Pallas’s cat, or the manul cat (Otocolobus manul), is a small felid native to the grasslands and steppes of central Asia. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources. These threats, combined withO. manul’s zoo collection popularity and value in evolutionary biology, necessitate improvement of species genomic resources. We used standalone nanopore sequencing to assemble a 2.5 Gb, 61-contig nuclear assembly and 17,097 bp mitogenome forO. manul. The primary nuclear assembly had 56x sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score forCarnivora-specific genes. High genome collinearity withinFelidaepermitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Manul contigs spanned all 19 felid chromosomes with an inferred total gap length of less than 400 kilobases. Modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; sixty-one differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. The assembled mitogenome successfully resolved existing discordance betweenFelinaenuclear and mtDNA phylogenies. All assembly drafts were generated from 158 Gb of sequence using seven minION flow cells.

Pallas's cat, or the manul cat (Otocolobus manul), is a small felid native to the grasslands and steppes of central Asia. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources. These threats, combined with O. manul’s zoo collection popularity and value in evolutionary biology, necessitate improvement of species genomic resources. We used standalone nanopore sequencing to assemble a 2.5 Gb, 61-contig nuclear assembly and 17097 bp mitogenome for O. manul. The primary nuclear assembly had 56× sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score for Carnivora-specific genes. High genome collinearity within Felidae permitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Manul contigs spanned all 19 felid chromosomes with an inferred total gap length of less than 400 kilobases. Modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; 61 differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. The assembled mitogenome successfully resolved existing discordance between Felinae nuclear and mtDNA phylogenies. All assembly drafts were generated from 158 Gb of sequence using seven minION flow cells.

Inorganic arsenic (iAs) is one of the largest toxic exposures to impact humanity worldwide. Exposure to iAs during pregnancy may disrupt the proper remodeling of the epigenome of F1 developing offspring and potentially their F2 grand-offspring via disruption of fetal primordial germ cells (PGCs). There is a limited understanding between the correlation of disease phenotype and methylation profile within offspring of both generations and whether it persists to adulthood. Our study aims to understand the intergenerational effects of in utero iAs exposure on the epigenetic profile and onset of disease phenotypes within F1 and F2 adult offspring, despite the lifelong absence of direct arsenic exposure within these generations. We exposed F0 female mice (C57BL6/J) to the following doses of iAs in drinking water 2 weeks before pregnancy until the birth of the F1 offspring: 1, 10, 245, and 2300 ppb. We found sex-and dose-specific changes in weight and body composition that persist from early time to adulthood within both generations. Fasting blood glucose challenge suggests iAs exposure causes dysregulation of glucose metabolism, revealing generational, exposure, and sex-specific differences. Toward understanding the mechanism, genome-wide DNA methylation data highlights exposure-specific patterns in liver, finding dysregulation within genes associated with cancer, T2D, and obesity. We also identified regions containing persistently differentially methylated CpG sites between F1 and F2 generations. Our results indicate the F1 developing embryos and their PGCs, which will result in F2 progeny, retain epigenetic damage established during the prenatal period and are associated with adult metabolic dysfunction.

Genetic data from non-model species can inform ecology and physiology, giving insight into a species’ distribution and abundance as well as their responses to changing environments, all of which are important for species conservation and management. Moreover, reduced sequencing costs and improved long-read sequencing technology allows researchers to readily generate genomic resources for non-model species. Here, we apply Oxford Nanopore long-read sequencing and low-coverage (∼1x) whole genome short-read sequencing technology (Illumina) to assemble a genome and examine population genetics of an abundant tropical and subtropical fish, the hardhead silverside (Atherinomorus stipes). These fish are found in shallow coastal waters and are frequently included in ecological models because they serve as abundant prey for commercially and ecologically important species. Despite their importance in sub-tropical and tropical ecosystems, little is known about their population connectivity and genetic diversity. Our A. stipes genome assembly is about 1.2 Gb with comparable repetitive element content (∼47%), number of protein duplication events, and DNA methylation patterns to other teleost fish species. Among five sampled populations spanning 43  km of South Florida and the Florida Keys, we find little population structure suggesting high population connectivity.

The cecidomyiid fly, soybean gall midge, Resseliella maxima Gagné, is a recently discovered insect that feeds on soybean plants in the Midwest US. Resseliella maxima larvae feed on soybean stems which may induce plant death and can cause considerable yield losses, making it an important agricultural pest. From three pools of 50 adults each, we used long-read nanopore sequencing to assemble a R. maxima reference genome. The final genome assembly is 206 Mb with 64.88X coverage, consisting of 1009 contigs with an N50 size of 714 kb. The assembly is high quality with a BUSCO score of 87.8%. Genome-wide GC level is 31.60% and DNA methylation was measured at 1.07%. The R. maxima genome is comprised of 21.73% repetitive DNA, which is in line with other cecidomyiids. Protein prediction annotated 14,798 coding genes with 89.9% protein BUSCO score. Mitogenome analysis indicated that R. maxima assembly is a single circular contig of 15,301 bp and shares highest identity to the mitogenome of the Asian rice gall midge, Orseolia oryzae Wood-Mason. The R. maxima genome has one of the highest completeness levels for a cecidomyiid and will provide a resource for research focused on the biology, genetics, and evolution of cecidomyiids, as well as plant-insect interactions in this important agricultural pest.

