Common phylogenomic approaches for recovering phylogenies are often time-consuming and require annotations for orthologous gene relationships that are not always available. In contrast, alignment-free phylogenomic approaches typically use structure and oligomer frequencies to calculate pairwise distances between species. We have developed an approach to quickly calculate distances between species based on codon aversion.

There are nine herpesviruses known to infect humans, of which Epstein-Barr virus (EBV) is the most widely distributed (>90% of adults infected). This ubiquitous virus is implicated in a variety of cancers and autoimmune diseases. Previous analyses of the EBV genome revealed numerous regions with evidence of generating unusually stable and conserved RNA secondary structures and led to the discovery of a novel class of EBV non-coding (nc)RNAs: the stable intronic sequence (sis)RNAs. To gain a better understanding of the roles of RNA structure in EBV biology and pathogenicity, we revisit EBV using recently developed tools for genome-wide motif discovery and RNA structural characterization. This corroborated previous results and revealed novel motifs with potential functionality; one of which has been experimentally validated. Additionally, since many herpesviruses increasingly rival the seroprevalence of EBV (VZV, HHV-6 and HHV-7 being the most notable), analyses were expanded to include all sequenced human Herpesvirus RefSeq genomes, allowing for genomic comparisons. In total 10 genomes were analyzed, for EBV (types 1 and 2), HCMV, HHV-6A, HHV-6B, HHV-7, HSV-1, HSV-2, KSHV, and VZV. All resulting data were archived in the RNAStructuromeDB (https://structurome.bb.iastate.edu/herpesvirus) to make them available to a wide array of researchers.

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

Mitogen-activated protein kinase (MAPK) cascades are involved with signal transduction in almost every aspect of plant growth and development, as well as biotic and abiotic stress responses. The evolutionary analysis of MAPKs and MKKs in individual or entire plant species has been reported, but the evolutionary patterns in the diverse inbred lines of Brachypodium distachyon are still unclear.

The RIPper (http://theripper.hawk.rocks) is a set of web-based tools designed for analyses of Repeat-Induced Point (RIP) mutations in the genome sequences of Ascomycota. The RIP pathway is a fungal genome defense mechanism that is aimed at identifying repeated and duplicated motifs, into which it then introduces cytosine to thymine transition mutations. RIP thus serves to deactivate and counteract the deleterious consequences of selfish or mobile DNA elements in fungal genomes. The occurrence, genetic context and frequency of RIP mutations are widely used to assess the activity of this pathway in genomic regions of interest. Here, we present a bioinformatics tool that is specifically fashioned to automate the investigation of changes in RIP product and substrate nucleotide frequencies in fungal genomes.

Cytokinins (CKs) are involved in determining the final grain yield in wheat. Multiple gene families are responsible for the controlled production of CKs in plants, including isopentenyl transferases for de novo synthesis, zeatin O-glucosyltransferases for reversible inactivation, β-glucosidases for reactivation, and CK oxidases/dehydrogenases for permanent degradation. Identifying and characterizing the genes of these families is an important step in furthering our understanding of CK metabolism. Using bioinformatics tools, we identified four new TaIPT, four new TaZOG, and 25 new TaGLU genes in common wheat. All of the genes harbored the characteristic conserved domains of their respective gene families. We renamed TaCKX genes on the basis of their true orthologs in rice and maize to remove inconsistencies in the nomenclature. Phylogenetic analysis revealed the early divergence of monocots from dicots, and the gene duplication event after speciation was obvious. Abscisic acid-, auxin-, salicylic acid-, sulfur-, drought- and light-responsive cis-regulatory elements were common to most of the genes under investigation. Expression profiling of CK metabolic gene families was carried out at the seedlings stage in AA genome donor of common wheat. Exogenous application of phytohormones (6-benzylaminopurine, salicylic acid, indole-3-acetic acid, gibberellic acid, and abscisic acid) for 3 h significantly upregulated the transcript levels of all four gene families, suggesting that plants tend to maintain CK stability. A 6-benzylaminopurine-specific maximum fold-change was observed for TuCKX1 and TuCKX3 in root and shoot tissues, respectively; however, the highest expression level was observed in the TuGLU gene family, indicating that the reactivation of the dormant CK isoform is the quickest way to counter external stress. The identification of new CK metabolic genes provides the foundation for their in-depth functional characterization and for elucidating their association with grain yield.

