De-novo long-read genome assembly and annotation of the luna moth (Actias luna) fully resolves repeat-rich silk genes.

We present the first long-read de-novo -assembly and annotation of the luna moth (Actias luna) and provide the full characterization of heavy chain fibroin (h-fibroin)--, a long and highly repetitive gene (>20 Kbp) essential in silk fiber production. There are more than 160,000 described species of moths and butterflies (Lepidoptera), but only within the last five years have we begun to recover high-quality annotated whole genomes across the order which capture h-fibroin. Using PacBio HiFi reads, we produce the first high-quality long-read reference genome for this species. The assembled genome has a length of 532 Mbp, a contig N50 of 16.8 Mbp, an L50 of 14 contigs, and 99.4% completeness (BUSCO). Our annotation using Bombyx mori protein and A.luna RNAseq evidence captured a total of 20,866 genes at 98.9% completeness with 10,267 functionally annotated proteins and a full-length h-fibroin annotation of 2,679 amino acid residues.

AgAnimalGenomes: browsers for viewing and manually annotating farm animal genomes.

Current genome sequencing technologies have made it possible to generate highly contiguous genome assemblies for non-model animal species. Despite advances in genome assembly methods, there is still room for improvement in the delineation of specific gene features in the genomes. Here we present genome visualization and annotation tools to support seven livestock species (bovine, chicken, goat, horse, pig, sheep, and water buffalo), available in a new resource called AgAnimalGenomes. In addition to supporting the manual refinement of gene models, these browsers provide visualization tracks for hundreds of RNAseq experiments, as well as data generated by the Functional Annotation of Animal Genomes (FAANG) Consortium. For species with predicted gene sets from both Ensembl and RefSeq, the browsers provide special tracks showing the thousands of protein-coding genes that disagree across the two gene sources, serving as a valuable resource to alert researchers to gene model issues that may affect data interpretation. We describe the data and search methods available in the new genome browsers and how to use the provided tools to edit and create new gene models.

The Complete Genome Sequence of Actias luna (Saturniidae, Lepidoptera), the luna moth.

Actias luna, the luna moth, is a Nearctic species in the family Saturniidae, the giant silk moths. Known for its large size, bright green wings and elongated tails, it is found in Eastern North America, from east of the Great Plains in the United States, and from Saskatchewan eastward through central Quebec to Nova Scotia in Canada. We present the complete genome sequence of this species. Raw read data and the assembled genome are available in Genbank.

Comparison of detection methods and genome quality when quantifying nuclear mitochondrial insertions in vertebrate genomes.

The integration of mitochondrial genome fragments into the nuclear genome is well documented, and the transfer of these mitochondrial nuclear pseudogenes (numts) is thought to be an ongoing evolutionary process. With the increasing number of eukaryotic genomes available, genome-wide distributions of numts are often surveyed. However, inconsistencies in genome quality can reduce the accuracy of numt estimates, and methods used for identification can be complicated by the diverse sizes and ages of numts. Numts have been previously characterized in rodent genomes and it was postulated that they might be more prevalent in a group of voles with rapidly evolving karyotypes. Here, we examine 37 rodent genomes, and an additional 26 vertebrate genomes, while also considering numt detection methods. We identify numts using DNA:DNA and protein:translated-DNA similarity searches and compare numt distributions among rodent and vertebrate taxa to assess whether some groups are more susceptible to transfer. A combination of protein sequence comparisons (protein:translated-DNA) and BLASTN genomic DNA searches detect 50% more numts than genomic DNA:DNA searches alone. In addition, higher-quality RefSeq genomes produce lower estimates of numts than GenBank genomes, suggesting that lower quality genome assemblies can overestimate numts abundance. Phylogenetic analysis shows that mitochondrial transfers are not associated with karyotypic diversity among rodents. Surprisingly, we did not find a strong correlation between numt counts and genome size. Estimates using DNA: DNA analyses can underestimate the amount of mitochondrial DNA that is transferred to the nucleus.

Hymenoptera Genome Database: new genomes and annotation datasets for improved go enrichment and orthologue analyses.

We report an update of the Hymenoptera Genome Database (HGD; http://HymenopteraGenome.org), a genomic database of hymenopteran insect species. The number of species represented in HGD has nearly tripled, with fifty-eight hymenopteran species, including twenty bees, twenty-three ants, eleven wasps and four sawflies. With a reorganized website, HGD continues to provide the HymenopteraMine genomic data mining warehouse and JBrowse/Apollo genome browsers integrated with BLAST. We have computed Gene Ontology (GO) annotations for all species, greatly enhancing the GO annotation data gathered from UniProt with more than a ten-fold increase in the number of GO-annotated genes. We have also generated orthology datasets that encompass all HGD species and provide orthologue clusters for fourteen taxonomic groups. The new GO annotation and orthology data are available for searching in HymenopteraMine, and as bulk file downloads.

