Abstract DNA methylation is an important epigenetic regulator of gene expression. 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) play distinct roles in epigenetic regulation, however the ability to distinguish between 5mC and 5hmC is often lost as they are frequently read as a combined signal in traditional sequencing approaches. NEBNext Enzymatic Methyl-seq v2 (EM-seq v2) and NEBNext Enzymatic 5hmC-seq (E5hmC-seq) are enzymatic approaches used to investigate cytosine methylation. EM-seq v2 detects both 5mC and 5hmC whereas E5hmC-seq detects only 5hmC. The first step for each method protects either 5mC and/or 5hmC from deamination by APOBEC enzyme. Cytosines are deaminated to uracil in EM-seq v2 whereas for E5hmC-seq, 5mC and cytosine bases are converted to thymine and uracil respectively. Using a subtractive data analysis approach, whereby E5hmC-seq data is subtracted from EM-seq data, the locations of 5mC and 5hmC can be decoupled at single base resolution. EM-seq v2 libraries and E5hmC-seq libraries were prepared using 0.1 ng to 200 ng of genomic DNA. Both methods leave DNA intact and result in superior sequencing libraries with longer insert sizes, lower duplication rates and minimal GC bias as well as accurately detecting methylation down to 0.1 ng DNA. These methods are streamlined due to reduced library preparation time as well as the removal of a cleanup step. Coverage of genomic features such as CpG islands and enhancers, for both methods were maintained to 1 ng but dropped as expected with the 0.1 ng inputs. EM-seq v2 and E5hmC-seq provides consistent methylation metrics for inputs ranging from 0.1 ng to 200 ng. EM-seq v2 and E5hmC-seq libraries provide accurate measurements across inputs from 0.1 ng – 200 ng with expected methylation, insert sizes and minimal GC bias. In addition, combining E5hmC-seq and EM-seq data to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease. Citation Format: Vaishnavi panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Matthew Campbell, Laura Blum, Bradley Langhorst, Louise Williams. NEBNext® enzymatic solutions for DNA methylation profiling at picogram scale [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B026.
Advancements in genome sequencing and assembly techniques have increased the documentation of structural variants in wild organisms. Of these variants, chromosomal inversions are especially prominent due to their large size and active recombination suppression between alternative homokaryotypes. This suppression enables the 2 forms of the inversion to be maintained and allows the preservation of locally adapted alleles. The Barramundi Perch (BP; Lates calcarifer) is a widespread species complex with 3 main genetic lineages located in the biogeographic regions of Australia and New Guinea (AUS + NG), Southeast Asia (SEA), and the Indian Subcontinent (IND). BP are typically considered to be a protandrous sequential hermaphrodite species that exhibits catadromy. Freshwater occupancy and intraspecific variation in life history (e.g. partially migratory populations) exist and provide opportunities for strongly divergent selection associated with, for example, salinity tolerance, swimming ability, and marine dispersal. Herein, we utilize genomic data generated from all 3 genetic lineages to identify and describe 3 polymorphic candidate chromosomal inversions. These candidate chromosomal inversions appear to be fixed for ancestral variants in the IND lineage and for inverted versions in the AUS + NG lineage and exhibit variation in all 3 inversions in the SEA lineage. BP have a diverse portfolio of life history options that includes migratory strategy as well as sexual system (i.e. hermaphroditism and gonochorism). We propose that the some of the life history variabilities observed in BP may be linked to inversions and, in doing so, we present genetic data that might be useful in enhancing aquaculture production and population management.