Frankliniella occidentalis (Pergande) is an invasive pest that endangers a wide variety of horticultural and agronomic crops. HSP70 is the most important member of the heat shock protein (HSP) family and plays an important role in insect thermal tolerance. In this study, a new gene encoding HSP70 from F. occidentalis, Fohsp706, was selected from the F. occidentalis transcriptome exposed to thermal stress (40 °C) and cloned by RT-PCR and RACE. Further characterization indicated that Fohsp706 localizes to the cytoplasm and does not contain introns. Quantitative real-time reverse transcriptase PCR indicated that Fohsp706 expression was significantly up-regulated by thermal stress; furthermore, there were significant differences in Fohsp706 expression in adults and second instar nymphs after heat stress. Our results indicated that Fohsp706 contributes to thermotolerance in F. occidentalis and provides another example of how this pest adapts to unfavorable environmental conditions.

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET, CMT, DRM and DNMT2. Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana, including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 (SmDNMT2) to 5,034 bp (SmMET1). The intron number of SmC5-MTases varies between 7 (SmDRM1) and 20 (SmCMT1 and SmCMT2b). These features were similar to their counterparts from Arabidopsis. Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases, implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases, including SmMET1, SmCMT1, SmCMT2a, SmCMT2b, SmCMT3 and SmDRM1, were salicylic acid-responsive, suggesting the involvement of SmC5-MTases in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants.

The CYP75 gene family plays an important role in flavonoid biosynthesis in plants. Little is known about the evolution of the gene family within the grape family. Here, we extracted the CYP75 genes from transcriptome data of 15 grape species and 36 representative genomes from other plants to explore the evolutionary history of the CYP75 gene family in Vitaceae. The structure of the CYP75 protein sequences is highly conserved with the variation mainly occurring in the N terminal and the middle region. The evolutionary analyses suggested classifying the CYP75 gene family into three groups in Vitaceae, namely Vitaceae A1, Vitaceae A2 and Vitaceae B. The Vitaceae A1 and A2 belong to the CYP75A subfamily and the Vitaceae B belongs to the CYP75B subfamily. Within the Vitaceae A1, most Vitaceae taxa present only one copy of the CYP75A protein sequence except for Vitis vinifera with a high number of sequences, which might have originated through recent gene duplications after its split from the other species. Vitaceae A2 contain only CYP75A sequences from Vitaceae sister to one from Camellia sinensis, probably representing a relict lineage. The CYP75B proteins were found to be dominated in Vitaceae and other angiosperms. Our results provide important insights into understanding the evolutionary history of the CYP75 gene family in Vitaceae and other angiosperms.

Biological control agents (BCA) are beneficial organisms that are applied to protect plants from pests. Many fungi of the genus Trichoderma are successful BCAs but the underlying mechanisms are not yet fully understood. Trichoderma cf. atroviride strain LU132 is a remarkably effective BCA compared to T. cf. atroviride strain LU140 but these strains were found to be highly similar at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between separately isolated strains prompted us to undertake a genome comparison study in order to identify DNA polymorphisms. We further investigated if the polymorphisms had functional effects on the phenotypes. The two strains were clearly identified as individuals, exhibiting different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Genome sequencing identified only one non-synonymous single nucleotide polymorphism (SNP) between the strains. Based on this SNP, we successfully designed and validated an RFLP protocol that can be used to differentiate LU132 from LU140 and other Trichoderma strains. This SNP changed the amino acid sequence of SERF, encoded by the previously undescribed single copy gene "small EDRK-rich factor" (serf). A deletion of serf in the two strains did not lead to identical phenotypes, suggesting that, in addition to the single functional SNP between the nearly clonal Trichoderma cf. atroviride strains, other non-genomic factors contribute to their phenotypic variation. This finding is significant as it shows that genomics is an extremely useful but not exhaustive tool for the study of biocontrol complexity and for strain typing.

Mollusk shell mineralization is a tightly controlled process made by shell matrix proteins (SMPs). However, the study of SMPs has been limited to a few model species. In this study, the N66 mRNA of the pearl oyster Pinctada mazatlanica was cloned and functionally characterized. The full sequence of the N66 mRNA comprises 1,766 base pairs, and encodes one N66 protein. A sequence analysis revealed that N66 contained two carbonic anhydrase (CA) domains, a NG domain and several glycosylation sites. The sequence showed similarity to the CA VII but also with its homolog protein nacrein. The native N66 protein was isolated from the shell and identified by mass spectrometry, the peptide sequence matched to the nucleotide sequence obtained. Native N66 is a glycoprotein with a molecular mass of 60-66 kDa which displays CA activity and calcium carbonate precipitation ability in presence of different salts. Also, a recombinant form of N66 was produced in Escherichia coli, and functionally characterized. The recombinant N66 displayed higher CA activity and crystallization capability than the native N66, suggesting that the lack of posttranslational modifications in the recombinant N66 might modulate its activity.

