Transcribed microsatellite allele lengths are often correlated with gene expression in natural sunflower populations.

Microsatellites are common in genomes of most eukaryotic species. Due to their high mutability, an adaptive role for microsatellites has been considered. However, little is known concerning the contribution of microsatellites towards phenotypic variation. We used populations of the common sunflower (Helianthus annuus) at two latitudes to quantify the effect of microsatellite allele length on phenotype at the level of gene expression. We conducted a common garden experiment with seed collected from sunflower populations in Kansas and Oklahoma followed by an RNA-Seq experiment on 95 individuals. The effect of microsatellite allele length on gene expression was assessed across 3,325 microsatellites that could be consistently scored. Our study revealed 479 microsatellites at which allele length significantly correlates with gene expression (eSTRs). When irregular allele sizes not conforming to the motif length were removed, the number of eSTRs rose to 2,379. The percentage of variation in gene expression explained by eSTRs ranged from 1%-86% when controlling for population and allele-by-population interaction effects at the 479 eSTRs. Of these eSTRs, 70.4% are in untranslated regions (UTRs). A gene ontology (GO) analysis revealed that eSTRs are significantly enriched for GO terms associated with cis- and trans-regulatory processes. Our findings suggest that a substantial number of transcribed microsatellites can influence gene expression.

Transcriptome profiles of sunflower reveal the potential role of microsatellites in gene expression divergence.

The mechanisms by which natural populations generate adaptive genetic variation are not well understood. Some studies propose that microsatellites can function as drivers of adaptive variation. Here, we tested a potentially adaptive role for transcribed microsatellites with natural populations of the common sunflower (Helianthus annuus L.) by assessing the enrichment of microsatellites in genes that show expression divergence across latitudes. Seeds collected from six populations at two distinct latitudes in Kansas and Oklahoma were planted and grown in a common garden. Morphological measurements from the common garden demonstrated that phenotypic variation among populations is largely explained by underlying genetic variation. An RNA-Seq experiment was conducted with 96 of the individuals grown in the common garden and differentially expressed (DE) transcripts between the two latitudes were identified. A total number of 825 DE transcripts were identified. DE transcripts and nondifferentially expressed (NDE) transcripts were then scanned for microsatellites. The abundance of different motif lengths and types in both groups were estimated. Our results indicate that DE transcripts are significantly enriched with mononucleotide repeats and significantly depauperate in trinucleotide repeats. Further, the standardized mononucleotide repeat motif A and dinucleotide repeat motif AG were significantly enriched within DE transcripts while motif types, C, AT, ACC and AAC in DE transcripts, are significantly differentiated in microsatellite tract length between the two latitudes. The tract length differentiation at specific microsatellite motif types across latitudes and their enrichment within DE transcripts indicate a potential functional role for transcribed microsatellites in gene expression divergence in sunflower.

Phylogeography and genetic structure of endemic Acmispon argophyllus and A. dendroideus (Fabaceae) across the California Channel Islands.

PREMISE OF THE STUDY: Taxa inhabiting the California Channel Islands exhibit variation in their degree of isolation, but few studies have considered patterns across the entire archipelago. We studied phylogeography of insular Acmispon argophyllus and A. dendroideus to determine whether infraspecific taxa are genetically divergent and to elucidate patterns of diversification across these islands. METHODS: DNA sequences were collected from nuclear (ADH) and plastid genomes (rpL16, ndhA, psbD-trnT) from >450 samples on the Channel Islands and California. We estimated population genetic diversity and structure, phylogenetic patterns among populations, and migration rates, and tested for population growth. KEY RESULTS: Populations of northern island A. argophyllus var. niveus are genetically distinct from conspecific populations on southern islands. On the southern islands, A. argophyllus var. argenteus populations on Santa Catalina are phylogenetically distinct from populations of var. argenteus and var. adsurgens on the other southern islands. For A. dendroideus, we found the varieties to be monophyletic. Populations of A. dendroideus var. traskiae on San Clemente are genetically differentiated from other conspecific populations, whereas populations on the northern islands and Santa Catalina show varying degrees of gene flow. Evidence of population growth was found in both species. CONCLUSIONS: Oceanic barriers between islands have had a strong influence on population genetic structure in both Acmispon species, although the species have differing phylogeographic patterns. This study provides a contrasting pattern of dispersal on a near island system that does not follow a strict stepping-stone model, commonly found on isolated island systems.

CRISPR-Cas9 homology-independent targeted integration of exons 1-19 restores full-length dystrophin in mice.

Duchenne muscular dystrophy is an X-linked disorder typically caused by out-of-frame mutations in the DMD gene. Most of these are deletions of one or more exons, which can theoretically be corrected through CRISPR-Cas9-mediated knockin. Homology-independent targeted integration is a mechanism for achieving such a knockin without reliance on homology-directed repair pathways, which are inactive in muscle. We designed a system based on insertion into intron 19 of a DNA fragment containing a pre-spliced mega-exon encoding DMD exons 1-19, along with the MHCK7 promoter, and delivered it via a pair of AAV9 vectors in mice carrying a Dmd exon 2 duplication. Maximal efficiency was achieved using a Cas9:donor adeno-associated virus (AAV) ratio of 1:5, with Cas9 under the control of the SPc5-12 promoter. This approach achieved editing of 1.4% of genomes in the heart, leading to 30% correction at the transcript level and restoration of 11% of normal dystrophin levels. Treatment efficacy was lower in skeletal muscles. Sequencing additionally revealed integration of fragmentary and recombined AAV genomes at the target site. These data provide proof of concept for a gene editing system that could restore full-length dystrophin in individuals carrying mutations upstream of intron 19, accounting for approximately 25% of Duchenne muscular dystrophy patients.