We investigate a pantropical sub-family and genus of damselfishes, the sergeant-majors (Pomacentridae: Abudefdufinae: Abudefduf), to identify the tempo and mechanisms of speciation in the lineage.We examine sequence capture data from 500 loci and 20 species, with multiple individuals sampled from across the geographic ranges of widespread species.Utilizing a maximum likelihood framework, as well as a timecalibrated Bayesian phylogeny, the following key questions are addressed: What is the historical tempo of speciation?What are the relative contributions of vicariant, peripatric and parapatric speciation to sergeant-major diversity?How is speciation related to major variation in trophic ecology?The approximately 20 species of sergeant-majors fall into three main lineages.The ancestral condition appears to be benthivory, which is predominant in two lineages comprising six species.The remaining species of sergeantmajors, of which there are at least 15, fall within a clade composed entirely of planktivores.This clade is sister to a benthivore clade that includes a species, A. notatus, in transition to planktivory.Most speciation of sergeant-majors, which appeared ~24 million years ago, occurred in the last 10 million years.Present distributional patterns indicate vicariant speciation precipitated by the closure of land barriers between both sides of the Atlantic and the Pacific, and the emergence of land between the Indian and Pacific Oceans.Within this backdrop, frequent oscillations in sea level over the last 10 million years also appear to have generated conditions suitable for both peripatric and vicariant speciation, and most speciation within the genus appears linked to these changes in sea level.Diversification within the genus has been concentrated in planktivorous seargeant-majors rather than benthivores.The root cause is unclear, but does not appear to be related to differences in dispersal potential, which is greater in the planktivorous species due to the ability of their post-larval juveniles to raft with floating debris.This
Pleistocene climatic instability had profound and diverse effects on the distribution and abundance of Arctic organisms revealed by variation in phylogeographic patterns documented in extant Arctic populations. To better understand the effects of geography and paleoclimate on Beringian freshwater fishes, we examined genetic variability in the genus Dallia (blackfish: Esociformes: Esocidae). The genus Dallia groups between one and three nominal species of small, cold- and hypoxia-tolerant freshwater fishes restricted entirely in distribution to Beringia from the Yukon River basin near Fairbanks, Alaska westward including the Kuskokwim River basin and low-lying areas of Western Alaska to the Amguema River on the north side of the Chukotka Peninsula and Mechigmen Bay on the south side of the Chukotka Peninsula. The genus has a non-continuous distribution divided by the Bering Strait and the Brooks Range. We examined the distribution of genetic variation across this range to determine the number and location of potential sub-refugia within the greater Beringian refugium as well as the roles of the Bering land bridge, Brooks Range, and large rivers within Beringia in shaping the current distribution of populations of Dallia. Our analyses were based on DNA sequence data from two nuclear gene introns (S7 and RAG1) and two mitochondrial genome fragments from nineteen sampling locations. These data were examined under genetic clustering and coalescent frameworks to identify sub-refugia within the greater Beringia refugium and to infer the demographic history of different populations of Dallia.We identified up to five distinct genetic clusters of Dallia. Four of these genetic clusters are present in Alaska: (1) Arctic Coastal Plain genetic cluster found north of the Brooks Range, (2) interior Alaska genetic cluster placed in upstream locations in the Kuskokwim and Yukon river basins, (3) a genetic cluster found on the Seward Peninsula, and (4) a coastal Alaska genetic cluster encompassing downstream Kuskokwim River and Yukon River basin sample locations and samples from Southwest Alaska not in either of these drainages. The Chukotka samples are assigned to their own genetic cluster (5) similar to the coastal Alaska genetic cluster. The clustering and ordination analyses implemented in Discriminant Analysis of Principal Components (DAPC) and STRUCTURE showed mostly concordant groupings and a high degree of differentiation among groups. The groups of sampling locations identified as genetic clusters correspond to geographic areas divided by likely biogeographic barriers including the Brooks Range and the Bering Strait. Estimates of sequence diversity (θ) are highest in the Yukon River and Kuskokwim River drainages near the Bering Sea. We also infer asymmetric migration rates between genetic clusters. The isolation of Dallia on the Arctic Coastal Plain of Alaska is associated with very low estimated migration rates between the coastal Alaska genetic cluster and the Arctic Coastal Plain genetic cluster.Our results support a scenario with multiple aquatic sub-refugia in Beringia during the Pleistocene and the preservation of that structure in extant populations of Dallia. An inferred historical presence of Dallia across the Bering land bridge explains the similarities in the genetic composition of Dallia in West Beringia and western coastal Alaska. In contrast, historic and contemporary isolation across the Brooks Range shaped the distinctiveness of present day Arctic Coastal Plain Dallia. Overall this study uncovered a high degree of genetic structuring among populations of Dallia supporting the idea of multiple Beringian sub-refugia during the Pleistocene and which appears to be maintained to the present due to the strictly freshwater nature and low dispersal ability of this genus.