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss.

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.

MaizeMine: A Data Mining Warehouse for the Maize Genetics and Genomics Database.

MaizeMine is the data mining resource of the Maize Genetics and Genome Database (MaizeGDB; http://maizemine.maizegdb.org). It enables researchers to create and export customized annotation datasets that can be merged with their own research data for use in downstream analyses. MaizeMine uses the InterMine data warehousing system to integrate genomic sequences and gene annotations from the Zea mays B73 RefGen_v3 and B73 RefGen_v4 genome assemblies, Gene Ontology annotations, single nucleotide polymorphisms, protein annotations, homologs, pathways, and precomputed gene expression levels based on RNA-seq data from the Z. mays B73 Gene Expression Atlas. MaizeMine also provides database cross references between genes of alternative gene sets from Gramene and NCBI RefSeq. MaizeMine includes several search tools, including a keyword search, built-in template queries with intuitive search menus, and a QueryBuilder tool for creating custom queries. The Genomic Regions search tool executes queries based on lists of genome coordinates, and supports both the B73 RefGen_v3 and B73 RefGen_v4 assemblies. The List tool allows you to upload identifiers to create custom lists, perform set operations such as unions and intersections, and execute template queries with lists. When used with gene identifiers, the List tool automatically provides gene set enrichment for Gene Ontology (GO) and pathways, with a choice of statistical parameters and background gene sets. With the ability to save query outputs as lists that can be input to new queries, MaizeMine provides limitless possibilities for data integration and meta-analysis.

Bovine Genome Database: new annotation tools for a new reference genome.

The Bovine Genome Database (BGD) (http://bovinegenome.org) has been the key community bovine genomics database for more than a decade. To accommodate the increasing amount and complexity of bovine genomics data, BGD continues to advance its practices in data acquisition, curation, integration and efficient data retrieval. BGD provides tools for genome browsing (JBrowse), genome annotation (Apollo), data mining (BovineMine) and sequence database searching (BLAST). To augment the BGD genome annotation capabilities, we have developed a new Apollo plug-in, called the Locus-Specific Alternate Assembly (LSAA) tool, which enables users to identify and report potential genome assembly errors and structural variants. BGD now hosts both the newest bovine reference genome assembly, ARS-UCD1.2, as well as the previous reference genome, UMD3.1.1, with cross-genome navigation and queries supported in JBrowse and BovineMine, respectively. Other notable enhancements to BovineMine include the incorporation of genomes and gene annotation datasets for non-bovine ruminant species (goat and sheep), support for multiple assemblies per organism in the Regions Search tool, integration of additional ontologies and development of many new template queries. To better serve the research community, we continue to focus on improving existing tools, developing new tools, adding new datasets and encouraging researchers to use these resources.

Lepidoptera genomes: current knowledge, gaps and future directions.

Butterflies and moths (Lepidoptera) are one of the most ecologically diverse and speciose insect orders. With recent advances in genomics, new Lepidoptera genomes are regularly being sequenced, and many of them are playing principal roles in genomics studies, particularly in the fields of phylo-genomics and functional genomics. Thus far, assembled genomes are only available for <10 of the 43 Lepidoptera superfamilies. Nearly all are model species, found in the speciose clade Ditrysia. Community support for Lepidoptera genomics is growing with successful management and dissemination of data and analytical tools in centralized databases. With genomic studies quickly becoming integrated with ecological and evolutionary research, the Lepidoptera community will unquestionably benefit from new high-quality reference genomes that are more evenly distributed throughout the order.

Positive Selection in Rapidly Evolving Plastid-Nuclear Enzyme Complexes.

Rates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid-nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic coevolution between pathogens and host immune genes. We discuss a possible role of plastid-nuclear conflict as a novel cause of accelerated evolution.

Most partial domains in proteins are alignment and annotation artifacts.

