Identification of Putative Biosynthetic Gene Clusters for Tolyporphins in Multiple Filamentous Cyanobacteria

Tolyporphins A–R are unusual tetrapyrrole macrocycles produced by the non-axenic filamentous cyanobacterium HT-58-2. A putative biosynthetic gene cluster for biosynthesis of tolyporphins (here termed BGC-1) was previously identified in the genome of HT-58-2. Here, homology searching of BGC-1 in HT-58-2 led to identification of similar BGCs in seven other filamentous cyanobacteria, including strains Nostoc sp. 106C, Nostoc sp. RF31YmG, Nostoc sp. FACHB-892, Brasilonema octagenarum UFV-OR1, Brasilonema octagenarum UFV-E1, Brasilonema sennae CENA114 and Oculatella sp. LEGE 06141, suggesting their potential for tolyporphins production. A similar gene cluster (BGC-2) also was identified unexpectedly in HT-58-2. Tolyporphins BGCs were not identified in unicellular cyanobacteria. Phylogenetic analysis based on 16S rRNA and a common component of the BGCs, TolD, points to a close evolutionary history between each strain and their respective tolyporphins BGC. Though identified with putative tolyporphins BGCs, examination of pigments extracted from three cyanobacteria has not revealed the presence of tolyporphins. Overall, the identification of BGCs and potential producers of tolyporphins presents a collection of candidate cyanobacteria for genetic and biochemical analysis pertaining to these unusual tetrapyrrole macrocycles.

Large-scale annotation and evolution analysis of miRNA in insects

Insects are among the most diverse and successful groups of animals and exhibit great morphological diversity and complexity. The innovation of wings and metamorphosis are some examples of the fascinating biological evolution of insects. Most miRNAs contribute to canalization by conferring robustness to gene networks and thus increase the heritability of important phenotypes. Though previous studies have demonstrated how miRNAs regulate important phenotypes, little is still known about miRNA evolution in insects. Here, we used both small RNA-seq data and homology searching methods to annotate the miRNA repertoires of 152 arthropod species, including 135 insects and 17 non-insect arthropods. We identified 16,212 miRNA genes, and classified them into highly-conserved (62), insect-conserved (90) and lineage-specific (354) miRNA families. The phylogenetic relationship of miRNA binary presence/absence dynamics implies that homoplastic loss of conserved miRNA families tends to occur in far-related morphologically-simplified taxa, including scale insects (Coccoidea) and twisted-wing insects (Strepsiptera), leading to inconsistent phylogenetic tree reconstruction. The common ancestor of Insecta shares 62 conserved miRNA families, of which five were rapidly gained in the early winged-insects (Pterygota). We also detected extensive miRNA losses in Paraneoptera that are correlated with morphological reduction, and miRNA gains in early Endopterygota around the time holometabolous metamorphosis appeared. This was followed by abundant miRNA gains in Hymenoptera and Lepidoptera. In summary, we provide a comprehensive dataset and a detailed evolutionary analysis of miRNAs in insects. These data will be important for future studies on miRNA functions associated with insect morphological innovation and trait biodiversity.

Map Based Cloning of a Dominant Rust Resistance Gene and Mapping of its Duplicated Paralogues in Cultivated Groundnut (Arachis hypogaea L.)

Understanding the mechanism and nature of resistance genes in crop plants is essential for its use in new breeding techniques. Previously, a dominant rust resistance gene was fine-mapped within a 1.2 cM interval in chromosome A03 of groundnut. Here, the rust resistance gene, VG9514-Rgene was isolated through map based cloning. Sequencing of the gene from resistant and susceptible plants revealed non-synonymous mutations in the TIR, NBS and LRR region of R-protein. Genetic mapping of these SNPs-based markers confirmed the position of VG9514-Rgene in between FRS 72 and SSR_GO340445 markers in chromosome A03. Homology searching identified four homologous R-genes in groundnut genome. Of them, Arahy.R8KUIR, Arahy.T6DCA5 and Arahy.ZZ0VZ9 are paralogues. These paralogous genes had several small InDels. Mapping of these InDels-based markers revealed tandem duplication of these paralogous R-genes at distal portion of chromosome A03. Ka/Ks calculation revealed that this unique VG9514-Rgene had undergone positive selection. Homology based structure modelling of this R-protein revealed a typical consensus three dimensional folding of TIR-NBS-LRR protein. Non-synonymous mutations in susceptible version of R-protein were mapped in this protein model and found E268Q mutation in hhGRExE motif, Y309F in RNBS-A motif and I579T in MHD motif of NB-ARC domain are probable candidates for loss of function.

