterozygotes at all of the nuclear loci examined, suggesting that duplicate gene loss is not extensive in M. sookensis. To assess the possibility that hybridization among M. sookensis has contributed to genetic diversity, we conducted crossing experiments within M. sookensis. We found that M. sookensis of independent origin are highly interfertile, suggesting that crossing barriers do not exist within M. sookensis, and that hybridization among M. sookensis may result in new recombinant genotypes. Together, the data suggest that although recurrent origins may be common, they can contribute to genetic diversity without contributing to reproductive isolation among independently arisen polyploid lineages., (© 2012 Blackwell Publishing Ltd.)

Identifying the genetic basis of parallelism reveals the means by which evolution repeats itself and shows what aspects-if any-may be predictable. The recently tetraploid luteus group of Mimulus contains five species native to central Chile, three of which have evolved extensive red floral pigmentation using at least two distinct loci . Here we show that the parallel evolution of petal lobe anthocyanin (PLA) pigmentation in M. cupreus and M. luteus var. variegatus occurred via separate yet strikingly similar mechanisms. In each case, a dominant, single-locus gain of pigmentation maps to a genomic region (pla1 and pla2, respectively) containing adjacent, apparently recently duplicated paralogs of MYB anthocyanin-regulating transcription factors. Interestingly, candidate genes in pla1 and pla2 are themselves related by an older duplication. In both cases, pla genotype cosegregates with expression of multiple genes in the anthocyanin biosynthetic pathway, revealing a mechanism of coordinated trans-regulatory expression changes across functionally related enzyme-encoding genes. We conclude that in this instance, evolution has repeated itself with marked consistency. Duplication has enabled that repetition to occur using two physically independent but functionally similar loci, highlighting the importance of genomic complexity to the evolutionary process., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Speciation is characterized by the evolution of reproductive isolation between two groups of organisms. Understanding the process of speciation requires the quantification of barriers to reproductive isolation, dissection of the genetic mechanisms that contribute to those barriers and determination of the forces driving the evolution of those barriers. Through a comprehensive analysis involving 19 pairs of plant taxa, we assessed the strength and patterns of asymmetry of multiple prezygotic and postzygotic reproductive isolating barriers. We then reviewed contemporary knowledge of the genetic architecture of reproductive isolation and the relative role of chromosomal and genic factors in intrinsic postzygotic isolation. On average, we found that prezygotic isolation is approximately twice as strong as postzygotic isolation, and that postmating barriers are approximately three times more asymmetrical in their action than premating barriers. Barriers involve a variable number of loci, and chromosomal rearrangements may have a limited direct role in reproductive isolation in plants. Future research should aim to understand the relationship between particular genetic loci and the magnitude of their effect on reproductive isolation in nature, the geographical scale at which plant speciation occurs, and the role of different evolutionary forces in the speciation process.

Knowledge regarding the origin and maintenance of hybrid zones is critical for understanding the evolutionary outcomes of natural hybridization. To evaluate the contribution of historical contact vs. long-distance gene flow in the formation of a broad hybrid zone in central and northern Georgia that involves Aesculus pavia, A. sylvatica, and A. flava, three cpDNA regions (matK, trnD-trnT, and trnH-trnK) were analyzed. The maternal inheritance of cpDNA in Aesculus was confirmed via sequencing of matK from progeny of controlled crosses. Restriction site analyses identified 21 unique haplotypes among 248 individuals representing 29 populations from parental species and hybrids. Haplotypes were sequenced for all cpDNA regions. Restriction site and sequence data were subjected to phylogeographic and population genetic analyses. Considerable cpDNA variation was detected in the hybrid zone, as well as ancestral cpDNA polymorphism; furthermore, the distribution of haplotypes indicates limited interpopulation gene flow via seeds. The genealogy and structure of genetic variation further support the historical presence of A. pavia in the Piedmont, although they are at present locally extinct. In conjunction with previous allozyme studies, the cpDNA data suggest that the hybrid zone originated through historical local gene flow, yet is maintained by periodic long-distance pollen dispersal.

Variation in inflorescence development patterns is a central factor in the evolutionary ecology of plants. The genetic architectures of 13 traits associated with inflorescence developmental timing, architecture, rosette morphology, and fitness were investigated in Arabidopsis thaliana, a model plant system. There is substantial naturally occurring genetic variation for inflorescence development traits, with broad sense heritabilities computed from 21 Arabidopsis ecotypes ranging from 0.134 to 0.772. Genetic correlations are significant for most (64/78) pairs of traits, suggesting either pleiotropy or tight linkage among loci. Quantitative trait locus (QTL) mapping indicates 47 and 63 QTL for inflorescence developmental traits in Ler x Col and Cvi x Ler recombinant inbred mapping populations, respectively. Several QTL associated with different developmental traits map to the same Arabidopsis chromosomal regions, in agreement with the strong genetic correlations observed. Epistasis among QTL was observed only in the Cvi x Ler population, and only between regions on chromosomes 1 and 5. Examination of the completed Arabidopsis genome sequence in three QTL regions revealed between 375 and 783 genes per region. Previously identified flowering time, inflorescence architecture, floral meristem identity, and hormone signaling genes represent some of the many candidate genes in these regions.