For a long time, turtles of the family Geoemydidae have been considered exceptional because representatives of this family were thought to possess a wide variety of sex determination systems. In the present study, we cytogenetically studied Geoemyda spengleri and G. japonica and re-examined the putative presence of sex chromosomes in Pangshura smithii. Karyotypes were examined by assessing the occurrence of constitutive heterochromatin, by comparative genome hybridization and in situ hybridization with repetitive motifs, which are often accumulated on differentiated sex chromosomes in reptiles. We found similar karyotypes, similar distributions of constitutive heterochromatin and a similar topology of tested repetitive motifs for all three species. We did not detect differentiated sex chromosomes in any of the species. For P. smithii, a ZZ/ZW sex determination system, with differentiated sex chromosomes, was described more than 40 years ago, but this finding has never been re-examined and was cited in all reviews of sex determination in reptiles. Here, we show that the identification of sex chromosomes in the original report was based on the erroneous pairing of chromosomes in the karyogram, causing over decades an error cascade regarding the inferences derived from the putative existence of female heterogamety in geoemydid turtles.

Perna viridis and P. canaliculus are economically and ecologically important species of shellfish. In this study, the complete ribosomal DNA (rDNA) unit sequences of these species were determined for the first time. The gene order, 18S rRNA-internal transcribed spacer (ITS) 1-5.8S rRNA-ITS2-28S rRNA-intergenic spacer (IGS), was similar to that observed in other eukaryotes. The lengths of the P. viridis and P. canaliculus rDNA sequences ranged from 8,432 to 8,616 bp and from 7,597 to 7,610 bp, respectively, this variability was mainly attributable to the IGS region. The putative transcription termination site and initiation site were confirmed. Perna viridis and P. canaliculus rDNA contained two (length: 93 and 40 bp) and one (length: 131 bp) repeat motifs, respectively. Individual intra-species differences mainly involved the copy number of repeat units. In P. viridis, three cytosine-guanine (CpG) sites with sizes of 440, 1,075 and 537 bp were found to cover nearly the entire IGS sequence, whereas in P. canaliculus, two CpG islands with sizes of 361 and 484 bp were identified. The phylogenetic trees constructed with maximum likelihood and neighbour-joining methods and based on ITS sequences were identical and included three major clusters. Species of the same genus were easily clustered together.

The plant hormone auxin regulates numerous aspects of plant growth and development, and small auxin-up RNA (SAUR) is the largest family of early auxin response genes in higher plants. SAUR has been implicated in the regulation of multiple biological processes. However, no comprehensive analysis of SAUR genes has been reported in Lycium ruthenicum. L. ruthenicum is a thorny shrub with very pronounced salt and drought tolerance, and studies have shown that stem thorns are related to drought tolerance in L. ruthenicum. In this study, the identification, phylogenetic analysis, and conserved motif prediction of SAUR genes were extensively explored. Furthermore, the tissue expression patterns of selected SAUR genes were assayed with quantitative real-time polymerase chain reaction (RT-qPCR). A total of 33 putative LrSAURs were identified and divided into three clusters in a phylogenetic tree of L. ruthenicum. MEME analysis identified 10 motifs in L. ruthenicum, and the results suggested that motif 1 and motif 3 were widely distributed. Analyzing the transcriptome data of stem thorns at four developmental stages indicated that LrSAURs were differentially expressed in L. ruthenicum, and could be divided into six expression patterns. The RT-qPCR analysis of 21 genes showed that LrSAUR2, LrSAUR8, LrSAUR9, LrSAUR11, LrSAUR12, and LrSAUR19 were mainly expressed in stems and stem thorns, and may be related to stem thorn development.