Identification, expression, and artificial selection of silkworm epigenetic modification enzymes

Background Understanding the genetic basis of phenotype variations during domestication and breeding is of great interest. Epigenetics and epigenetic modification enzymes (EMEs) may play a role in phenotypic variations; however, no comprehensive study has been performed to date. Domesticated silkworm (Bombyx mori) may be utilized as a model in determining how EMEs influence domestication traits. Results We identified 44 EMEs in the genome of silkworm (Bombyx mori) using homology searching. Phylogenetic analysis showed that genes in a subfamily among different animals were well clustered, and the expression pattern of EMEs is constant among Bombyx mori, Drosophila melanogaster, and Mus musculus. These are most highly expressed in brain, early embryo, and internal genitalia. By gene-related selective sweeping, we identified five BmEMEs under artificial selection during the domestication and breeding of silkworm. Among these selected genes, BmSuv4–20 and BmDNMT2 harbor selective mutations in their upstream regions that alter transcription factor-binding sites. Furthermore, these two genes are expressed higher in the testis and ovary of domesticated silkworm compared to wild silkworms, and correlations between their expression pattern and meiosis of the sperm and ova were observed. Conclusions The domestication of silkworm has induced artificial selection on epigenetic modification markers that may have led to phenotypic changes during domestication. We present a novel perspective to understand the genetic basis underlying animal domestication and breeding.

Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys-like (Rickettsiales: Anaplasmataceae) in water buffalo from eight provinces of Thailand

Background Anaplasmosis, an animal disease caused by rickettsial bacteria in the genus Anaplasma, is of considerable economic importance in livestock animals in many countries worldwide. The objectives of this study were to determine the identity, prevalence, and geographic distribution of Ehrlichia and Anaplasma in naturally infected water buffalo in Thailand using PCR amplification and sequencing of the 16S ribosomal RNA and heat shock protein groEL genes. A total of 456 buffalo blood samples from Thailand were investigated. Species identification and genetic differentiation of intra-population and inter-population with the global isolates were conducted based on nucleotide sequences. Interplay between the infection and host factors was also assessed. Results Overall, 41% of water buffalo were found to be infected with rickettsial organisms in the family Anaplasmataceae, but Ehrlichia spp., Neorickettsia spp., and Wolbachia spp. were not found in any of the sequenced samples in this study. Female buffalo were more frequently infected with bacteria in the family Anaplasmataceae than males [71 out of 176 females (40.3%) versus 11 out of 47 males (23.4%)]. The Odds Ratio value indicated that the risk of infection for female buffalo was 2.2-fold higher than that for males (p < 0.05). We detected three haplotypes of A. marginale 16S rRNA gene and they were placed in a clade that was closely related to the A. marginale in buffalo in China; and cattle in Thailand, Uganda, and China. Homology searching of groEL sequences against the GenBank™ database using the BLASTn algorithm revealed that the obtained sequences had a high percentage similarity (98.36–99.62%) to A. platys sequences. The groEL sequences of three A. platys-like isolates were clustered in the same clade as the A. platys from the tick Rhipicephalus microplus in China. Conclusions Our data showed that the apparently healthy buffalo were naturally infected by bacteria in the family Anaplasmataceae at a relatively high prevalence. We also report the finding of A. platys-like infections in water buffalo in Thailand for the first time. Water buffalo serving as the reservoir host of anaplasmosis is of concern for managing the disease control and prevention in ruminants.

Analysis of conserved miRNAs in cynomolgus macaque genome using small RNA sequencing and homology searching