BACKGROUND: Protein domains are commonly used to assess the functional roles and evolutionary relationships of proteins and protein families. Here, we use the Pfam protein family database to examine a set of candidate partial domains. Pfam protein domains are often thought of as evolutionarily indivisible, structurally compact, units from which larger functional proteins are assembled; however, almost 4% of Pfam27 PfamA domains are shorter than 50% of their family model length, suggesting that more than half of the domain is missing at those locations. To better understand the structural nature of partial domains in proteins, we examined 30,961 partial domain regions from 136 domain families contained in a representative subset of PfamA domains (RefProtDom2 or RPD2). RESULTS: We characterized three types of apparent partial domains: split domains, bounded partials, and unbounded partials. We find that bounded partial domains are over-represented in eukaryotes and in lower quality protein predictions, suggesting that they often result from inaccurate genome assemblies or gene models. We also find that a large percentage of unbounded partial domains produce long alignments, which suggests that their annotation as a partial is an alignment artifact; yet some can be found as partials in other sequence contexts. CONCLUSIONS: Partial domains are largely the result of alignment and annotation artifacts and should be viewed with caution. The presence of partial domain annotations in proteins should raise the concern that the prediction of the protein's gene may be incomplete. In general, protein domains can be considered the structural building blocks of proteins.

Cytonuclear interactions and relaxed selection accelerate sequence evolution in organelle ribosomes.

Many mitochondrial and plastid protein complexes contain subunits that are encoded in different genomes. In animals, nuclear-encoded mitochondrial proteins often exhibit rapid sequence evolution, which has been hypothesized to result from selection for mutations that compensate for changes in interacting subunits encoded in mutation-prone animal mitochondrial DNA. To test this hypothesis, we analyzed nuclear genes encoding cytosolic and organelle ribosomal proteins in flowering plants. The model angiosperm genus Arabidopsis exhibits low organelle mutation rates, typical of most plants. Nevertheless, we found that (nuclear-encoded) subunits of organelle ribosomes in Arabidopsis have higher amino acid sequence polymorphism and divergence than their counterparts in cytosolic ribosomes, suggesting that organelle ribosomes experience relaxed functional constraint. However, the observed difference between organelle and cytosolic ribosomes was smaller than in animals and could be partially attributed to rapid evolution in N-terminal organelle-targeting peptides that are not involved in ribosome function. To test the role of organelle mutation more directly, we used transcriptomic data from an angiosperm genus (Silene) with highly variable rates of organelle genome evolution. We found that Silene species with unusually fast-evolving mitochondrial and plastid DNA exhibited increased amino acid sequence divergence in ribosomal proteins targeted to the organelles but not in those that function in cytosolic ribosomes. Overall, these findings support the hypothesis that rapid organelle genome evolution has selected for compensatory mutations in nuclear-encoded proteins. We conclude that coevolution between interacting subunits encoded in different genomic compartments within the eukaryotic cell is an important determinant of variation in rates of protein sequence evolution.

A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae).

In flowering plants, plastid genomes are generally conserved, exhibiting slower rates of sequence evolution than the nucleus and little or no change in structural organization. However, accelerated plastid genome evolution has occurred in scattered angiosperm lineages. For example, some species within the genus Silene have experienced a suite of recent changes to their plastid genomes, including inversions, shifts in inverted repeat boundaries, large indels, intron losses, and rapid rates of amino acid sequence evolution in a subset of protein genes, with the most extreme divergence occurring in the protease gene clpP. To investigate the relationship between the rates of sequence and structural evolution, we sequenced complete plastid genomes from three species (Silene conoidea, S. paradoxa, and Lychnis chalcedonica), representing independent lineages within the tribe Sileneae that were previously shown to have accelerated rates of clpP evolution. We found a high degree of parallel evolution. Elevated rates of amino acid substitution have occurred repeatedly in the same subset of plastid genes and have been accompanied by a recurring pattern of structural change, including cases of identical inversions and intron loss. This "syndrome" of changes was not observed in the closely related outgroup Agrostemma githago or in the more slowly evolving Silene species that were sequenced previously. Although no single mechanism has yet been identified to explain the correlated suite of changes in plastid genome sequence and structure that has occurred repeatedly in angiosperm evolution, we discuss a possible mixture of adaptive and non-adaptive forces that may be responsible.

Simultaneous extraction of high-quality RNA and DNA from small tissue samples.

Purification of high-quality DNA and RNA from a single sample is becoming increasingly important for studies seeking both genomic and transcriptomic data. We compare different methods for isolating DNA and RNA from fish embryos (Gulf killifish; Fundulus grandis) and describe an optimal technique to extract high-quality DNA and RNA from a single embryo. The optimal method utilizes a chaotropic buffer and spin column technology. From embryos weighing approximately 4 mg, we were able to isolate an average of 6.1 microg of DNA and 1.1 microg of RNA per sample. Relative amounts of DNA and RNA can be adjusted as needed per study. Although these extraction trials were conducted on fish embryos, they can be potentially applied to small samples that typically do not yield high concentrations of nucleic acids.