Pi7(t), a dominant blast resistance gene derived from the rice cultivar Moroberekan, confers complete resistance against the fungal pathogen Magnaporthe grisea. Pi7(t) previously was positioned on chromosome 11 by restriction fragment length polymorphism (RFLP) mapping of a recombinant inbred line population. One derivative of this population, recombinant inbred line (RIL)29, was designated as the representative line for Pi7(t). A segregating F2 population was created from RIL29 in order to determine the location of Pi7(t). The new mapping data indicate a position for Pi7(t) 30 centimorgans distal to the original location. Pi7(t) shares a common position with the previously mapped Pi1 M. grisea resistance gene. RIL29 carries DNA not derived from either parent used to create the RIL population at the newly assigned Pi7(t) locus. RFLP analysis has identified a possible donor source.
Phylogenetic inference based on evidence from DNA sequences has led to significant strides in the development of a stable and robustly supported framework for the vertebrate tree of life. To date, the bulk of those advances have relied on sequence data from a small number of genome regions that have proven unable to produce satisfactory answers to consistently recalcitrant phylogenetic questions. Here, we re-examine phylogenetic relationships among early-branching euteleostean fish lineages classically grouped in the Protacanthopterygii using DNA sequence data surrounding ultraconserved elements. We report and examine a dataset of thirty-four OTUs with 17,957 aligned characters from fifty-three nuclear loci. Phylogenetic analysis is conducted both in concatenated and joint gene trees and species tree estimation frameworks. Both analytical frameworks yield supporting evidence for existing hypotheses of relationship for the placement of Lepidogalaxias salamandroides , monophyly of the Stomiatii and the presence of an esociform + salmonid clade. Lepidogalxias salamandroides and the Esociformes + Salmoniformes are successive sister lineages to all other euteleosts in the two analysis types receiving high support values for this arrangement. However, inter-relationships of Argentiniformes, Stomiatii and Neoteleostei remain uncertain as they varied by analysis type while receiving strong and contradictory indices of support. Topological differences between analysis types are apparent within the Ostarioclupeomorpha and the percomorph taxa in the data set. Our results identify concordant areas with strong support for relationships within and between early-branching euteleost lineages but they also reveal limitations in the ability of larger datasets to conclusively resolve other aspects of that phylogeny.
Abstract Fluctuating environmental pressures can challenge organisms by repeatedly shifting the optimum phenotype. Two contrasting evolutionary strategies to cope with these fluctuations are 1) evolution of the mean phenotype to follow the optimum (adaptive tracking) or 2) diversifying phenotypes so that at least some individuals have high fitness in the current fluctuation (bet-hedging). Bet-hedging could underlie stable differences in the behavior of individuals that are present even when genotype and environment are held constant. Instead of being simply ‘noise,’ behavioral variation across individuals may reflect an evolutionary strategy of phenotype diversification. Using geographically diverse wild-derived fly strains and high-throughput assays of individual preference, we tested whether thermal preference variation in Drosophila melanogaster could reflect a bet-hedging strategy. We also looked for evidence that populations from different regions differentially adopt bet-hedging or adaptive-tracking strategies. Computational modeling predicted regional differences in the relative advantage of bet-hedging, and we found patterns consistent with that in regional variation in thermal preference heritability. In addition, we found that temporal patterns in mean preference support bet-hedging predictions and that there is a genetic basis for thermal preference variability. Our empirical results point to bet-hedging in thermal preference as a potentially important evolutionary strategy in wild populations.