MicroRNAs (miRNAs) are important regulators that fine-tune diverse cellular activities. Cynomolgus macaques (Macaca fascicularis) are used extensively in biomedical and pharmaceutical research; however, substantially fewer miRNAs have been identified in this species than in humans. Consequently, we investigated conserved miRNA profiles in cynomolgus macaques by homology searching and small RNA sequencing. In total, 1,455 high-confidence miRNA gene loci were identified, 408 of which were also confirmed by RNA sequencing, including 73 new miRNA loci reported in cynomolgus macaques for the first time. Comparing miRNA expression with age, we found a positive correlation between sequence conservation and expression levels during miRNA evolution. Additionally, we found that the miRNA gene locations in cynomolgus macaque genome were very flexible. Most were embedded in intergenic spaces or introns and clustered together. Several miRNAs were found in certain gene locations, including 64 exon-resident miRNAs, six splice-site-overlapping miRNAs (SO-miRNAs), and two pairs of distinct mirror miRNAs. We also identified 78 miRNA clusters, 68 of which were conserved in the human genome, including 10 large miRNA clusters predicted to regulate diverse developmental and cellular processes in cynomolgus macaque. Thus, this study not only expands the number of identified miRNAs in cynomolgus macaques but also provides clues for future research on the differences in miRNA repertoire between macaques and humans.

CRISPR sequences are sometimes erroneously translated and can contaminate public databases with spurious proteins containing spaced repeats

The genomics era is resulting in the generation of a plethora of biological sequences that are usually stored in public databases. There are many computational tools that facilitate the annotation of these sequences, but sometimes they produce mistakes that enter the databases and can be propagated when erroneous data are used for secondary analyses, such as gene prediction or homology searching. While developing a computational gene finder based on protein-coding sequences, we discovered that the reference UniProtKB protein database is contaminated with some spurious sequences translated from DNA containing clustered regularly interspaced short palindromic repeats. We therefore encourage developers of prokaryotic computational gene finders and protein database curators to consider this source of error.

Assessing the performance of different approaches for functional and taxonomic annotation of metagenomes

Background Metagenomes can be analysed using different approaches and tools. One of the most important distinctions is the way to perform taxonomic and functional assignment, choosing between the use of assembly algorithms or the direct analysis of raw sequence reads instead by homology searching, k-mer analysys, or detection of marker genes. Many instances of each approach can be found in the literature, but to the best of our knowledge no evaluation of their different performances has been carried on, and we question if their results are comparable. Results We have analysed several real and mock metagenomes using different methodologies and tools, and compared the resulting taxonomic and functional profiles. Our results show that database completeness (the representation of diverse organisms and taxa in it) is the main factor determining the performance of the methods relying on direct read assignment either by homology, k-mer composition or similarity to marker genes, while methods relying on assembly and assignment of predicted genes are most influenced by metagenomic size, that in turn determines the completeness of the assembly (the percentage of read that were assembled). Conclusions Although differences exist, taxonomic profiles are rather similar between raw read assignment and assembly assignment methods, while they are more divergent for methods based on k-mers and marker genes. Regarding functional annotation, analysis of raw reads retrieves more functions, but it also makes a substantial number of over-predictions. Assembly methods are more advantageous as the size of the metagenome grows bigger.

Functional interaction between Mus81-Mms4 and Rad52 in Saccharomyces cerevisiae

Mus81-Mms4 is a well conserved DNA structure–specific endonuclease and efficiently cleaves different DNA structures that could arise during the repair of stalled/blocked replication forks and homologous recombination repair. Rad52 is an ezyme that stimulates main steps of DNA sequence-homology searching. In this study, we proved that Rad52 and Mus81-Mms4 possess a species-specific functional interaction, indicating that Rad52 and Mus81-Mms4 collaborate in processing of homologous recombination intermediates.

Fast and sensitive protein sequence homology searches using hierarchical cluster BLAST

The throughput of DNA sequencing continues to increase, allowing researchers to analyze genomes of interest at greater depths. An unintended consequence of this data deluge is the increased cost of analyzing these datasets. As a result, genome and metagenome annotation pipelines are left with a few options: (i) search against smaller reference databases, (ii) use faster, but less sensitive, algorithms to assess sequence similarities, or (iii) invest in computing hardware specifically designed to improve BLAST searches such as GPGPU systems and/or large CPU-rich clusters.We present a pipeline that improves the speed of amino acid sequence homology searches with a minimal decrease in sensitivity and specificity by searching against hierarchical clusters. Briefly, the pipeline requires two homology searches: the first search is against a clustered version of the database and the second is against sequences belonging to clusters with a hit from the first search. We tested this method using two assembled viral metagenomes and three databases (Swiss-Prot, Metagenomes Online, and UniRef100). Hierarchical cluster homology searching proved to be 12-times faster than BLASTp and produced alignments that were nearly identical to BLASTp (precision=0.99; recall=0.97). This approach is ideal when searching large collections of sequences against large databases.