Molecular analyses of mitochondrial pseudogenes within the nuclear genome of arvicoline rodents.

Nuclear sequences of mitochondrial origin (numts) are common among animals and plants. The mechanism(s) by which numts transfer from the mitochondrion to the nucleus is uncertain, but their insertions may be mediated in part by chromosomal repair mechanisms. If so, then lineages where chromosomal rearrangements are common should be good models for the study of numt evolution. Arvicoline rodents are known for their karyotypic plasticity and numt pseudogenes have been discovered in this group. Here, we characterize a 4 kb numt pseudogene in the arvicoline vole Microtus rossiaemeridionalis. This sequence is among the largest numts described for a mammal lacking a completely sequenced genome. It encompasses three protein-coding and six tRNA pseudogenes that span approximately 25% of the entire mammalian mitochondrial genome. It is bordered by a dinucleotide microsatellite repeat and contains four transposable elements within its sequence and flanking regions. To determine the phylogenetic distribution of this numt among the arvicolines, we characterized one of the mitochondrial pseudogenes (cytochrome b) in 21 additional arvicoline species. Average rates of nucleotide substitution in this arvicoline pseudogene are estimated as 2.3 x 10(-8) substitutions/per site/per year. Furthermore, we performed comparative analyses among all species to estimate the age of this mitochondrial transfer at nearly 4 MYA, predating the origin of most arvicolines.

Extensive mitochondrial DNA transfer in a rapidly evolving rodent has been mediated by independent insertion events and by duplications.

Mitochondrial DNA translocations to the nucleus (numt pseudogenes) are pervasive among eukaryotes, but copy number within the nuclear genome varies widely among taxa. As an increasing number of genomes are sequenced in their entirety, the origins, transfer mechanisms and insertion sites of numts are slowly being characterized. We investigated mitochondrial transfers within a genetically diverse rodent lineage and here report 15 numts totaling 21.8 kb that are harbored within the nuclear genome of the vole Microtus rossiaemeridionalis. The 15 numts total 21.8 kb and range from 0.39 to over 3.0 kb in length. Phylogenetic analyses revealed that these numts resulted from three independent insertions to the nucleus, two of which were followed by subsequent nuclear duplication events. The dates of the two translocations that led to subsequent duplications were estimated at 1.97 and 1.19 MYA, which coincide with the origin and radiation of the genus Microtus. Numt sequence data from five Microtus species were used to estimate an average rate of nucleotide substitution as 2.6x10(-8) subs/site/yr. This substitution rate is higher than in many other mammals, but is concordant with the elevated rate of mtDNA substitution in this lineage. Our data suggest that numt translocation in Microtus is more extensive than in either Mus or in Rattus, consistent with the elevated rate of speciation, karyotypic rearrangement, and mitochondrial DNA evolution in Microtus.

Accelerated molecular evolution in Microtus (Rodentia) as assessed via complete mitochondrial genome sequences.

Microtus is one of the most taxonomically diverse mammalian genera, including over 60 extant species. These rodents have evolved rapidly, as the genus originated less than 2 million years ago. If these numbers are taken at face value, then an average of 30 microtine speciation events have occurred every million years. One explanation for the rapid rate of cladogenesis in Microtus could be the karyotypic differentiation exhibited across the genus: diploid numbers range from 17 to 64. Despite the striking chromosomal variability within Microtus, phenotypic variation is unremarkable. To determine whether nucleotide substitution rates are also elevated in voles, we sequenced the entire mitochondrial DNA (mtDNA) genome of the Eurasian sibling vole (Microtus rossiaemeridionalis). We compared this genome to another previously sequenced vole mtDNA genome (Microtus kikuchii) and performed pairwise sequence comparisons with the mtDNA genomes of ten additional mammalian genera. We found that microtine mtDNA genomes are evolving more rapidly than any other mammalian lineage we sampled, as gauged by the rate of nucleotide substitution across the entire mtDNA genome as well as at each individual protein-coding gene. Additionally, we compared substitution rates within the cytochrome b gene to seven other rodent genera and found that Microtus mtDNA is evolving fastest. The root cause of accelerated evolution in Microtus remains uncertain, but merits further investigation.