The cecidomyiid fly, soybean gall midge,Resseliella maximaGagn, is a recently discovered insect that feeds on soybean plants in the Midwest US.Resseliella maximalarvae feed on soybean stems which may induce plant death and can cause considerable yield losses, making it an important agricultural pest. From three pools of 50 adults each, we used long-read nanopore sequencing to assemble aR. maximareference genome. The final genome assembly is 206 Mb with 64.88X coverage, consisting of 1009 contigs with an N50 size of 714 kb. The assembly is high quality with a BUSCO score of 87.8%. Genome-wide GC level is 31.60% and DNA methylation was measured at 1.07%. TheR. maximagenome is comprised of 21.73% repetitive DNA, which is in line with other cecidomyiids. Protein prediction annotated 14,798 coding genes with 89.9% protein BUSCO score. Mitogenome analysis indicated thatR. maximaassembly is a single circular contig of 15,301 bp and shares highest identity to the mitogenome of the Asian rice gall midge,Orseolia oryzae(Wood-Mason). TheR. maximagenome has one of the highest completeness levels for a cecidomyiid and will provide a resource for research focused on the biology, genetics, and evolution of cecidomyiids, as well as plant-insect interactions in this important agricultural pest.

5-aza-2'-deoxycytidine (decitabine), is a chemotherapeutic DNA methyltransferase (DNMT) inhibitor widely used to treat myelodysplastic syndrome and acute myeloid leukemias. Decitabine’s anti-neoplastic activity is thought to result from inhibition of DNMTs leading to passive demethylation of 5’methylcytosines (5mC) in rapidly dividing tissues, resulting in cell death. However, we previously reported paradoxical effects on DNA methylation by decitabine in somatic tissues. Given the potential for lasting damage to DNA methylation in reproductive tissues from even short courses of decitabine in reproductive age humans, we chose to characterize its long-term effects here. Mice were treated with two clinically relevant doses of decitabine (0.15 mg/kg, 0.35 mg/kg) for 7 weeks and DNA methylation was assessed within female reproductive tract tissues. We found methylated cytosines within the ovary to be the least sensitive to decitabine exposure at both doses, whereas the uterus and the oviduct exhibited higher 5mC dysregulation, surprisingly biased towards hypermethylation at the 0.35 mg/kg dose. We identified the sites of differential methylation; revealing specific genes and pathways involved in cell differentiation, development, communication, and cell signaling that were universally altered in all tissues. In addition to our differential methylation data, we identified dysregulated transcription and pathways using RNAseq analyses. Overall, our findings show decitabine exposure causes an epigenetic insult to DNA methylation within female reproductive tissues. Our data provides evidence that further evaluation is needed to fully establish the long-term phenotypic effects post-decitabine exposure.

5-aza-2'-deoxycytidine (decitabine), is a chemotherapeutic DNA methyltransferase (DNMT) inhibitor widely used to treat myelodysplastic syndrome and acute myeloid leukemias. Decitabine’s anti-neoplastic activity is thought to result from inhibition of DNMTs leading to passive demethylation of 5’methylcytosines (5mC) in rapidly dividing tissues, resulting in cell death. However, we previously reported paradoxical effects on DNA methylation by decitabine in somatic tissues. Given the potential for lasting damage to DNA methylation in reproductive tissues from even short courses of decitabine in reproductive age humans, we chose to characterize its long-term effects here. Mice were treated with two clinically relevant doses of decitabine (0.15 mg/kg, 0.35 mg/kg) for 7 weeks and DNA methylation was assessed within female reproductive tract tissues. We found methylated cytosines within the ovary to be the least sensitive to decitabine exposure at both doses, whereas the uterus and the oviduct exhibited higher 5mC dysregulation, surprisingly biased towards hypermethylation at the 0.35 mg/kg dose. We identified the sites of differential methylation; revealing specific genes and pathways involved in cell differentiation, development, communication, and cell signaling that were universally altered in all tissues. In addition to our differential methylation data, we identified dysregulated transcription and pathways using RNAseq analyses. Overall, our findings show decitabine exposure causes an epigenetic insult to DNA methylation within female reproductive tissues. Our data provides evidence that further evaluation is needed to fully establish the long-term phenotypic effects post-decitabine exposure.