Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.

Abstract— Wake Atoll is an isolated chain of three islets located in the Western Pacific. Included in its endemic flora is a representative of the genus Gossypium colloquially referred to as Wake Island cotton. Stanley G. Stephens pointed out that “Wake Island cotton does not resemble closely either the Caribbean or other Pacific forms.” Taking into consideration morphological distinctions, the geographic isolation of Wake Atoll, and newly generated molecular data presented here, we conclude that the cottons of Wake Atoll do in fact represent a new species of Gossypium, here named Gossypium stephensii . This name is chosen to commemorate the eminent natural historian, evolutionary geneticist, and cotton biologist, S. G. Stephens.

Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits). Starting with genotyping-by-sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well-characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19- to 151-fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets.

The performance of hybridization capture combined with next-generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient-domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187-fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient-domestic dromedaries with 17–95% length coverage and 1.27–47.1-fold read depths for the covered regions. Using whole-genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1–1.06-fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.

In ancient cultures without a writing system, it is difficult to infer the basis of status and rank. Here the authors analyse ancient DNA from nine presumed elite individuals buried successively over a 300-year period at Chaco Canyon, and show evidence of matrilineal rela…

Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the “common deletion” that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5′ repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.

Although hybridisation through genome duplication is well known, hybridisation without genome duplication (homoploid hybrid speciation, HHS) is not. Few well-documented cases have been reported. A possible instance of HHS in Medicago prostrata Jacq. was suggested previously, based on only two genes and one individual. We tested whether this species was formed through HHS by sampling eight nuclear loci and 22 individuals, with additional individuals from related species, using gene capture and Illumina sequencing. Phylogenetic inference and coalescent simulations were performed to infer the causes of gene tree incongruence. We found no evidence that phylogenetic differences among M. prostrata individuals were the result of HHS. Instead, an autopolyploid origin of tetraploids with introgression from tetraploids of the M. sativa complex is likely. We argue that tetraploid M. prostrata individuals constitute a new species, characterised by a partially non-overlapping distribution and distinctive alleles (from the M. sativa complex). No gene flow from tetraploid to diploid M. prostrata is apparent, suggesting partial reproductive isolation. Thus, speciation via autopolyploidy appears to have been reinforced by introgression. This raises the intriguing possibility that introgressed alleles may be responsible for the increased range exploited by tetraploid M. prostrata with respect to that of the diploids.

The microhylid frog genus Kaloula is an adaptive radiation spanning the edge of the Asian mainland and multiple adjacent island archipelagos, with much of the clade’s diversity associated with an endemic Philippine radiation. Relationships among clades from the Philippines, however, remain unresolved. With ultraconserved element (UCE) and mitogenomic data, we identified highly supported differences in topology and areas of poor resolution, for each marker set. Using the UCE data, we then identified possible instances of contemporary hybridization, past introgression, and incomplete lineage sorting (ILS) within the Philippine Kaloula. Using a simulation approach, and an estimate of the Philippine Kaloula clade origin (12.7—21.0 mya), we demonstrate that an evolutionary history including inferred instances of hybridization, introgression, and ILS leads to phylogenetic reconstructions that show concordance with results from the observed mitogenome and UCE data. In the process of validating a complex evolutionary scenario in the Philippine Kaloula, we provide the first demonstration of the efficacy of UCE data for phylogenomic studies of anuran amphibians.

The Adelaide geosyncline, a mountainous region in central southern Australia, is purported to be an important continental refugium for Mediterranean and semi-arid Australian biota, yet few population genetic studies have been conducted to test this theory. Here, we focus on a plant species distributed widely throughout the region, the narrow-leaf hopbush, Dodonaea viscosa ssp. angustissima, and examine its genetic diversity and population structure. We used a hybrid-capture target enrichment technique to selectively sequence over 700 genes from 89 individuals across 17 sampling locations. We compared 815 single nucleotide polymorphisms among individuals and populations to investigate population genetic structure. Three distinct genetic clusters were identified; a Flinders/Gammon ranges cluster, an Eastern cluster, and a Kangaroo Island cluster. Higher genetic diversity was identified in the Flinders/Gammon Ranges cluster, indicating that this area is likely to have acted as a refugium during past climate oscillations. We discuss these findings and consider the historical range dynamics of these populations. We also provide methodological considerations for population genomics studies that aim to use novel genomic approaches (such as target capture methods) on non-model systems. The application of our findings to restoration of this species across the region are also considered.