In addition to encoding RNA primary structures, genomes also encode RNA secondary and tertiary structures that play roles in gene regulation and, in the case of RNA viruses, genome replication. Methods for the identification of functional RNA structures in genomes typically rely on scanning analysis windows, where multiple partially-overlapping windows are used to predict RNA structures and folding metrics to deduce regions likely to form functional structure. Separate structural models are produced for each window, where the step size can greatly affect the returned model. This makes deducing unique local structures challenging, as the same nucleotides in each window can be alternatively base paired. We are presenting here a new approach where all base pairs from analysis windows are considered and weighted by favorable folding. This results in unique base pairing throughout the genome and the generation of local regions/structures that can be ranked by their propensity to form unusually thermodynamically stable folds. We applied this approach to the Zika virus (ZIKV) and HIV-1 genomes. ZIKV is linked to a variety of neurological ailments including microcephaly and Guillain-Barré syndrome and its (+)-sense RNA genome encodes two, previously described, functionally essential structured RNA regions. HIV, the cause of AIDS, contains multiple functional RNA motifs in its genome, which have been extensively studied. Our approach is able to successfully identify and model the structures of known functional motifs in both viruses, while also finding additional regions likely to form functional structures. All data have been archived at the RNAStructuromeDB (www.structurome.bb.iastate.edu), a repository of RNA folding data for humans and their pathogens.

Next-Generation Sequencing (NGS) technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Several Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. In this study, we describe the biological characteristics, whole genome sequences, and annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), which were isolated from Egyptian soil samples. Growth analyses indicated that the phages BM5 and BM10 have a shorter latent period (25 and 30 min, respectively) and a smaller burst size (103 and 117 PFU, respectively), in comparison to what is typical for Bacillus phages. The genome sizes of the phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with modular organization. Bioinformatic analyses of these genomes enabled the assignment of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of the BM5 and BM10 genome structures, in conjunction with other B. megaterium bacteriophages, revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7, respectively), within the L-cluster, and display different lifestyles (lysogenic and lytic, respectively). Moreover, sequenced phages encode proteins associated with Bacillus pathogenesis. In addition, BM5 does not contain any tRNA sequences, whereas BM10 genome codes for 17 tRNAs.

Catalase (CAT) is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by degradation of hydrogen peroxide to oxygen and water. In the present study, the complete CAT cDNA sequence of Zostera marina was identified through expressed sequence tags (EST) analysis and the rapid amplification of cDNA ends (RACE) technique. The nucleotide sequence of ZmCAT cDNA consisted of 1,816 bp with a 1,434 bp open reading frame (ORF), encoding a polypeptide of 477 amino acid residues, which possessed significant homology to other known plant CATs. The molecular mass of the predicted protein was 55.3 kDa with an estimated isoelectric point of 6.40. Phylogenetic analysis showed that ZmCAT was closely related to CAT from gramineous species. In response to temperature stress, H2O2 and MDA contents in Z. marina increased significantly with cold stress (<10 °C) and heat stress (>25 °C). ZmCAT expression was significantly upregulated at temperatures from 5 to 10 °C and then gradually downregulated, reaching its lowest expression at 30 °C. Recombinant ZmCAT protein exhibited strong antioxidant activity over a wide temperature range, with the highest rZmCAT activity observed at 25 °C and a higher relative activity retained even with heat stress. All these results indicated that ZmCAT was a member of the plant CAT family and involved in minimizing oxidative damage effects in Z. marina under temperature stress.

The identification of functional sequence variations in regulatory DNA regions is one of the major challenges of modern genetics. Here, we report results of a combined multifactor analysis of properties characterizing functional sequence variants located in promoter regions of genes.

The suppressor of the cytokine signaling (SOCS) family of proteins play an essential role in inhibiting cytokine receptor signaling by regulating immune signal pathways. Although SOCS gene functions have been examined extensively, no comprehensive study has been performed on this gene family's molecular evolution in reptiles. In this study, we identified eight canonical SOCS genes using recently-published reptilian genomes. We used phylogenetic analysis to determine that the SOCS genes had highly conserved evolutionary dynamics that we classified into two types. We identified positive SOCS4 selection signals in whole reptile lineages and SOCS2 selection signals in the crocodilian lineage. Selective pressure analyses using the branch model and Z-test revealed that these genes were under different negative selection pressures compared to reptile lineages. We also concluded that the nature of selection pressure varies across different reptile lineages on SOCS3, and the crocodilian lineage has experienced rapid evolution. Our results may provide a theoretical foundation for further analyses of reptilian SOCS genes' functional and molecular mechanisms, as well as their roles in reptile growth and development.

Nudibranchia is an under-studied taxonomic group of gastropods, including more than 3,000 species with colourful and extravagant body shapes and peculiar predatory and defensive strategies. Although symbiosis with bacteria has been reported, no data are available for the nudibranch microbiome nor regarding viruses possibly associated with these geographically widespread species.