Multiomics in primary and metastatic breast tumors from the AURORA US network finds microenvironment and epigenetic drivers of metastasis.

The AURORA US Metastasis Project was established with the goal to identify molecular features associated with metastasis. We assayed 55 females with metastatic breast cancer (51 primary cancers and 102 metastases) by RNA sequencing, tumor/germline DNA exome and low-pass whole-genome sequencing and global DNA methylation microarrays. Expression subtype changes were observed in ~30% of samples and were coincident with DNA clonality shifts, especially involving HER2. Downregulation of estrogen receptor (ER)-mediated cell-cell adhesion genes through DNA methylation mechanisms was observed in metastases. Microenvironment differences varied according to tumor subtype; the ER+/luminal subtype had lower fibroblast and endothelial content, while triple-negative breast cancer/basal metastases showed a decrease in B and T cells. In 17% of metastases, DNA hypermethylation and/or focal deletions were identified near HLA-A and were associated with reduced expression and lower immune cell infiltrates, especially in brain and liver metastases. These findings could have implications for treating individuals with metastatic breast cancer with immune- and HER2-targeting therapies.

Variable length chloroplast markers for population genetic studies in Acmispon (Fabaceae).

PREMISE OF THE STUDY: To estimate genetic structure, chloroplast loci containing length-variable regions were developed for two legumes, Acmispon argophyllus and A. dendroideus. • METHODS AND RESULTS: Primers for 14 chloroplast loci containing repeat regions were developed from the chloroplast genome sequence of the legume Lotus japonicus and tested in Acmispon. Nine loci exhibited polymorphism in Acmispon, with up to six alleles per locus. Gene diversity ranged from 0 to 0.775 in A. argophyllus and 0.142 to 0.766 in A. dendroideus. The primers also amplified in other Acmispon species. Sequencing of the fragments revealed discordance between fragment sizes and underlying sequence for three loci containing complex repeat regions. • CONCLUSIONS: Although genotypes were easily generated and sized, sequencing may be more informative of genetic variation in loci with complex repeat regions. These loci exhibit substantial variation and should be useful for understanding genetic structure associated with seed dispersal in Acmispon.

Evaluating the adaptive evolutionary convergence of carnivorous plant taxa through functional genomics.

Carnivorous plants are striking examples of evolutionary convergence, displaying complex and often highly similar adaptations despite lack of shared ancestry. Using available carnivorous plant genomes along with non-carnivorous reference taxa, this study examines the convergence of functional overrepresentation of genes previously implicated in plant carnivory. Gene Ontology (GO) coding was used to quantitatively score functional representation in these taxa, in terms of proportion of carnivory-associated functions relative to all functional sequence. Statistical analysis revealed that, in carnivorous plants as a group, only two of the 24 functions tested showed a signal of substantial overrepresentation. However, when the four carnivorous taxa were analyzed individually, 11 functions were found to be significant in at least one taxon. Though carnivorous plants collectively may show overrepresentation in functions from the predicted set, the specific functions that are overrepresented vary substantially from taxon to taxon. While it is possible that some functions serve a similar practical purpose such that one taxon does not need to utilize both to achieve the same result, it appears that there are multiple approaches for the evolution of carnivorous function in plant genomes. Our approach could be applied to tests of functional convergence in other systems provided on the availability of genomes and annotation data for a group.

Posterior predictive checks of coalescent models: P2C2M, an R package.

Bayesian inference operates under the assumption that the empirical data are a good statistical fit to the analytical model, but this assumption can be challenging to evaluate. Here, we introduce a novel r package that utilizes posterior predictive simulation to evaluate the fit of the multispecies coalescent model used to estimate species trees. We conduct a simulation study to evaluate the consistency of different summary statistics in comparing posterior and posterior predictive distributions, the use of simulation replication in reducing error rates and the utility of parallel process invocation towards improving computation times. We also test P2C2M on two empirical data sets in which hybridization and gene flow are suspected of contributing to shared polymorphism, which is in violation with the coalescent model: Tamias chipmunks and Myotis bats. Our results indicate that (i) probability-based summary statistics display the lowest error rates, (ii) the implementation of simulation replication decreases the rate of type II errors, and (iii) our r package displays improved statistical power compared to previous implementations of this approach. When probabilistic summary statistics are used, P2C2M corroborates the assumption that genealogies collected from Tamias and Myotis are not a good fit to the multispecies coalescent model. Taken as a whole, our findings argue that an assessment of the fit of the multispecies coalescent model should accompany any phylogenetic analysis that estimates a species tree.

A review of the prevalence, utility, and caveats of using chloroplast simple sequence repeats for studies of plant biology.

Microsatellites occur in all plant genomes and provide useful markers for studies of genetic diversity and structure. Chloroplast microsatellites (cpSSRs) are frequently targeted because they are more easily isolated than nuclear microsatellites. Here, we quantified the frequency and uses of cpSSRs based on a literature review of over 400 studies published 1995-2013. These markers are an important and economical tool for plant biologists and continue to be used alongside modern genomics approaches to study genetic diversity and structure, evolutionary history, and hybridization in native and agricultural species. Studies using species-specific primers reported a greater number of polymorphic loci than those employing universal primers. A major disadvantage to cpSSRs is fragment size homoplasy; therefore, we documented its occurrence at several cpSSR loci within and between species of Acmispon (Fabaceae). Based on our empirical data set, we recommend targeted sequencing of a subset of samples combined with fragment genotyping as a cost-efficient, data-rich approach to the use of cpSSRs and as a test of homoplasy. The availability of genomic resources for plants aids in the development of primers for new study systems, thereby enhancing the utility of cpSSRs across plant biology.